Odin (ANKS1A) is a Src family kinase target in colorectal cancer cells

Background

Src family kinases (SFK) are implicated in the development of some colorectal cancers (CRC). One SFK member, Lck, is not detectable in normal colonic epithelium, but becomes aberrantly expressed in a subset of CRCs. Although SFK have been extensively studied in fibroblasts and different types of immune cells, their physical and functional targets in many epithelial cancers remain poorly characterised.

Results

64 CRC cell lines were tested for expression of Lck. SW620 CRC cells, which express high levels of Lck and also contain high basal levels of tyrosine phosphorylated (pY) proteins, were then analysed to identify novel SFK targets. Since SH2 domains of SFK are known to often bind substrates after phosphorylation by the kinase domain, the LckSH2 was compared with 14 other SH2s for suitability as affinity chromatography reagent. Mass spectrometric analyses of LckSH2-purified pY proteins subsequently identified several proteins readily known as SFK kinase substrates, including cortactin, Tom1L1 (SRCASM), GIT1, vimentin and AFAP1L2 (XB130). Additional proteins previously reported as substrates of other tyrosine kinase were also detected, including the EGF and PDGF receptor target Odin. Odin was further analysed and found to contain substantially less pY upon inhibition of SFK activity in SW620 cells, indicating that it is a formerly unknown SFK target in CRC cells.

Conclusion

Rapid identification of known and novel SFK targets in CRC cells is feasible with SH2 domain affinity chromatography. The elucidation of new SFK targets like Odin in epithelial cancer cells is expected to lead to novel insight into cancer cell signalling mechanisms and may also serve to indicate new biomarkers for monitoring tumor cell responses to drug treatments.     

Crystal structure of methylenetetrahydromethanopterin reductase (Mer) in complex with coenzyme F420: Architecture of the F420/FMN binding site of enzymes within the nonprolyl cis-peptide containing bacterial luciferase family

Methylenetetratetrahydromethanopterin reductase (Mer) is involved in CO(2) reduction to methane in methanogenic archaea and catalyses the reversible reduction of methylenetetrahydromethanopterin (methylene-H(4)MPT) to methyl-H(4)MPT with coenzyme F(420)H(2), which is a reduced 5'-deazaflavin. Mer was recently established as a TIM barrel structure containing a nonprolyl cis-peptide bond but the binding site of the substrates remained elusive. We report here on the crystal structure of Mer in complex with F(420) at 2.6 A resolution. The isoalloxazine ring is present in a pronounced butterfly conformation, being induced from the Re-face of F(420) by a bulge that contains the non-prolyl cis-peptide bond. The bindingmode of F(420) is very similar to that in F(420)-dependent alcohol dehydrogenase Adf despite the low sequence identity of 21%. Moreover, binding of F(420) to the apoenzyme was only associated with minor conformational changes of the polypeptide chain. These findings allowed us to build an improved model of FMN into its binding site in bacterial luciferase, which belongs to the same structural family as Mer and Adf and also contains a nonprolyl cis-peptide bond in an equivalent position.     

Dominant role for TL1A/DR3 pathway in IL-12 plus IL-18-induced IFN-gamma production by peripheral blood and mucosal CCR9+ T lymphocytes

