PIDD death-domain phosphorylation by ATM controls prodeath versus prosurvival PIDDosome signaling.

Biochemical evidence implicates the death-domain (DD) protein PIDD as a molecular switch capable of signaling cell survival or death in response to genotoxic stress. PIDD activity is determined by binding-partner selection at its DD: whereas recruitment of RIP1 triggers prosurvival NF-κB signaling, recruitment of RAIDD activates proapoptotic caspase-2 via PIDDosome formation. However, it remains unclear how interactor selection, and thus fate decision, is regulated at the PIDD platform. We show that the PIDDosome functions in the "Chk1-suppressed" apoptotic response to DNA damage, a conserved ATM/ATR-caspase-2 pathway antagonized by Chk1. In this pathway, ATM phosphorylates PIDD on Thr788 within the DD. This phosphorylation is necessary and sufficient for RAIDD binding and caspase-2 activation. Conversely, nonphosphorylatable PIDD fails to bind RAIDD or activate caspase-2, and engages prosurvival RIP1 instead. Thus, ATM phosphorylation of the PIDD DD enables a binary switch through which cells elect to survive or die upon DNA injury.

Calibration of time domain reflectometry (DTR) for soil moisture measurements in cultivated peat soils

Tiivistelmä: TDR-mittausten kalibrointi viljeltyjen turvemaiden kosteuden mittaamiseen

Molecular basis of coagulation factor V deficiency caused by the R1698W inter-domain mutation.

Coagulation factor V (FV) deficiency is characterised by variable bleeding phenotypes and heterogeneous mutations. To add new insights into the FV genotype-phenotype relationship, we characterised the R1698W change in the A3 domain, at the poorly investigated interface with the A2 domain. The FV R1698W mutation was responsible for a markedly reduced expression level (10% of FV-WT) and specific activity in thrombin generation (0.39). Interestingly, the FVa1698W showed rapid activity decay upon activation due to increased dissociation rate between the heavy and light chains. The importance of the size and charge of the residue at position 1698 was investigated by three additional recombinant mutants, FVR1698A, FVR1698Q, and FVR1698E. FVR1698A and FVR1698Q expression (30 and 45% of FV-WT), specific activity (both 0.57) and stability were all reduced. Noticeably, FVR1698E showed normal activity and stability despite poor expression (10% of FV-WT). These data indicate the essential role of R1698 for normal biosynthetic process and support local flexibility for positively or negatively charged residues to produce stable and functional A3-A2 domain interactions. Their experimental alteration produces a gradient of FV defects, which help to interpret the wide spectrum of phenotypes in FV-deficient patients.

A template-assembled synthetic protein surface mimetic of the von Willebrand factor A1 domain inhibits botrocetin-induced platelet aggregation.

Platelet adhesion, the initial step of platelet activation, is mediated by the interaction of von Willebrand factor (VWF) with its platelet receptor, the GPIb-IX complex. The binding of VWF to GPIb-IX is induced either by increased shear stress or by exogenous modulators, such as botrocetin. At a molecular level, this interaction takes place between the A1 domain of VWF and the GPIb alpha chain of the GPIb-IX complex. We report here the design and functional characteristics of a VWF template-assembled synthetic protein (TASP), a chimeric four-helix-bundle TASP scaffold mimicking the surface of the A1 domain. Twelve residues located on helices alpha 3 and alpha 4 in the native A1 domain were grafted onto a surface formed by two neighboring helices of the TASP. VWF TASP was found to inhibit specifically botrocetin-induced platelet aggregation and to bind both botrocetin and GPIb alpha. However, in contrast to the native A1 domain, VWF TASP did not bind simultaneously to both ligands. Modeling studies revealed that the relative orientation of the alpha helices in VWF TASP led to a clash of bound botrocetin and GPIb alpha. These results demonstrate that a chimeric four-helix-bundle TASP as a scaffold offers a suitable surface for presenting crucial residues of the VWF A1 domain; the potential of the TASP approach for de novo protein design and mimicry is thereby illustrated.

A novel dominant mutation of the Nav1.4 alpha-subunit domain I leading to sodium channel myotonia.

