High-throughput mapping of a dynamic signaling network in mammalian cells

Signaling pathways transmit information through protein interaction networks that are dynamically regulated by complex extracellular cues. We developed LUMIER (for luminescence-based mammalian interactome mapping), an automated high-throughput technology, to map protein-protein interaction networks systematically in mammalian cells and applied it to the transforming growth factor-beta (TGFbeta) pathway. Analysis using self-organizing maps and k-means clustering identified links of the TGFbeta pathway to the p21-activated kinase (PAK) network, to the polarity complex, and to Occludin, a structural component of tight junctions. We show that Occludin regulates TGFbeta type I receptor localization for efficient TGFbeta-dependent dissolution of tight junctions during epithelial-to-mesenchymal transitions.     

Targeting of cyclic AMP degradation to beta 2-adrenergic receptors by beta-arrestins

Catecholamines signal through the beta2-adrenergic receptor by promoting production of the second messenger adenosine 3',5'-monophosphate (cAMP). The magnitude of this signal is restricted by desensitization of the receptors through their binding to beta-arrestins and by cAMP degradation by phosphodiesterase (PDE) enzymes. We show that beta-arrestins coordinate both processes by recruiting PDEs to activated beta2-adrenergic receptors in the plasma membrane of mammalian cells. In doing so, the beta-arrestins limit activation of membrane-associated cAMP-activated protein kinase by simultaneously slowing the rate of cAMP production through receptor desensitization and increasing the rate of its degradation at the membrane.     

Seasonal speedup along the western flank of the Greenland Ice Sheet

It has been widely hypothesized that a warmer climate in Greenland would increase the volume of lubricating surface meltwater reaching the ice-bedrock interface, accelerating ice flow and increasing mass loss. We have assembled a data set that provides a synoptic-scale view, spanning ice-sheet to outlet-glacier flow, with which to evaluate this hypothesis. On the ice sheet, these data reveal summer speedups (50 to 100%) consistent with, but somewhat larger than, earlier observations. The relative speedup of outlet glaciers, however, is far smaller (<15%). Furthermore, the dominant seasonal influence on Jakobshavn Isbrae's flow is the calving front's annual advance and retreat. With other effects producing outlet-glacier speedups an order of magnitude larger, seasonal melt's influence on ice flow is likely confined to those regions dominated by ice-sheet flow.     

TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder characterized pathologically by ubiquitinated TAR DNA binding protein (TDP-43) inclusions. The function of TDP-43 in the nervous system is uncertain, and a mechanistic role in neurodegeneration remains speculative. We identified neighboring mutations in a highly conserved region of TARDBP in sporadic and familial ALS cases. TARDBPM337V segregated with disease within one kindred and a genome-wide scan confirmed that linkage was restricted to chromosome 1p36, which contains the TARDBP locus. Mutant forms of TDP-43 fragmented in vitro more readily than wild type and, in vivo, caused neural apoptosis and developmental delay in the chick embryo. Our evidence suggests a pathophysiological link between TDP-43 and ALS.     

Cross-species identification of Mendel's I locus

A key gene involved in plant senescence, mutations of which partially disable chlorophyll catabolism and confer stay-green leaf and cotyledon phenotypes, has been identified in Pisum sativum, Arabidopsis thaliana, and Festuca pratensis by using classical and molecular genetics and comparative genomics. A stay-green locus in F. pratensis is syntenically equivalent to a similar stay-green locus on rice chromosome 9. Functional testing in Arabidopsis of a homolog of the rice candidate gene revealed (i) senescence-associated gene expression and (ii) a stay-green phenotype after RNA interference silencing. Genetic mapping in pea demonstrated cosegregation with the yellow/green cotyledon polymorphism (I/i) first reported by Gregor Mendel in 1866.     

Evaluation of spectrophotometric and HPLC methods for shikimic acid determination in plants: models in glyphosate-resistant and -susceptible crops

Endogenous shikimic acid determinations are routinely used to assess the efficacy of glyphosate in plants. Numerous analytical methods exist in the public domain for the detection of shikimic acid, yet the most commonly cited comprise spectrophotometric and high-pressure liquid chromatography (HPLC) methods. This paper compares an HPLC and two spectrophotometric methods (Spec 1 and Spec 2) and assesses the effectiveness in the detection of shikimic acid in the tissues of glyphosate-treated plants. Furthermore, the study evaluates the versatility of two acid-based shikimic acid extraction methods and assesses the longevity of plant extract samples under different storage conditions. Finally, Spec 1 and Spec 2 are further characterized with respect to (1) the capacity to discern between shikimic acid and chemically related alicyclic hydroxy acids, (2) the stability of the chromophore (t1/2), (3) the detection limits, and (4) the cost and simplicity of undertaking the analytical procedure. Overall, spectrophotometric methods were more cost-effective and simpler to execute yet provided a narrower detection limit compared to HPLC. All three methods were specific to shikimic acid and detected the compound in the tissues of glyphosate-susceptible crops, increasing exponentially in concentration within 24 h of glyphosate application and plateauing at approximately 72 h. Spec 1 estimated more shikimic acid in identical plant extract samples compared to Spec 2 and, likewise, HPLC detection was more effective than spectrophotometric determinations. Given the unprecedented global adoption of glyphosate-resistant crops and concomitant use of glyphosate, an effective and accurate assessment of glyphosate efficacy is important. Endogenous shikimic acid determinations are instrumental in corroborating the efficacy of glyphosate and therefore have numerous applications in herbicide research and related areas of science as well as resolving many commercial issues as a consequence of glyphosate utilization.     

