Intron-derived small RNAs for silencing viral RNAs in mosquito cells

Aedes aegypti and Ae. albopictus are the main vectors of mosquito-borne viruses of medical and veterinary significance. Many of these viruses have RNA genomes. Exogenously provided, e.g. transgene encoded, small RNAs could be used to inhibit virus replication, breaking the transmission cycle. We tested, in Ae. aegypti and Ae. albopictus cell lines, reporter-based strategies for assessing the ability of two types of small RNAs to inhibit a chikungunya virus (CHIKV) derived target. Both types of small RNAs use a Drosophila melanogaster pre-miRNA-1 based hairpin for their expression, either with perfect base-pairing in the stem region (shRNA-like) or containing two mismatches (miRNA-like). The pre-miRNA-1 stem loop structure was encoded within an intron; this allows co-expression of one or more proteins, e.g. a fluorescent protein marker tracking the temporal and spatial expression of the small RNAs in vivo. Three reporter-based systems were used to assess the relative silencing efficiency of ten shRNA-like siRNAs and corresponding miRNA-like designs. Two systems used a luciferase reporter RNA with CHIKV RNA inserted either in the coding sequence or within the 3’ UTR. A third reporter used a CHIKV derived split replication system. All three reporters demonstrated that while silencing could be achieved with both miRNA-like and shRNA-like designs, the latter were substantially more effective. Dcr-2 was required for the shRNA-like siRNAs as demonstrated by loss of inhibition of the reporters in Dcr-2 deficient cell lines. These positive results in cell culture are encouraging for the potential use of this pre-miRNA-1-based system in transgenic mosquitoes.     

Complementary genetic and genomic approaches help characterize the linkage group I seed protein QTL in soybean

Background

Cotton fibers (produced by Gossypium species) are the premier natural fibers for textile production. The two tetraploid species, G. barbadense (Gb) and G. hirsutum (Gh), differ significantly in their fiber properties, the former having much longer, finer and stronger fibers that are highly prized. A better understanding of the genetics and underlying biological causes of these differences will aid further improvement of cotton quality through breeding and biotechnology. We evaluated an inter-specific Gh × Gb recombinant inbred line (RIL) population for fiber characteristics in 11 independent experiments under field and glasshouse conditions. Sites were located on 4 continents and 5 countries and some locations were analyzed over multiple years.

Results

The RIL population displayed a large variability for all major fiber traits. QTL analyses were performed on a per-site basis by composite interval mapping. Among the 651 putative QTLs (LOD > 2), 167 had a LOD exceeding permutation based thresholds. Coincidence in QTL location across data sets was assessed for the fiber trait categories strength, elongation, length, length uniformity, fineness/maturity, and color. A meta-analysis of more than a thousand putative QTLs was conducted with MetaQTL software to integrate QTL data from the RIL and 3 backcross populations (from the same parents) and to compare them with the literature. Although the global level of congruence across experiments and populations was generally moderate, the QTL clustering was possible for 30 trait x chromosome combinations (5 traits in 19 different chromosomes) where an effective co-localization of unidirectional (similar sign of additivity) QTLs from at least 5 different data sets was observed. Most consistent meta-clusters were identified for fiber color on chromosomes c6, c8 and c25, fineness on c15, and fiber length on c3.

Conclusions

Meta-analysis provided a reliable means of integrating phenotypic and genetic mapping data across multiple populations and environments for complex fiber traits. The consistent chromosomal regions contributing to fiber quality traits constitute good candidates for the further dissection of the genetic and genomic factors underlying important fiber characteristics, and for marker-assisted selection.     

Mutations in the fumarate hydratase gene cause hereditary leiomyomatosis and renal cell cancer in families in North America

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is an autosomal dominant disorder characterized by smooth-muscle tumors of the skin and uterus and/or renal cancer. Although the identification of germline mutations in the fumarate hydratase (FH) gene in European families supports it as the susceptibility gene for HLRCC, its role in families in North America has not been studied. We screened for germline mutations in FH in 35 families with cutaneous leiomyomas. Sequence analysis revealed mutations in FH in 31 families (89%). Twenty different mutations in FH were identified, of which 18 were novel. Of these 20 mutations, 2 were insertions, 5 were small deletions that caused frameshifts leading to premature truncation of the protein, and 13 were missense mutations. Eleven unrelated families shared a common mutation: R190H. Eighty-one individuals (47 women and 34 men) had cutaneous leiomyomas. Ninety-eight percent (46/47) of women with cutaneous leiomyomas also had uterine leiomyomas. Eighty-nine percent (41/46) of women with cutaneous and uterine leiomyomas had a total hysterectomy, 44% at age < or =30 years. We identified 13 individuals in 5 families with unilateral and solitary renal tumors. Seven individuals from four families had papillary type II renal cell carcinoma, and another individual from one of these families had collecting duct carcinoma of the kidney. The present study shows that mutations in FH are associated with HLRCC in North America. HLRCC is associated with clinically significant uterine fibroids and aggressive renal tumors. The present study also expands the histologic spectrum of renal tumors and FH mutations associated with HLRCC.     