BACKGROUND: Mutations in SCN4A may lead to myotonia. METHODS: Presentation of a large family with myotonia, including molecular studies and patch clamp experiments using human embryonic kidney 293 cells expressing wild-type and mutated channels. RESULTS: In a large family with historic data on seven generations and a clear phenotype, including myotonia at movement onset, with worsening by cold temperature, pregnancy, mental stress, and especially after rest after intense physical activity, but without weakness, the phenotype was linked with the muscle sodium channel gene (SCN4A) locus, in which a novel p.I141V mutation was found. This modification is located within the first transmembrane segment of domain I of the Na(v)1.4 alpha subunit, a region where no mutation has been reported so far. Patch clamp experiments revealed a mutation-induced hyperpolarizing shift (-12.9 mV) of the voltage dependence of activation, leading to a significant increase (approximately twofold) of the window current amplitude. In addition, the mutation shifted the voltage dependence of slow inactivation by -8.7 mV and accelerated the entry to this state. CONCLUSIONS: We propose that the gain-of-function alteration in activation leads to the observed myotonic phenotype, whereas the enhanced slow inactivation may prevent depolarization-induced paralysis.

Transcription factor CTF1 acts as a chromatin domain boundary that shields human telomeric genes from silencing.

Telomeres are associated with chromatin-mediated silencing of genes in their vicinity. However, how epigenetic markers mediate mammalian telomeric silencing and whether specific proteins may counteract this effect are not known. We evaluated the ability of CTF1, a DNA- and histone-binding transcription factor, to prevent transgene silencing at human telomeres. CTF1 was found to protect a gene from silencing when its DNA-binding sites were interposed between the gene and the telomeric extremity, while it did not affect a gene adjacent to the telomere. Protein fusions containing the CTF1 histone-binding domain displayed similar activities, while mutants impaired in their ability to interact with the histone did not. Chromatin immunoprecipitation indicated the propagation of a hypoacetylated histone structure to various extents depending on the telomere. The CTF1 fusion protein was found to recruit the H2A.Z histone variant at the telomeric locus and to restore high histone acetylation levels to the insulated telomeric transgene. Histone lysine trimethylations were also increased on the insulated transgene, indicating that these modifications may mediate expression rather than silencing at human telomeres. Overall, these results indicate that transcription factors can act to delimit chromatin domain boundaries at mammalian telomeres, thereby blocking the propagation of a silent chromatin structure.

p53-induced protein with a death domain (PIDD) isoforms differentially activate nuclear factor-kappaB and caspase-2 in response to genotoxic stress

Cells respond to DNA damage in a complex way and the fate of damaged cells depends on the balance between pro- and antiapoptotic signals. This is of crucial importance in cancer as genotoxic stress is implied both in oncogenesis and in classical tumor therapies. p53-induced protein with a death domain (PIDD), initially described as a p53-inducible gene, is one of the molecular switches able to activate a survival or apoptotic program. Two isoforms of PIDD, PIDD (isoform 1) and LRDD (isoform 2), have already been reported and we describe here a third isoform. These three isoforms are differentially expressed in tissues and cell lines. Genotoxic stress only affects PIDD isoform 3 mRNA levels, whereas isoforms 1 and 2 mRNA levels remain unchanged. All isoforms are capable of activating nuclear factor-kappaB in response to genotoxic stress, but only isoform 1 interacts with RIP-associated ICH-1/CED-3 homologous protein with a death domain and activates caspase-2. Isoform 2 counteracts the pro-apoptotic function of isoform 1, whereas isoform 3 enhances it. Thus, the differential splicing of PIDD mRNA leads to the formation of at least three proteins with antagonizing/agonizing functions, thereby regulating cell fate in response to DNA damage

Experimental cerebral malaria develops independently of caspase recruitment domain-containing protein 9 signaling.

The outcome of infection depends on multiple layers of immune regulation, with innate immunity playing a decisive role in shaping protection or pathogenic sequelae of acquired immunity. The contribution of pattern recognition receptors and adaptor molecules in immunity to malaria remains poorly understood. Here, we interrogate the role of the caspase recruitment domain-containing protein 9 (CARD9) signaling pathway in the development of experimental cerebral malaria (ECM) using the murine Plasmodium berghei ANKA infection model. CARD9 expression was upregulated in the brains of infected wild-type (WT) mice, suggesting a potential role for this pathway in ECM pathogenesis. However, P. berghei ANKA-infected Card9(-/-) mice succumbed to neurological signs and presented with disrupted blood-brain barriers similar to WT mice. Furthermore, consistent with the immunological features associated with ECM in WT mice, Card9(-/-) mice revealed (i) elevated levels of proinflammatory responses, (ii) high frequencies of activated T cells, and (iii) CD8(+) T cell arrest in the cerebral microvasculature. We conclude that ECM develops independently of the CARD9 signaling pathway.