The TNF-like cytokine TL1A augments IFN-gamma production by anti-CD3 plus anti-CD28 and IL-12/IL-18-stimulated peripheral blood (PB) T cells. However, only a small subset of PB T cells respond to TL1A stimulation with IFN-gamma production. PB CCR9+ T cells represent a small subset of circulating T cells with mucosal T cell characteristics and a Th1/Tr1 cytokine profile. In the current study, we show that TL1A enhanced IFN-gamma production by TCR- or CD2/CD28-stimulated CCR9(+)CD4+ PB T cells. However, TL1A had the most pronounced effect on augmenting IFN-gamma production by IL-12/IL-18-primed CCR9(+)CD4+ PB T cells. TL1A enhanced both the percentage and the mean fluorescence intensity of IFN-gamma in CCR9(+)CD4+ T cells as assessed by intracellular cytokine staining. IL-12 plus IL-18 up-regulated DR3 expression in CCR9(+)CD4+ T cells but had negligible effect on CCR9(-)CD4+ T cells. CCR9(+)CD4+ T cells isolated from the small intestine showed a 37- to 105-fold enhancement of IFN-gamma production when TL1A was added to the IL-12/IL18 cytokine combination. Cell membrane-expressed TL1A was preferentially expressed in CCR9(+)CD4+ PB T cells, and a blocking anti-TL1A mAb inhibited IFN-gamma production by cytokine-primed CCR9(+)CD4+ T cells by approximately 50%. Our data show that the TL1A/DR3 pathway plays a dominant role in the ultimate level of cytokine-induced IFN-gamma production by CCR9+ mucosal and gut-homing PB T cells and could play an important role in Th1-mediated intestinal diseases, such as Crohn's disease, where increased expression of IL-12, IL-18, TL1A, and DR3 converge in the inflamed intestinal mucosa.     

The EF-Hand Ca2+ Binding Protein MICU Choreographs Mitochondrial Ca2+ Dynamics in Arabidopsis

Plant organelle function must constantly adjust to environmental conditions, which requires dynamic coordination. Ca²⁺ signaling may play a central role in this process. Free Ca²⁺ dynamics are tightly regulated and differ markedly between the cytosol, plastid stroma, and mitochondrial matrix. The mechanistic basis of compartment-specific Ca²⁺ dynamics is poorly understood. Here, we studied the function of At-MICU, an EF-hand protein of Arabidopsis thaliana with homology to constituents of the mitochondrial Ca²⁺ uniporter machinery in mammals. MICU binds Ca²⁺ and localizes to the mitochondria in Arabidopsis. In vivo imaging of roots expressing a genetically encoded Ca²⁺ sensor in the mitochondrial matrix revealed that lack of MICU increased resting concentrations of free Ca²⁺ in the matrix. Furthermore, Ca²⁺ elevations triggered by auxin and extracellular ATP occurred more rapidly and reached higher maximal concentrations in the mitochondria of micu mutants, whereas cytosolic Ca²⁺ signatures remained unchanged. These findings support the idea that a conserved uniporter system, with composition and regulation distinct from the mammalian machinery, mediates mitochondrial Ca²⁺ uptake in plants under in vivo conditions. They further suggest that MICU acts as a throttle that controls Ca²⁺ uptake by moderating influx, thereby shaping Ca²⁺ signatures in the matrix and preserving mitochondrial homeostasis. Our results open the door to genetic dissection of mitochondrial Ca²⁺ signaling in plants.     

Quantification of Gram-negative sulphate-reducing bacteria in rice field soil by 16S rRNA gene-targeted real-time PCR

For the quantification of Gram-negative sulphate reducers in rice fields, 11 real-time PCR assays were established targeting 16S rRNA genes combined with SybrGreen detection. Three of these assays were specific for the "main" groups, i.e. the Desulfovibrionaceae, the Desulfobacteraceae and Desulfobulbus sp., whereas eight assays were developed for subgroups within the first two main groups. The detection limits of the assays were between 2 x 10(5) and 4 x 10(3) targets g(-1) (wet weight) or less than 0.02% of the eubacterial 16S rDNA targets in bulk soil, rhizosphere soil and rice root DNA extracts. Analysis of soil spiked with defined cell numbers of sulphate-reducing bacteria showed good correlation of measured target numbers to amended cells. In rice field bulk and rhizosphere soil, the Desulfobacteraceae were the predominant main group with target numbers of 6.4 x 10(7) (+/-1.0 x 10(7)) and 7.5 x 10(7) (+/-1.7 x 10(7)), respectively. Within this group the Desulforhabdus/Synthrophobacter assemblage and Desulfobacterium sp. were predominant. At the rice roots, the three main groups were abundant in similar numbers (approx. 1.0 x 10(8)) indicating that the relative abundance of the Desulfovibrionaceae and also of Desulfobulbus sp. was increased, relatively to the Desulfobacteraceae. Within the Desulfovibrionaceae the subgroup was predominant that was detected by assay DSV-II. This assay detects many from rice field soil isolated Desulfovibrio-strains and molecular retrieved sequences. Therefore these organisms that were already detected in the rice field environment by isolation and by molecular techniques are indeed best adapted to the conditions provided by the rice roots.     

