Potential of grasses and rhizosphere bacteria for bioremediation of diesel-contaminated soils

The techniques available for the remediation of environmental accidents involving petroleum hydrocarbons are generally high-cost solutions. A cheaper, practical and ecologically relevant alternative is the association of plants with microorganisms that contribute to the degradation and removal of hydrocarbons from the soil. The growth of three tropical grass species (Brachiaria brizantha, Brachiaria decumbens and Paspalum notatum) and the survival of root-associated bacterial communities was evaluated at different diesel oil concentrations. Seeds of three grass species were germinated in greenhouse and at different doses of diesel (0, 2.5, 5 and 10 g kg-1 soil). Plants were grown for 10 weeks with periodic assessment of germination, growth (fresh and dry weight), height, and number of bacteria in the soil (pots with or without plants). Growth and biomass of B. decumbens and P. notatum declined significantly when planted in diesel-oil contaminated soils. The presence of diesel fuel did not affect the growth of B. brizantha, which was highly tolerant to this pollutant. Bacterial growth was significant (p < 0.05) and the increase was directly proportional to the diesel dose. Bacteria growth in diesel-contaminated soils was stimulated up to 5-fold by the presence of grasses, demonstrating the positive interactions between rhizosphere and hydrocarbon-degrading bacteria in the remediation of diesel-contaminated soils.

Semiquantitative RT-PCR measurement of gene expression in rat tissues including a correction for varying cell size and number

Background: Current methodology of gene expression analysis limits the possibilities of comparison between cells/tissues of organs in which cell size and/or number changes as a consequence of the study (e.g. starvation). A method relating the abundance of specific mRNA copies per cell may allow direct comparison or different organs and/or changing physiological conditions. Methods: With a number of selected genes, we analysed the relationship of the number of bases and the fluorescence recorded at a present level using cDNA standards. A lineal relationship was found between the final number of bases and the length of the transcript. The constants of this equation and those of the relationship between fluorescence and number of bases in cDNA were determined and a general equation linking the length of the transcript and the initial number of copies of mRNA was deduced for a given pre-established fluorescence setting. This allowed the calculation of the concentration of the corresponding mRNAs per g of tissue. The inclusion of tissue RNA and the DNA content per cell, allowed the calculation of the mRNA copies per cell. Results: The application of this procedure to six genes: Arbp, cyclophilin, ChREBP, T4 deiodinase 2, acetyl-CoA carboxylase 1 and IRS-1, in liver and retroperitoneal adipose tissue of food-restricted rats allowed precise measures of their changes irrespective of the shrinking of the tissue, the loss of cells or changes in cell size, factors that deeply complicate the comparison between changing tissue conditions. The percentage results obtained with the present methods were essentially the same obtained with the delta-delta procedure and with individual cDNA standard curve quantitative RT-PCR estimation. Conclusion: The method presented allows the comparison (i.e. as copies of mRNA per cell) between different genes and tissues, establishing the degree of abundance of the different molecular species tested.

Seed-specific silencing of OsMRP5 reduces seed phytic acid and weight in rice.

Phytic acid (PA) is poorly digested by humans and monogastric animals and negatively affects human/animal nutrition and the environment. Rice mutants with reduced PA content have been developed but are often associated with reduced seed weight and viability, lacking breeding value. In the present study, a new approach was explored to reduce seed PA while attaining competitive yield. The OsMRP5 gene, of which mutations are known to reduce seed PA as well as seed yield and viability, was down-regulated specifically in rice seeds by using an artificial microRNA driven by the rice seed specific promoter Ole18. Seed PA contents were reduced by 35.8-71.9% in brown rice grains of transgenic plants compared to their respective null plants (non-transgenic plants derived from the same event). No consistent significant differences of plant height or number of tillers per plant were observed, but significantly lower seed weights (up to 17.8% reduction) were detected in all transgenic lines compared to null plants, accompanied by reductions of seed germination and seedling emergence. It was observed that the silencing of the OsMRP5 gene increased the inorganic P (Pi) levels (up to 7.5 times) in amounts more than the reduction of PA-P in brown rice. This indicates a reduction in P content in other cellular compounds, such as lipids and nucleic acids, which may affect overall seed development. Put together, the present study demonstrated that seed specific silencing of OsMRP5 could significantly reduce the PA content and increase Pi levels in seeds; however, it also significantly lowers seed weight in rice. Discussions were made regarding future directions towards producing agronomically competitive and nutritionally valuable low PA rice.