The effect of blanket bog drainage on habitat condition and on sheep grazing, evidence from a Welsh upland bog

There has been increasing interest in potential benefits to be gained by restoring damaged peatlands, with the majority of funding currently being driven by the recovery of protected habitats. However, few data are available linking vegetation community declines with peatland drainage, and so the potential for recovery remains difficult to predict. Surprisingly, there is a similar paucity of research demonstrating the extent of drainage impacts on grazing conditions for livestock, despite this being a priority amongst land managers. We attempt to address these two knowledge gaps, first by investigating whether ditches in blanket bog habitat have improved or increased grazing for sheep, and second by assessing the impact of ditches on the condition of vegetation communities. At an extensively drained upland blanket bog in Wales, currently undergoing ditch blocking restoration, we collected vegetation and sheep occurrence data across five catchments and over 2 years. We present evidence that drained areas had remained relatively wet and were less preferred by sheep. Furthermore, our results show that while sheep preferred graminoid-rich areas, drainage did not increase the abundance of this species group. Drainage at the site has, however, resulted in some degradation in blanket bog vegetation adjacent to ditches; although at the landscape scale, historic high grazing levels appear to explain much of the current poor ecological condition of the site. We conclude that drain-blocking restoration should not reduce sheep grazing conditions beyond their current relatively poor state, and thus in this respect may not represent a threat to hill farming productivity.     

Vitamin D mushrooms: comparison of the composition of button mushrooms (Agaricus bisporus) treated postharvest with UVB light or sunlight

This study compared the compositional changes in mushrooms exposed to sunlight with those occurring after commercial ultraviolet (UV) light processing. Button mushrooms (75 kg) were processed in the presence or absence of UVB light; a third group was exposed to direct sunlight. Mushroom composition was evaluated using chemical analyses. Vitamin D concentrations were 5, 410, and 374 μg/100 g (dw) in control, UVB, and sunlight groups, respectively. On a dry weight basis, no significant changes in vitamin C, folate, vitamins B(6), vitamin B(5), riboflavin, niacin, amino acids, fatty acids, ergosterol, or agaritine were observed following UVB processing. Sunlight exposure resulted in a 26% loss of riboflavin, evidence of folate oxidation, and unexplained increases in ergosterol (9.5%). It was concluded that compositional effects of UVB light are limited to changes in vitamin D and show no detrimental changes relative to natural sunlight exposure and, therefore, provide important information relevant to the suitability and safety of UVB light technology for vitamin D enhanced mushrooms.     

Inheritance and physiological basis for 2,4-D resistance in prickly lettuce (Lactuca serriola L.)

Experiments were conducted to determine the inheritance and physiological basis for resistance to the synthetic auxinic herbicide (2,4-dichlorophenoxy)acetic acid (2,4-D) in a prickly lettuce biotype. Inheritance of 2,4-D resistance in prickly lettuce is governed by a single codominant gene. Absorption and translocation were conducted using (14)C-2,4-D applied to 2,4-D-resistant and -susceptible biotypes. At 96 h after treatment (HAT), the resistant biotype absorbed less applied 2,4-D and retained more 2,4-D in the treated portion of the leaf compared to the susceptible biotype. The resistant biotype translocated less applied 2,4-D to leaves above the treated leaf and crown at 96 HAT compared to the susceptible biotype. No difference in the rate of metabolism of 2,4-D was observed between the two biotypes. Resistance to 2,4-D appears to originate from a reduced growth deregulatory and overstimulation response compared to the susceptible biotype, resulting in lower translocation of 2,4-D in the resistant prickly lettuce biotype.     

Spatial heterogeneity in the relocation of added C within the structure of an upland grassland soil

A pulse of ¹³CO₂ was added to the above ground vegetation in an upland grassland to determine the effects of faunal diversity on the flux of carbon to the surface horizons of the soil. Faunal diversity was manipulated by liming and biocide treatments for three years prior to the pulse addition. The relocation of ¹³C within roots and rhizosphere soil was determined by analysis of samples of bulk soil and of specific features identified on soil thin sections on four dates after the addition of the ¹³CO₂ pulse. Analysis of bulk soils showed only a small enrichment in ¹³C and no significant effects of the treatments. Analysis by isotope ratio mass spectrometry of the products of in situ laser combustion of root material and aggregates formed from faunal excrement showed that the distribution of the newly photosynthesised ¹³C is very localised, with large spatial variability in soil and root δ¹³C at scales of less than 1 mm. δ¹³C values ranged from the natural abundance level of around −28‰ to −4.9‰ in roots and to −8.4‰ in aggregates. The small pulse and large spatial variability masked any effects of the liming and biocide treatments in these soils. However, the variability in the relocation of newly photosynthesised carbon may help to explain the large spatial variability found in bacterial numbers at the sub-mm scale within soils and emphasises the importance of the accessibility of substrates to decomposers in undisturbed structured soils.