The Cbl proteins are a family of proteins found in metazoans from nematodes to vertebrates. These proteins have several highly conserved domains including an N-terminal tyrosine kinase binding (TKB) 1 domain and a RING finger (1-9). The three mammalian Cbl proteins,2,[6][7][8], are tyrosine-phosphorylated upon activation of a wide variety of growth factor receptors, and they associate with many signaling proteins via SH2 and SH3 interactions (reviewed in Ref. 10 and 11). These diverse interactions modulate signaling through many pathways (10,11). Recent work has shown that c-Cbl-and Cblb-deficient mice have hyperplastic tissues, consistent with a negative regulatory role in cellular proliferation for Cbl proteins (12-15). Together, these data indicate that the Cbl proteins are important regulators of intracellular signaling and consequently of cell function and development.Cbl proteins are negative regulators of epidermal growth factor receptor (EGFR) signaling. This was first shown by genetic studies in Caenorhabditis elegans, which demonstrated that Sli-1 (the C. elegans Cbl homologue) is a negative regulator of the Let-23 receptor tyrosine kinase (the EGFR homologue) in vulva development (3, 16). The Drosophila Cbl protein (D-Cbl) has been shown to associate with the EGFR, and overexpression of D-Cbl in the eye of Drosophila embryos inhibits EGFR-dependent photoreceptor cell development (4, 5). Several studies have shown that mammalian Cbl proteins become phosphorylated and recruited to the EGFR upon stimulation (11, 17) and that they inhibit EGFR function (7, 18 -20).The mechanism underlying the negative regulation of activated tyrosine kinases by Cbl proteins has recently been described. Cbl proteins function as ubiquitin protein ligases, which mediate the ubiquitination of activated tyrosine kinases including the EGFR and target them for degradation (20 -31). Ubiquitination of proteins occurs via the sequential activation and conjugation of ubiquitin to target proteins by the ubiquitinactivating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin protein ligase (E3) (32). The E3 confers specificity to the ubiquitination process. An increasing number of RING finger proteins has been demonstrated to function as E3 proteins or as part of E3 complexes, and in each of them the RING finger is essential to this activity (33-43). The highly conserved TKB and RING finger domains of Cbl proteins are essential and sufficient for their E3 activity, and together these domains target the ubiquitination of activated tyrosine kinases such as the EGFR (20 -31).Here, we show that EGF activation induces a coordinated degradation of the EGFR, Cbl proteins, and other proteins of the EGFR signaling complex. These results suggest that Cbl proteins regulate degradation of multiple proteins in the active EGFR-signaling complex. EXPERIMENTAL PROCEDURESExpression Constructs-The expression plasmid for HA epitopetagged Cbl-b, c-Cbl, and the control vector (pCEFL) have been previously described (18). HA epitope-tagged C...

Rare diseases (RD) patient registries are powerful instruments that help develop clinical research, facilitate the planning of appropriate clinical trials, improve patient care, and support healthcare management. They constitute a key information system that supports the activities of European Reference Networks (ERNs) on rare diseases. A rapid proliferation of RD registries has occurred during the last years and there is a need to develop guidance for the minimum requirements, recommendations and standards necessary to maintain a high-quality registry. In response to these heterogeneities, in the framework of RD-Connect, a European platform connecting databases, registries, biobanks and clinical bioinformatics for rare disease research, we report on a list of recommendations, developed by a group of experts, including members of patient organizations, to be used as a framework for improving the quality of RD registries. This list includes aspects of governance, Findable, Accessible, Interoperable and Reusable (FAIR) data and information, infrastructure, documentation, training, and quality audit. The list is intended to be used by established as well as new RD registries. Further work includes the development of a toolkit to enable continuous assessment and improvement of their organizational and data quality.

There is a growing international agreement on the need to provide greater access to research data and bio-specimen collections to optimize their long-term value and exploit their potential for health discovery and validation. This is especially evident for rare disease research. Currently, the rising value of data and bio-specimen collections does not correspond with an equal increase in data/sample-sharing and data/sample access. Contradictory legal and ethical frameworks across national borders are obstacles to effective sharing: more specifically, the absence of an integrated model proves to be a major logistical obstruction. The Charter intends to amend the obstacle by providing both the ethical foundations on which data sharing should be based, as well as a general Material and Data Transfer Agreement (MTA/DTA). This Charter is the result of a careful negotiation of different stakeholders' interest and is built on earlier consensus documents and position statements, which provided the general international legal framework. Further to this, the Charter provides tools that may help accelerate sharing. The Charter has been formulated to serve as an enabling tool for effective and transparent data and bio-specimen sharing and the general MTA/DTA constitutes a mechanism to ensure uniformity of access across projects and countries, and may be regarded as a consistent basic agreement for addressing data and material sharing globally. The Charter is forward looking in terms of emerging issues from the perspective of a multi-stakeholder group, and where possible, provides strategies that may address these issues.