Stereologic estimates of total spinophilin-immunoreactive spine number in area 9 and the CA1 field: Relationship with the progression of Alzheimer's disease

The loss of presynaptic markers is thought to represent a strong pathologic correlate of cognitive decline in Alzheimer's disease (AD). Spinophilin is a postsynaptic marker mainly located to the heads of dendritic spines. We assessed total numbers of spinophilin-immunoreactive puncta. in the CA I and CA3 fields of hippocampus and area 9 in 18 elderly individuals with various degrees of cognitive decline. The decrease in spinophilin-immunoreactivity was significantly related to both Braak neurofibrillary tangle (NFT) staging and clinical severity but not A beta deposition staging. The total number of spinophilin-immunoreactive puncta in CA I field and area 9 were significantly related to MMSE scores and predicted 23.5 and 61.9% of its variability. The relationship between total number of spinophilin-immunoreactive puncta in CA I field and MMSE scores did not persist when adjusting for Braak NFT staging. In contrast, the total number of spinophilin-immunoreactive puncta in area 9 was still significantly related to the cognitive outcome explaining an extra 9.6% of MMSE and 25.6% of the Clinical Dementia Rating scores variability. Our data suggest that neocortical dendritic spine loss is an independent parameter to consider in AD clinicopathologic correlations.

Identification and validation of copy number variants using SNP genotyping arrays from a large clinical cohort.

BACKGROUND: Genotypes obtained with commercial SNP arrays have been extensively used in many large case-control or population-based cohorts for SNP-based genome-wide association studies for a multitude of traits. Yet, these genotypes capture only a small fraction of the variance of the studied traits. Genomic structural variants (GSV) such as Copy Number Variation (CNV) may account for part of the missing heritability, but their comprehensive detection requires either next-generation arrays or sequencing. Sophisticated algorithms that infer CNVs by combining the intensities from SNP-probes for the two alleles can already be used to extract a partial view of such GSV from existing data sets. RESULTS: Here we present several advances to facilitate the latter approach. First, we introduce a novel CNV detection method based on a Gaussian Mixture Model. Second, we propose a new algorithm, PCA merge, for combining copy-number profiles from many individuals into consensus regions. We applied both our new methods as well as existing ones to data from 5612 individuals from the CoLaus study who were genotyped on Affymetrix 500K arrays. We developed a number of procedures in order to evaluate the performance of the different methods. This includes comparison with previously published CNVs as well as using a replication sample of 239 individuals, genotyped with Illumina 550K arrays. We also established a new evaluation procedure that employs the fact that related individuals are expected to share their CNVs more frequently than randomly selected individuals. The ability to detect both rare and common CNVs provides a valuable resource that will facilitate association studies exploring potential phenotypic associations with CNVs. CONCLUSION: Our new methodologies for CNV detection and their evaluation will help in extracting additional information from the large amount of SNP-genotyping data on various cohorts and use this to explore structural variants and their impact on complex traits.

Linear relations between writhe and minimal crossing number in Conway families of ideal knots and links

We showed earlier how to predict the writhe of any rational knot or link in its ideal geometric configuration, or equivalently the average of the 3D writhe over statistical ensembles of random configurations of a given knot or link (Cerf and Stasiak 2000 Proc. Natl Acad. Sci. USA 97 3795). There is no general relation between the minimal crossing number of a knot and the writhe of its ideal geometric configuration. However, within individual families of knots linear relations between minimal crossing number and writhe were observed (Katritch et al 1996 Nature 384 142). Here we present a method that allows us to express the writhe as a linear function of the minimal crossing number within Conway families of knots and links in their ideal configuration. The slope of the lines and the shift between any two lines with the same

Copy number variation of KIR genes influences HIV-1 control.

A genome-wide screen for large structural variants showed that a copy number variant (CNV) in the region encoding killer cell immunoglobulin-like receptors (KIR) associates with HIV-1 control as measured by plasma viral load at set point in individuals of European ancestry. This CNV encompasses the KIR3DL1-KIR3DS1 locus, encoding receptors that interact with specific HLA-Bw4 molecules to regulate the activation of lymphocyte subsets including natural killer (NK) cells. We quantified the number of copies of KIR3DS1 and KIR3DL1 in a large HIV-1 positive cohort, and showed that an increase in KIR3DS1 count associates with a lower viral set point if its putative ligand is present (p = 0.00028), as does an increase in KIR3DL1 count in the presence of KIR3DS1 and appropriate ligands for both receptors (p = 0.0015). We further provide functional data that demonstrate that NK cells from individuals with multiple copies of KIR3DL1, in the presence of KIR3DS1 and the appropriate ligands, inhibit HIV-1 replication more robustly, and associated with a significant expansion in the frequency of KIR3DS1+, but not KIR3DL1+, NK cells in their peripheral blood. Our results suggest that the relative amounts of these activating and inhibitory KIR play a role in regulating the peripheral expansion of highly antiviral KIR3DS1+ NK cells, which may determine differences in HIV-1 control following infection.