Influence of spatially dependent,modeled soil carbon emission factors on life‐cycle greenhouse gas emissions of corn and cellulosic ethanol

Converting land to biofuel feedstock production incurs changes in soil organic carbon (SOC) that can influence biofuel life‐cycle greenhouse gas (GHG) emissions. Estimates of these land use change (LUC) and life‐cycle GHG emissions affect biofuels' attractiveness and eligibility under a number of renewable fuel policies in the USA and abroad. Modeling was used to refine the spatial resolution and depth extent of domestic estimates of SOC change for land (cropland, cropland pasture, grassland, and forest) conversion scenarios to biofuel crops (corn, corn stover, switchgrass, Miscanthus, poplar, and willow) at the county level in the USA. Results show that in most regions, conversions from cropland and cropland pasture to biofuel crops led to neutral or small levels of SOC sequestration, while conversion of grassland and forest generally caused net SOC loss. SOC change results were incorporated into the Greenhouse Gases, Regulated Emissions, and Energy use in Transportation (GREET) model to assess their influence on life‐cycle GHG emissions of corn and cellulosic ethanol. Total LUC GHG emissions (g CO₂eq MJ^?1) were 2.1–9.3 for corn‐, ?0.7 for corn stover‐, ?3.4 to 12.9 for switchgrass‐, and ?20.1 to ?6.2 for Miscanthus ethanol; these varied with SOC modeling assumptions applied. Extending the soil depth from 30 to 100 cm affected spatially explicit SOC change and overall LUC GHG emissions; however, the influence on LUC GHG emission estimates was less significant in corn and corn stover than cellulosic feedstocks. Total life‐cycle GHG emissions (g CO₂eq MJ^?1, 100 cm) were estimated to be 59–66 for corn ethanol, 14 for stover ethanol, 18–26 for switchgrass ethanol, and ?7 to ?0.6 for Miscanthus ethanol. The LUC GHG emissions associated with poplar‐ and willow‐derived ethanol may be higher than that for switchgrass ethanol due to lower biomass yield.     

Chronic Treatment of Rats with SCH-23390 or Raclopride Does Not Affect the Concentrations of DARPP-32 or Its mRNA in Dopamine-Innervated Brain Regions

DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, Mr = 32,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is a neuronal phosphoprotein that is enriched in neurons which possess dopamine D1 receptors, particularly striatonigral neurons. In rat brain slices, the phosphorylation state of DARPP-32 is regulated by dopamine, acting through the dopamine D1 receptor and the adenylyl cyclase system. This study reports that chronic blockade (21 days) of either dopamine D1 receptors by SCH-23390 or dopamine D2 receptors by raclopride does not affect the concentrations of DARPP-32 in specific rat brain regions (striatum, thalamus, hippocampus, frontal cerebral cortical pole). Northern blot analysis indicates that the steady-state level of DARPP-32 mRNA in striatum is also unchanged by these treatments.     

The Influence of Hunting on Antipredator Behavior in Central African Monkeys and Duikers

Many animals can adjust their behavioral strategies to reduce predation risk. We investigated whether rain forest monkeys and duikers alter their antipredatory behavior in response to hunting by humans in southwestern Gabon. We compared monkey and duiker responses to human observers in an area where hunting is prohibited, to those in a nearby area where hunting pressure is moderate but spatially variable. The results of our study indicate that monkeys become more secretive when hunted, commencing alarm calls only when at a certain distance (typically > 50 m) from humans. We found no difference in monkey group size between hunted and no-hunting areas. In no-hunting areas, duikers often freeze in response to approaching observers, but in hunted areas they abandon this strategy and rapidly flee from humans. Duikers also whistle more often in areas where they are hunted frequently. Our findings have at least two important implications. First, behavioral observations of monkeys and duikers may be useful in gauging local hunting intensity in African rain forests. Second, duiker densities are likely to be overestimated in hunted areas, where they more readily flee and whistle, and underestimated in no-hunting areas, where they rely on freezing behavior to avoid detection. Because behavioral adaptations to hunting vary both among species and localities, these differences should be considered when attempting to derive population-density estimates for forest wildlife.     

