3D mapping of the SPRY2 domain of ryanodine receptor 1 by single-particle Cryo-EM

The type 1 skeletal muscle ryanodine receptor (RyR1) is principally responsible for Ca(2+) release from the sarcoplasmic reticulum and for the subsequent muscle contraction. The RyR1 contains three SPRY domains. SPRY domains are generally known to mediate protein-protein interactions, however the location of the three SPRY domains in the 3D structure of the RyR1 is not known. Combining immunolabeling and single-particle cryo-electron microscopy we have mapped the SPRY2 domain (S1085-V1208) in the 3D structure of RyR1 using three different antibodies against the SPRY2 domain. Two obstacles for the image processing procedure; limited amount of data and signal dilution introduced by the multiple orientations of the antibody bound in the tetrameric RyR1, were overcome by modifying the 3D reconstruction scheme. This approach enabled us to ascertain that the three antibodies bind to the same region, to obtain a 3D reconstruction of RyR1 with the antibody bound, and to map SPRY2 to the periphery of the cytoplasmic domain of RyR1. We report here the first 3D localization of a SPRY2 domain in any known RyR isoform.The authors want to thank the Brigham and Women’s Hospital Biomedical Research Institute (to MS), the Australian National Health and the Medical Research Council (471418 to AD, MC and PB), and the European Commission (Marie Curie Action PIOF-GA-2009-237120 to AP-M)

Ensuring the semantic correctness of a BAUML artifact-centric BPM

Context: Using models to represent business processes provides several advantages, such as facilitating the communication between the stakeholders or being able to check the correctness of the processes before their implementation. In contrast to traditional process modeling approaches, the artifact-centric approach treats data as a key element of the process, also considering the tasks or activities that are performed in it. Objective: This paper presents a way to verify and validate the semantic correctness of an artifact-centric business process model defined using a combination of UML and OCL models - a BAUML model. Method: We achieve our goal by presenting several algorithms that encode the initial models into first-order logic, which then allows to use an existing satisfiability checking tool to determine their correctness. Results: An approach to verify and validate an artifact-centric BPM specified in BAUML, which uses a combination of UML and OCL models. To do this, we provide a method to translate all BAUML components into a set of logic formulas. The result of this translation ensures that the only changes allowed are those specified in the model, and that those changes are taking place according the order established by the model. Having obtained this logic representation, these models can be validated by any existing reasoning method able to deal with negation of derived predicates. Moreover, we show how to automatically generate the relevant tests to validate the models. We also show the feasibility of our approach by implementing a prototype tool and applying it to a running example. Conclusion: It is feasible to ensure the semantic correctness of an artifact-centric business process model in practice.Peer ReviewedPostprint (author's final draft

FRET-Based Localization of Fluorescent Protein Insertions Within the Ryanodine Receptor Type 1

Fluorescent protein (FP) insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM) maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET) measurements were used to localize green fluorescent protein (GFP) insertions within the ryanodine receptor type 1 (RyR1), a large intracellular Ca2+ release channel that plays a key role in skeletal muscle excitation contraction coupling. A series of full-length His-tagged GFP-RyR1 fusion constructs were created, expressed in human embryonic kidney (HEK)-293T cells and then complexed with Cy3NTA, a His-tag specific FRET acceptor. FRET efficiency values measured from each GFP donor to Cy3NTA bound to each His tag acceptor site were converted into intermolecular distances and the positions of each inserted GFP were then triangulated relative to a previously published X-ray crystal structure of a 559 amino acid RyR1 fragment. We observed that the chromophoric centers of fluorescent proteins inserted into RyR1 can be located as far as 45 Å from their insertion sites and that the fused proteins can also be located in internal cavities within RyR1. These findings should prove useful in interpreting structural results obtained in cryo EM maps using fusions of small fluorescent proteins. More accurate point-to-point distance information may be obtained using complementary orthogonal labeling systems that rely on fluorescent probes that bind directly to amino acid side chains

Specifying artifact-centric business process models in UML: technical report

In recent years, the artifact-centric approach to process modeling has attracted a lot of attention. One of the research lines in this area is finding a suitable way to represent the dimensions in this approach. Bearing this in mind, this paper proposes a way to specify artifact-centric business process models by means of well-known UML diagrams, from a high-level of abstraction and with a technology-independent perspective. UML is a graphical language, widely used and with a precise semantics.Preprin

EU-Rent as an artifact-centric process model: technical report

Business process modeling using an artifact-centric approach has raised a significant interest over the last few years. This approach is usually stated in terms of the BALSA framework which defi nes the four dimensions of an artifact-centric business process model: Business Artifacts, Lifecycles, Services and Associations. One of the research challenges in this area is looking for diff erent diagrams to represent these dimensions. Bearing this in mind, this technical report shows how various UML diagrams can be used to represent all the elements in the BALSA framework by applying them to the EU-Rent case study.Preprin

A semantic model to fight social exclusion

This work presents a semantic model meant to help with the identification and prediction of individuals at risk of social exclusion. The model is based on the self-sufficiency matrix, a tool that evaluates a person's self-sufficiency in different areas, and that is used by Barcelona's City Council. Existing data sources can then be mapped to this model, in order to analyze, query, and visualize the data.This work is partially supported by the Semiotic project, funded by Ministerio de Economia, Industria, y Competitividad (TIN2016-78473-C3-2-R).Peer ReviewedPostprint (author's final draft

Structural Determinants of Skeletal Muscle Ryanodine Receptor Gating

Ryanodine receptor type 1 (RyR1) releases Ca2+ from intracellular stores upon nerve impulse to trigger skeletal muscle contraction. Effector binding at the cytoplasmic domain tightly controls gating of the pore domain of RyR1 to release Ca2+. However, the molecular mechanism that links effector binding to channel gating is unknown due to lack of structural data. Here, we used a combination of computational and electrophysiological methods and cryo-EM densities to generate structural models of the open and closed states of RyR1. Using our structural models, we identified an interface between the pore-lining helix (Tyr-4912–Glu-4948) and a linker helix (Val-4830–Val-4841) that lies parallel to the cytoplasmic membrane leaflet. To test the hypothesis that this interface controls RyR1 gating, we designed mutations in the linker helix to stabilize either the open (V4830W and T4840W) or closed (H4832W and G4834W) state and validated them using single channel experiments. To further confirm this interface, we designed mutations in the pore-lining helix to stabilize the closed state (Q4947N, Q4947T, and Q4947S), which we also validated using single channel experiments. The channel conductance and selectivity of the mutations that we designed in the linker and pore-lining helices were indistinguishable from those of WT RyR1, demonstrating our ability to modulate RyR1 gating without affecting ion permeation. Our integrated computational and experimental approach significantly advances the understanding of the structure and function of an unusually large ion channel

Evaluación de la relación entre guía dentaria y guía condilar. Parte II: estudio poblacional

Se presenta un estudio sobre una muestra de 32 individuos, 14 hombres y 18 mujeres, con una edad media de 21,5 años, a partir de los cuales se evalúa la interrelación funcional entre la guía dentaria (o Determinante anterior) y la guía condilar (o Determinante posterior) a través del estudio de correlación d~ ángulos en los movimientos de protrusión y lateralidad. En ángulo de la guía condilar se mide en registros axiográficos, mientras que los ángulos de la guía dentaria se miden con registros kinesiográficos. No se encuentra correlación lineal estadísticamente significativa entre los valores de los ángulos de la guía dentaria y de la guía condilar, tanto para el movimiento de protrusión como para los de lateralidad