Does the physical disector method provide an accurate estimation of sensory neuron number in rat dorsal root ganglia?

The physical disector is a method of choice for estimating unbiased neuron numbers; nevertheless, calibration is needed to evaluate each counting method. The validity of this method can be assessed by comparing the estimated cell number with the true number determined by a direct counting method in serial sections. We reconstructed a 1/5 of rat lumbar dorsal root ganglia taken from two experimental conditions. From each ganglion, images of 200 adjacent semi-thin sections were used to reconstruct a volumetric dataset (stack of voxels). On these stacks the number of sensory neurons was estimated and counted respectively by physical disector and direct counting methods. Also, using the coordinates of nuclei from the direct counting, we simulate, by a Matlab program, disector pairs separated by increasing distances in a ganglion model. The comparison between the results of these approaches clearly demonstrates that the physical disector method provides a valid and reliable estimate of the number of sensory neurons only when the distance between the consecutive disector pairs is 60 microm or smaller. In these conditions the size of error between the results of physical disector and direct counting does not exceed 6%. In contrast when the distance between two pairs is larger than 60 microm (70-200 microm) the size of error increases rapidly to 27%. We conclude that the physical dissector method provides a reliable estimate of the number of rat sensory neurons only when the separating distance between the consecutive dissector pairs is no larger than 60 microm.

Effect of arcelin protein on the biology of Zabrotes subfasciatus (Boheman 1833), in dry beans

Arcelin is a seed protein found in wild beans (Phaseolus vulgaris) which gives resistance to Mexican bean weevil, Zabrotes subfasciatus (Boheman 1833) (Coleoptera: Bruchidae). Studies were carried out with the objective of estimating the effect of four alleles of protein arcelin (Arc1, Arc2, Arc3 and Arc4) on the biology of Z. subfasciatus. The experiment was carried out in laboratory at Embrapa-Centro Nacional de Pesquisa de Arroz e Feijão, in Santo Antônio de Goiás, GO, Brazil, under non controlled conditions. The highest levels of antibiosis to Z. subfasciatus were observed in Arc1, with reduction in the number of eggs, number of emerged adults, adults longevity. In the line Arc2 only reduction in the number of emerged adults was observed. The lines Arc3 and Arc4 showed low efficiency on the reduction of progeny of Z. subfasciatus and effects in the longevity and egg-adult cycle were not detected. Insect sexual ratio was not altered by the presence of Arc1, Arc2, Arc3 and Arc4 in the seeds.

Toxicity of neem oil to Bemisia tabaci biotype B nymphs reared on dry bean

The objective of this work was to determine the most susceptible nymphal stage of Bemisia tabaci biotype B to neem (Azadirachta indica A. Juss.) oil applied to dry bean (Phaseolus vulgaris L.) in a screenhouse. A solution of commercial oil (Dalneem) extracted from neem seeds was sprayed directly on each nymphal instar at 0, 0.1, 0.25, 0.5, 1 and 2% concentrations for lethal concentration (LC) determination, and at 0, 0.5 and 1% concentrations for lethal time (LT) determination. The number of living and dead nymphs was recorded five days after spraying for LC determination, and daily during six days for LT determination. The LC50 estimated for fourth instar nymphs occurred at 0.56% concentration. For all instars, LC50 and LC95 were estimated at 0.32 and 2.78% concentrations, respectively. The estimated values of LT50 at 1% concentration were 2.46, 4.45, 3.02 and 6.98 days for the first to fourth instars, respectively. The LT50 occurred at five days for 0.5% and at four days for 1% concentration in all instars. A mortality rate of over 80% was observed on the 6th day for the first to third instars at 1% concentration. The first three nymphal stages were more susceptible to neem oil when compared to the fourth nymphal stage.

Identification of zygotic and nucellar seedlings in polyembryonic mango cultivars

The objective of this work was to evaluate the occurrence of polyembryony in the mango cultivars Manila and Ataulfo, and to determine whether seedlings cultured in vitro are zygotic or nucelar. Percentage of polyembryony was calculated and the number of embryos in 100 seeds of each cultivar was recorded. 'Manila' exhibited 97% polyembryony with 3.4 embryos per seed, while 'Ataulfo' had 95% polyembryony with 3.2 embryos per seed. Later, 20 seeds of each cultivar were established in vitro, and it was analyzed those in which all embryos germinated (12 seeds from 'Manila' and 7 from 'Ataulfo'). DNA was extracted from seedling leaf tissue, and its origin was identified with 14 RAPD primers. The polymorphic markers recognized the seedlings of sexual origin in seven of nine 'Manila' polyembryonic seeds, and in four of seven 'Ataulfo' ones. Also, in polyembryonic seeds not all zygotic seedlings were produced by small embryos located at the micropyle.