Application of semi-quantitative electrofishing to juvenile salmonid stock surveys

A semi-quantitative electrofishing technique is described, which has applications for assessing distribution and densities of 0+ salmonids in streams. Linear regressions of logarithmically-transformed data satisfactorily described the relationship between numbers of 0+ Atlantic salmon ( Salmo salar L.) and 0+ brown trout ( Salmo trutta L.) captured in a standard 5 min fishing and quantitative population estimates obtained using a commonly used method. Applications of the semi-quantitative technique to management are discussed and a categorization system proposed.     

Role of the MRP1/ABCC1 multidrug transporter protein in cancer

Multidrug resistance is a major obstacle to cancer treatment and leads to poor prognosis for the patient. Multidrug resistance-associated protein 1 (MRP1) transports a wide range of therapeutic agents as well as diverse physiological substrates and may play a role in the development of drug resistance in several cancers including those of the lung, breast and prostate, as well as childhood neuroblastoma. The majority of patients with neuroblastoma present with widely disseminated disease at diagnosis and despite intensive treatment, the prognosis for such patients is dismal. There is increasing evidence that MRP1 is a MYCN target gene involved in the development of multidrug resistance in neuroblastoma. Given the importance of MRP1 overexpression in neuroblastoma, MRP1 inhibition may be a clinically relevant approach to improving patient outcome in this disease.     

Metabolism of Caffeine and Related Purine Alkaloids in Leaves of Tea (Camellia sinensis L.)

Purine alkaloid catabolism pathways in young, mature and agedleaves of tea (Camellia sinensis L.) were investigated by incubatingleaf sections with ¹⁴C-labelled theobromine, caffeine, theophyllineand xanthine. Incorporation of label into CO₂ was determinedand methanol-soluble metabolites were analysed by high-performanceliquid chromatography-radiocounting and thin layer chro-matography.The data obtained demonstrate that theobromine is the immediateprecursor of caffeine, which accumulates in tea leaves becauseits conversion to theophylline is the rate limiting step inthe purine alkaloid catabolism pathway. The main fate of [8-¹⁴C]theophyllineincubated with mature and aged leaves, and to a lesser extentyoung leaves, is conversion to 3-methylxanthine and onto xanthinewhich is degraded to ¹⁴CO₂ via the purine catabolism pathway.However, with young leaves, sizable amounts of [8-¹⁴C]-theophyllinewere salvaged for the synthesis of caffeine via a 3-methylxanthine     

Rates of root and organism growth, soil conditions, and temporal and spatial development of the rhizosphere

BACKGROUND: Roots growing in soil encounter physical, chemical and biological environments that influence their rhizospheres and affect plant growth. Exudates from roots can stimulate or inhibit soil organisms that may release nutrients, infect the root, or modify plant growth via signals. These rhizosphere processes are poorly understood in field conditions. SCOPE AND AIMS: We characterize roots and their rhizospheres and rates of growth in units of distance and time so that interactions with soil organisms can be better understood in field conditions. We review: (1) distances between components of the soil, including dead roots remnant from previous plants, and the distances between new roots, their rhizospheres and soil components; (2) characteristic times (distance(2)/diffusivity) for solutes to travel distances between roots and responsive soil organisms; (3) rates of movement and growth of soil organisms; (4) rates of extension of roots, and how these relate to the rates of anatomical and biochemical ageing of root tissues and the development of the rhizosphere within the soil profile; and (5) numbers of micro-organisms in the rhizosphere and the dependence on the site of attachment to the growing tip. We consider temporal and spatial variation within the rhizosphere to understand the distribution of bacteria and fungi on roots in hard, unploughed soil, and the activities of organisms in the overlapping rhizospheres of living and dead roots clustered in gaps in most field soils. CONCLUSIONS: Rhizosphere distances, characteristic times for solute diffusion, and rates of root and organism growth must be considered to understand rhizosphere development. Many values used in our analysis were estimates. The paucity of reliable data underlines the rudimentary state of our knowledge of root-organism interactions in the field.