TaqMan Real-Time PCR Assays To Assess Arbuscular Mycorrhizal Responses to Field Manipulation of Grassland Biodiversity: Effects of Soil Characteristics,Plant Species Richness,and Functional Traits

Large-scale (temporal and/or spatial) molecular investigations of the diversity and distribution of arbuscular mycorrhizal fungi (AMF) require considerable sampling efforts and high-throughput analysis. To facilitate such efforts, we have developed a TaqMan real-time PCR assay to detect and identify AMF in environmental samples. First, we screened the diversity in clone libraries, generated by nested PCR, of the nuclear ribosomal DNA internal transcribed spacer (ITS) of AMF in environmental samples. We then generated probes and forward primers based on the detected sequences, enabling AMF sequence type-specific detection in TaqMan multiplex real-time PCR assays. In comparisons to conventional clone library screening and Sanger sequencing, the TaqMan assay approach provided similar accuracy but higher sensitivity with cost and time savings. The TaqMan assays were applied to analyze the AMF community composition within plots of a large-scale plant biodiversity manipulation experiment, the Jena Experiment, primarily designed to investigate the interactive effects of plant biodiversity on element cycling and trophic interactions. The results show that environmental variables hierarchically shape AMF communities and that the sequence type spectrum is strongly affected by previous land use and disturbance, which appears to favor disturbance-tolerant members of the genus Glomus. The AMF species richness of disturbance-associated communities can be largely explained by richness of plant species and plant functional groups, while plant productivity and soil parameters appear to have only weak effects on the AMF community.Arbuscular mycorrhizae are mutualistic associations between roots of plants and fungi that have been present for more than 400 million years (54). Approximately 80% of examined land plants (71), and almost all fungi of the phylum Glomeromycota (60), are capable of forming such associations. The main benefit of this relationship for plants is that it facilitates their acquisition of nutrients (especially P and N), while the fungus receives photoassimilates (7, 62). About 200 Glomeromycota species have been described to date, based on spore morphology (http://www.lrz-muenchen.de/∼schuessler/amphylo/amphylogeny.html), but there is increasing molecular evidence of significantly higher diversity in arbuscular mycorrhizal fungi (AMF) (10, 72).Diverse AMF communities have been detected in a wide range of plant communities (inter alia grasslands, boreal forests, and tropical communities; for an overview, see reference 48). Hence, AMF have been considered to be tolerant of wide ranges of ecological conditions and capable of associating with diverse plant partners. Identifying the factors regulating their community assemblages is challenging, but AMF community composition has been shown to be influenced by plant species diversity (e.g., see references 10, 22, and 33), and conversely, significant effects of AMF species and communities on the diversity and productivity of plant communities have been described (25, 68). Soil physicochemical parameters like phosphorus, nitrogen, and carbon availability (e.g., see references 4, 9, and 31); pH (17); moisture content (53); and disturbance (30) also reportedly influence AMF distribution. Hence, there is some support for niche theory, which presumes that two species of the same trophic level cannot coexist in a limited system and, if two species are present in such circumstances, one should become extinct (21). As a corollary, two cooccurring species must occupy niches that differ in some dimensions, e.g., plant hosts and/or soil properties (28). However, there are also indications that neutral ecological processes, as well as niche-defining parameters, may influence AMF diversity and community composition (17, 39). In contrast to niche theory, neutral theory (27) postulates that all individuals of every species at a given trophic level in a food web have ecological equivalence, and thus, all species within trophically defined communities can be regarded as open nonequilibrium assemblages that are solely shaped by dispersal and distinctions in spatiotemporal dimensions. According to the work of Hubbell (27), neutrality is defined at the level of individual organisms with identical probabilities of birth, death, migration, and speciation and not at the species level. In order to explore AMF communities more thoroughly and to test competing hypotheses, such as those raised by the niche and neutral theories, robust methods for high-throughput analyses of the communities are required.Recent investigations of variables that affect the structure of AMF communities have considered relationships between niche-defining dimensions, such as soil types (39) and pH gradients (17), and spatial variations in AMF community structure but not the role of plant diversity or functional traits of host plants. There have been several plant diversity manipulation experiments designed for coanalyzing multiple sets of ecological variables (e.g., the BIODEPTH and Cedar Creek projects) that would have been ideal for detailed examinations of effects of ecological variables on AMF, but previously reported AMF analyses in these experiments have been limited to counts of spores in a single study (11). However, not all AMF species regularly sporulate, and when present, spores poorly reflect AMF diversity (69), since active AMF occur as mycelia in roots and soils (e.g., see references 12 and 26). PCR-based molecular techniques enable much more rigorous characterization of AMF communities in these compartments (e.g., see references 26, 36, and 72), but assessments of broad spatial (42) and/or temporal (52) variations in AMF communities require analysis of large numbers of samples, which is not feasible using conventional PCR amplification followed by cloning and sequencing. This challenge can be potentially met by real-time PCR-based approaches, in which the AMF sequence types present in compartments of interest are first identified and then sequence type-specific probes are used for large-scale screening in real-time PCR TaqMan assays.In the study presented here, we explored AMF diversity in plots used in the Jena Experiment, a grassland plant diversity manipulation of 60 plant species representing four functional groups in 81 plots of 400 m² (56). The overall AMF diversity and community structure were first assessed by PCR amplification, cloning, and sequencing (55) of internal transcribed spacer (ITS) ribosomal DNA (rDNA) gene sequences in soil samples from 23 representative plots. Using the acquired data, we then developed sequence type-specific probes, which were applied in high-throughput real-time PCR TaqMan assays of samples from all 81 experimental plots, and the effects of 15 plant and soil variables on the AMF community assemblage were investigated.     