Germination and development of pecan cultivar seedlings by seed stratification

Abstract: The objective of this work was to evaluate the effect of seed stratification on germination rate, germination speed, and initial development of seedlings of six pecan (Carya illinoinensis) cultivars under subtropical climatic conditions in southern Brazil. For stratification, the seeds were placed in boxes with moist sand, in a cold chamber at 4°C, for 90 days. In the fourteenth week after sowing, the emergence speed index, total emergence, plant height, stem diameter, and number of leaves were evaluated. Seed stratification significantly improves the germination potential and morphological traits of the evaluated cultivars.

KCTD13 is a major driver of mirrored neuroanatomical phenotypes of the 16p11.2 copy number variant.

Copy number variants (CNVs) are major contributors to genetic disorders. We have dissected a region of the 16p11.2 chromosome--which encompasses 29 genes--that confers susceptibility to neurocognitive defects when deleted or duplicated. Overexpression of each human transcript in zebrafish embryos identified KCTD13 as the sole message capable of inducing the microcephaly phenotype associated with the 16p11.2 duplication, whereas suppression of the same locus yielded the macrocephalic phenotype associated with the 16p11.2 deletion, capturing the mirror phenotypes of humans. Analyses of zebrafish and mouse embryos suggest that microcephaly is caused by decreased proliferation of neuronal progenitors with concomitant increase in apoptosis in the developing brain, whereas macrocephaly arises by increased proliferation and no changes in apoptosis. A role for KCTD13 dosage changes is consistent with autism in both a recently reported family with a reduced 16p11.2 deletion and a subject reported here with a complex 16p11.2 rearrangement involving de novo structural alteration of KCTD13. Our data suggest that KCTD13 is a major driver for the neurodevelopmental phenotypes associated with the 16p11.2 CNV, reinforce the idea that one or a small number of transcripts within a CNV can underpin clinical phenotypes, and offer an efficient route to identifying dosage-sensitive loci.

Effect of preharvest spraying with thidiazuron on fruit quality and maturity of apples

Apple trees, cultivars Gala and Fuji, were sprayed at full bloom with thidiazuron (TDZ) at the doses of 0, 5, 10, or 20 g (a.i.) ha-1 and fruit were assessed for quality and maturity. In both cultivars, the increase of TDZ dose had detrimental effects on fruit quality, causing a reduction of fruit red surface and an increase of percentage of fruit that was asymmetrical and with calyx-end rot. TDZ caused an increment of calyx-end aperture that might have increased calyx-end rot. TDZ increased density and the length : diameter (L : D) ratio of the fruit. The number of viable seeds was not affected by TDZ. TDZ resulted in a substantial delay of fruit maturity in 'Gala' but not in 'Fuji'. TDZ also reduced soluble solids content (SSC) in 'Gala' and reduced titratable acidity in both cultivars.

Evaluation of substrates on the emergence of "araticum-de-terra-fria" (Annona emarginata (Schltdl.) H. Rainer) Seedlings

Choosing a substrate is the determinant factor for the seedling producer; thus, the aim of this study was to evaluate the effect of different types of substrates on the emergence of "araticum-de-terra-fria" (Annona emarginata (Schltdl.) H. Rainer) seedlings. The experiment was carried out in a greenhouse and the experimental design was in randomized blocks, with three treatments and five replicates of 72 seeds per plot. The treatments consisted of the following substrates: coconut fiber, vermiculite and Plantmax® Citrus. The number of emerged seedlings was weekly counted for 105 days. Data regarding seedling height were obtained, and the emergence velocity index and mean time, besides total emergence percentage and that over time were calculated. Results from total mean emergence percentage, seedling height, emergence velocity index (EVI), and mean emergence time (MET) were subjected to analysis of variance and means were compared by the Tukey's test at 5% significance. The curves concerning the emergence percentage over time were fit by the logistic growth equation for each treatment and the means of each parameter (A, B, C) were compared by the Duncan's test at 5% significance. The substrates vermiculite led to the highest values of emergence percentage differing from the PlantMax® Citrus, but not of the coconut fiber, however the vermiculite promoted seedling height in a shorter time; therefore, this substrate is recommended for the initial development of "araticum-de-terra-fria" (Annona emarginata (Schltdl.) H. Rainer) seedlings.