Human biospecimens are subject to a number of different collection, processing, and storage factors that can significantly alter their molecular composition and consistency. These biospecimen preanalytical factors, in turn, influence experimental outcomes and the ability to reproduce scientific results. Currently, the extent and type of information specific to the biospecimen preanalytical conditions reported in scientific publications and regulatory submissions varies widely. To improve the quality of research utilizing human tissues it is critical that information regarding the handling of biospecimens be reported in a thorough, accurate, and standardized manner. The Biospecimen Reporting for Improved Study Quality (BRISQ) recommendations outlined herein are intended to apply to any study in which human biospecimens are used. The purpose of reporting these details is to supply others, from researchers to regulators, with more consistent and standardized information to better evaluate, interpret, compare, and reproduce the experimental results. The BRISQ guidelines are proposed as an important and timely resource tool to strengthen communication and publications around biospecimen-related research and help reassure patient contributors and the advocacy community that the contributions are valued and respected.

The acetylation levels of lysine residues in nucleosomes, which are determined by the opposing activities of histone acetyltransferases (HATs) and deacetylases, play an important role in regulating chromatin-related processes, including transcription. We report that HMGN1, a nucleosomal binding protein that reduces the compaction of the chromatin fiber, increases the levels of acetylation of K14 in H3. The levels of H3K14ac in Hmgn1-/- cells are lower than in Hmgn1+/+ cells. Induced expression of wild-type HMGN1, but not of a mutant that does not bind to chromatin, in Hmgn1-/- cells elevates the levels of H3K14ac. In vivo, HMGN1 elevates the levels of H3K14ac by enhancing the action of HAT. In vitro, HMGN1 enhances the ability of PCAF to acetylate nucleosomal, but not free, H3. Thus, HMGN1 modulates the levels of H3K14ac by binding to chromatin. We suggest that HMGN1, and perhaps similar architectural proteins, modulates the levels of acetylation in chromatin by altering the equilibrium generated by the opposing enzymatic activities that continuously modify and de-modify the histone tails in nucleosomes.

Executive SummaryA movement to create a federated global patient registry containing core data and using a standardized vocabulary for as many as 7,000 rare diseases was launched at a workshop,

Most breast cancer cell lines are resistant to TNF-related apoptosis inducing ligand (TRAIL) induced apoptosis. In sensitive breast cancer cell lines TRAIL rapidly induces the cleavage and activation of caspases leading to the subsequent cleavage of downstream caspase substrates. In contrast, there is no caspase activation in the resistant cell lines. The transcription factor NF-KB can inhibit apoptosis induced by a variety of stimuli including activation of death receptors. We investigated whether NF-kappaB contributes to the resistance of breast cancer cells to TRAIL induced apoptosis. All of the resistant breast cancer cell lines expressed NF-kappaB and had detectable NF-kappaB activity in nuclear extracts prior to treatment with TRAIL. Upon TRAIL treatment, a significant increase in NF-kappaB activity was seen in most of the cell lines. To directly test if NF-kappaB activity contributes to the resistance of these cell lines to TRAIL, we transiently transfected the resistant cell lines with an inhibitor of NF-kappaB (IkappaBdeltaN) and measured TRAIL induced apoptosis in control and transfected cells. All of the resistant cell lines tested showed an increase in TRAIL induced apoptosis when transfected with the IKBdeltaN. These results demonstrate that TRAIL resistant breast cancer cells fail to rapidly activate the apoptotic machinery but they do activate NF-kappaB. Inhibition of NF-kappaB activity increases the sensitivity to TRAIL mediated apoptosis in resistant cells. These results suggest that agents which inhibit NF-kappaB should increase the clinical efficacy of TRAIL in breast cancer cells.

In the field of rare diseases, registries are considered power tool to develop clinical research, to facilitate the planning of appropriate clinical trials, to improve patient care and healthcare planning. Therefore high quality data of rare diseases registries is considered to be one of the most important element in the establishment and maintenance of a registry. Data quality can be defined as the totality of features and characteristics of data set that bear on its ability to satisfy the needs that result from the intended use of the data. In the context of registries, the 'product' is data, and quality refers to data quality, meaning that the data coming into the registry have been validated, and ready for use for analysis and research. Determining the quality of data is possible through data assessment against a number of dimensions: completeness, validity; coherence and comparability; accessibility; usefulness; timeliness; prevention of duplicate records. Many others factors may influence the quality of a registry: development of standardized Case Report Form and security/safety controls of informatics infrastructure. With the growing number of rare diseases registries being established, there is a need to develop a quality validation process to evaluate the quality of each registry. A clear description of the registry is the first step when assessing data quality or the registry evaluation system. Here we report a template as a guide for helping registry owners to describe their registry.