Identification of novel and cell type enriched cofactors of the transcription activation domain of RelA (p65 NF-kappaB).

RelA (NF-kappaB) is a transcription factor inducible by distinct stimuli in many different cell types. To find new cell type specific cofactors of NF-kappaB dependent transcription, we isolated RelA transcription activation domain binding proteins from the nuclear extracts of three different cell types. Analysis by electrophoresis and liquid chromatography tandem mass spectrometry identified several novel putative molecular partners. Some were strongly enriched in the complex formed from the nuclear extracts of specific cell types.

The role of MMP-9 and its fibronectin-like domain in tumor growth & invasion : certainties, controversies & discrepancies

Tumors are often compared to wounds that do not heal, where the crosstalk between tumor cells and their surrounding stroma is crucial at all stages of development, from the initial primary growth to metastasis. Similar to wound healing, fibroblasts in the tumor stroma differentiate into myofibroblasts, also referred to as "cancer-associated fibroblasts" (CAFs), primarily, but not exclusively, in response to transforming growth factor-ß (TGF-ß). Myofibroblasts in turn enhance tumor progression by remodeling the stroma. Among molecules implicated in stroma remodeling, matrix metalloproteinases (MMPs), and MMP-g in particular, play a prominent role. However, the mechanisms that regulate MMP-g activation and function remain poorly understood. Recent evidence indicates that tumor cell surface association of MMP-g is an important event in its activation, and more generally in tumor growth and invasion. In the present work we address the potential association of MMP-g activity with cell-surface recruitment to human fibroblasts. We show for the first time that recruitment of MMP-g to the MRC-5 fibroblast cell surface occurs through the fibronectin-like (FN) domain, shared only by MMP-g and MMP-2 among all the MMPs. Functional assays suggest that both the pro- and active form of MMP-g trigger a-smooth muscle actin (aSMA) expression in resting fibroblasts that reflects myofibroblast differentiation, possibly through TGF-ß activation. Moreover, the FN domain of MMP-g inhibits both MMP-g-induced TGF-ß activation and aSMA expression by sequestering MMP-g. Xenograft experiments in NOD/SCID mice using HT1080 fibrosarcoma or MDA-MD231 breast adenocarcinoma cells stably expressing the FN domain of MMP-g revealed no changes in primary tumor growth. However, in the context of metastasis, expression of the FN domain by these same tumor cells dramatically increased their metastatic proclivity whereas expression of wt MMP-g either promoted no change or actually reduced the number of metastases. We observed a decrease of an active form of MMP-g in MDA-MB231 cells overexpressing the FN domain suggesting that the FN domain may inhibit MMP-g activity in Tumors are often compared to wounds that do not heal, where the crosstalk between tumor cells and their surrounding stroma is crucial at all stages of development, from the initial primary growth to metastasis. Similar to wound healing, fibroblasts in the tumor stroma differentiate into myofibroblasts, also referred to as "cancer-associated fibroblasts" (CAFs), primarily, but not exclusively, in response to transforming growth factor-ß (TGF-ß). Myofibroblasts in turn enhance tumor progression by remodeling the stroma. Among molecules implicated in stroma remodeling, matrix metalloproteinases (MMPs), and MMP-g in particular, play a prominent role. However, the mechanisms that regulate MMP-g activation and function remain poorly understood. Recent evidence indicates that tumor cell surface association of MMP-g is an important event in its activation, and more generally in tumor growth and invasion. In the present work we address the potential association of MMP-g activity with cell-surface recruitment to human fibroblasts. We show for the first time that recruitment of MMP-g to the MRC-5 fibroblast cell surface occurs through the fibronectin-like (FN) domain, shared only by MMP-g and MMP-2 among all the MMPs. Functional assays suggest that both the pro- and active form of MMP-g trigger a-smooth muscle actin (aSMA) expression in resting fibroblasts that reflects myofibroblast differentiation, possibly through TGF-ß activation. Moreover, the FN domain of MMP-g inhibits both MMP-g-induced TGF-ß activation and aSMA expression by sequestering MMP-g. Xenograft experiments in NOD/SCID mice using HT1080 fibrosarcoma or MDA-MD231 breast adenocarcinoma cells stably expressing the FN domain of MMP-9 revealed no changes in primary tumor growth. However, in the context of metastasis, expression of the FN domain by these same tumor cells dramatically increased their metastatic proclivity whereas expression of wt MMP-g either promoted no change or actually reduced the number of metastases. We observed a decrease of an active form of MMP-9 in MDA-MB231 cells overexpressing the FN domain suggesting that the FN domain may inhibit MMP-9 activity in those cells and therefore prevent MMP-9-induced activation of TGF-b, which results in increased invasion. Curiously, xenografts of SW480 colorectal adenocarcinoma cells stably expressing the FN domain of MMP-9 displayed reduced growth at both the primary (subcutaneous) injection site and the lungs of NOD/SCID mice, in experimental metastasis assays, whilst the same cells overexpressing wt MMP-9 showed enhanced growth and dissemination. Gelatin zymography of conditioned medium revealed that these effects may be due to the FN domain, which displaces MMP-9 from SW480 cell surface. These observations suggest a dual role of MMP-9 and its FN domain in primary tumor growth and metastasis, underscoring the notion that the effect of MMP-9 on tumor cells may depend on the cell type and highlighting possible protective effects of MMPs in tumor progression.