Aptamer-based multiplexed proteomic technology for biomarker discovery

Background

The interrogation of proteomes (“proteomics”) in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology and medicine.

Methodology/Principal Findings

We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 µL of serum or plasma). Our current assay measures 813 proteins with low limits of detection (1 pM median), 7 logs of overall dynamic range (∼100 fM–1 µM), and 5% median coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding signature of DNA aptamer concentrations, which is quantified on a DNA microarray. Our assay takes advantage of the dual nature of aptamers as both folded protein-binding entities with defined shapes and unique nucleotide sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to rapidly discover unique protein signatures characteristic of various disease states.

Conclusions/Significance

We describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine.     

Dispersal failure contributes to plant losses in NW Europe

The ongoing decline of many plant species in Northwest Europe indicates that traditional conservation measures to improve the habitat quality, although useful, are not enough to halt diversity losses. Using recent databases, we show for the first time that differences between species in adaptations to various dispersal vectors, in combination with changes in the availability of these vectors, contribute significantly to explaining losses in plant diversity in Northwest Europe in the 20th century. Species with water- or fur-assisted dispersal are over-represented among declining species, while others (wind- or bird-assisted dispersal) are under-represented. Our analysis indicates that the 'colonization deficit' due to a degraded dispersal infrastructure is no less important in explaining plant diversity losses than the more commonly accepted effect of eutrophication and associated niche-based processes. Our findings call for measures that aim to restore the dispersal infrastructure across entire regions and that go beyond current conservation practices.