Dynamic control of selectivity in the ubiquitination pathway revealed by an ASP to GLU substitution in an intra-molecular salt-bridge network

Ubiquitination relies on a subtle balance between selectivity and promiscuity achieved through specific interactions between ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s). Here, we report how a single aspartic to glutamic acid substitution acts as a dynamic switch to tip the selectivity balance of human E2s for interaction toward E3 RING-finger domains. By combining molecular dynamic simulations, experimental yeast-two-hybrid screen of E2-E3 (RING) interactions and mutagenesis, we reveal how the dynamics of an internal salt-bridge network at the rim of the E2-E3 interaction surface controls the balance between an “open”, binding competent, and a “closed”, binding incompetent state. The molecular dynamic simulations shed light on the fine mechanism of this molecular switch and allowed us to identify its components, namely an aspartate/glutamate pair, a lysine acting as the central switch and a remote aspartate. Perturbations of single residues in this network, both inside and outside the interaction surface, are sufficient to switch the global E2 interaction selectivity as demonstrated experimentally. Taken together, our results indicate a new mechanism to control E2-E3 interaction selectivity at an atomic level, highlighting how minimal changes in amino acid side-chain affecting the dynamics of intramolecular salt-bridges can be crucial for protein-protein interactions. These findings indicate that the widely accepted sequence-structure-function paradigm should be extended to sequence-structure-dynamics-function relationship and open new possibilities for control and fine-tuning of protein interaction selectivity

BloodLink: Computer-based Decision Support for Blood Test Ordering; Assessment of the effect on physicians' test-ordering behavior

Requesting blood tests is an important aspect of the health care delivered by the general practitioner in The Netherlands. About three to four percent of the patients encounters with Dutch general practitioners result in the physician requesting blood tests, which is lower than in many other European countries. Although test ordering is limited to three to four percent of patients encounters with the general practitioner, the use of diagnostic tests in general practice, has known an overwhelming growth in the years behind. Physicians' use of blood tests, however, is not always appropriate. General practitioners are taught test ordering when training in hospitals before settling down in general practice. Hospital morbidity, however, is different from morbidity patterns in general practice. Appropriate test ordering panels in hospital settings, therefore, are not always appropriate for primary care. Nevertheless, general practitioners use these test panels, once taught, automatically in the primary care setting. Uncertainty and the desire not to miss a diagnosis stimulate the use of blood tests. Excessive and inappropriate test ordering is not only expensive but also may even add to the uncertainty by generating unexpected abnormal or false positive values. The use of blood tests may thus even increase uncertainty and stimu late further unnecessary diagnostic investigations. It is important, therefore, that once the decision to obtain blood tests has been made, appropriate test ordering is adhered to. Influencing this heuristic test-ordering behavior has proven to be difficult

Visual analytics in histopathology diagnostics: a protocol-based approach

Computer-Aided-Diagnosis (CAD) systems supporting the diagnostic process are widespread in radiology. Digital Pathology is still behind in the introduction of such solutions. Several studies investigated pathologists' behavior but only a few aimed to improve the diagnostic and report process with novel applications. In this work we designed and implemented a first protocol-based CAD viewer supported by visual analytics. The system targets the optimization of the diagnostic workflow in breast cancer diagnosis by means of three image analysis features that belong to the standard grading system (Nottingham Histologic Grade). A pathologist's routine was tracked during the examination of breast cancer tissue slides and diagnostic traces were analyzed from a qualitative perspective. Accordingly, a set of generic requirements was elicited to define the design and the implementation of the CAD-Viewer. A first qualitative evaluation conducted with five pathologists shows that the interface suffices the diagnostic workflow and diminishes the manual effort. We present promising evidence of the usefulness of our CAD-viewer and opportunities for its extension and integration in clinical practice. As a conclusion, the findings demonstrate that it is feasibile to optimize the Nottingham Grading workflow and, generally, the histological diagnosis by integrating computational pathology data with visual analytics techniques

Analysis of the practice guidelines of the Dutch College of General Practitioners with respect to the use of blood tests

OBJECTIVE: To determine the consistency among the practice guidelines of the Dutch College of General Practitioners with respect to the use of blood tests. METHODS: The authors evaluated 64 practice guidelines of the Dutch College of General Practitioners. For each guideline, they analyzed each sentence that contained a reference to a blood test to determine the clinical situation in which the test should be performed (the indication) and to determine the tests that should be performed in that situation (the recommended test). An incomplete recommendation refers to a guideline that mentioned a blood test but did not identify the indication for that test. An inconsistency refers to the situation in which one guideline recommended a certain test for a given indication whereas another guideline mentioned the same indication but did not recommend the same test. RESULTS: Twenty-seven practice guidelines mentioned blood tests. Of these, three explicitly recommended not to request blood tests. Five guidelines contained incomplete recommendations, and the authors encountered two inconsistencies among the guidelines. Twenty-three guidelines mentioned blood tests and allowed the authors to identify indications and recommended tests. CONCLUSION: The identification of indications and recommended tests allows evaluation of consistency among practice guidelines. Although some incomplete recommendations and inconsistencies were discovered, the majority of the guidelines provide clear and unambiguous recommendations for blood-test ordering in primary care