Semantic domains and supersens tagging for domain-specific ontology learning

In this paper we propose a novel unsupervised approach to learning domain-specific ontologies from large open-domain text collections. The method is based on the joint exploitation of Semantic Domains and Super Sense Tagging for Information Retrieval tasks. Our approach is able to retrieve domain specific terms and concepts while associating them with a set of high level ontological types, named supersenses, providing flat ontologies characterized by very high accuracy and pertinence to the domain.

Domain Adaptation in remote sensing: increasing the portability of land-cover classifiers

Among the types of remote sensing acquisitions, optical images are certainly one of the most widely relied upon data sources for Earth observation. They provide detailed measurements of the electromagnetic radiation reflected or emitted by each pixel in the scene. Through a process termed supervised land-cover classification, this allows to automatically yet accurately distinguish objects at the surface of our planet. In this respect, when producing a land-cover map of the surveyed area, the availability of training examples representative of each thematic class is crucial for the success of the classification procedure. However, in real applications, due to several constraints on the sample collection process, labeled pixels are usually scarce. When analyzing an image for which those key samples are unavailable, a viable solution consists in resorting to the ground truth data of other previously acquired images. This option is attractive but several factors such as atmospheric, ground and acquisition conditions can cause radiometric differences between the images, hindering therefore the transfer of knowledge from one image to another. The goal of this Thesis is to supply remote sensing image analysts with suitable processing techniques to ensure a robust portability of the classification models across different images. The ultimate purpose is to map the land-cover classes over large spatial and temporal extents with minimal ground information. To overcome, or simply quantify, the observed shifts in the statistical distribution of the spectra of the materials, we study four approaches issued from the field of machine learning. First, we propose a strategy to intelligently sample the image of interest to collect the labels only in correspondence of the most useful pixels. This iterative routine is based on a constant evaluation of the pertinence to the new image of the initial training data actually belonging to a different image. Second, an approach to reduce the radiometric differences among the images by projecting the respective pixels in a common new data space is presented. We analyze a kernel-based feature extraction framework suited for such problems, showing that, after this relative normalization, the cross-image generalization abilities of a classifier are highly increased. Third, we test a new data-driven measure of distance between probability distributions to assess the distortions caused by differences in the acquisition geometry affecting series of multi-angle images. Also, we gauge the portability of classification models through the sequences. In both exercises, the efficacy of classic physically- and statistically-based normalization methods is discussed. Finally, we explore a new family of approaches based on sparse representations of the samples to reciprocally convert the data space of two images. The projection function bridging the images allows a synthesis of new pixels with more similar characteristics ultimately facilitating the land-cover mapping across images.

A time-domain method for the analysis of thermal impedance response preserving the convolution form

The study of the thermal behavior of complex packages as multichip modules (MCM¿s) is usually carried out by measuring the so-called thermal impedance response, that is: the transient temperature after a power step. From the analysis of this signal, the thermal frequency response can be estimated, and consequently, compact thermal models may be extracted. We present a method to obtain an estimate of the time constant distribution underlying the observed transient. The method is based on an iterative deconvolution that produces an approximation to the time constant spectrum while preserving a convenient convolution form. This method is applied to the obtained thermal response of a microstructure as analyzed by finite element method as well as to the measured thermal response of a transistor array integrated circuit (IC) in a SMD package.