Medical and eHealth systems are progressively realized in the context of standardized architectures that support safety and ease the integration of the heterogeneous (and often proprietary) medical devices and sensors. The Integrated Clinical Environment (ICE) architecture appeared recently with the goal of becoming a common framework for defining the structure of the medical applications as concerns the safe integration of medical devices and sensors. ICE is simply a high level architecture that defines the functional blocks that should be part of a medical system to support interoperability. As a result, the underlying communication backbone is broadly undefined as concerns the enabling software technology (including the middleware) and associated algorithms that meet the ICE requirements of the flexible integration of medical devices and services. Supporting the on line composition of services in a medical system is also not part of ICE; however, supporting this behavior would enable flexible orchestration of functions  on the fly. iLandis one of the few software technologies that supports on line service composition and reconfiguration, ensuring time-bounded transitions across different service orchestrations; it supports the design, deployment and on line reconfiguration of applications, which this paper applies to service-based eHealth domains. This paper designs the integration between ICE architecture and iLand middleware to enhance the capabilities of ICE with on line service composition and the time-bounded reconfiguration of medical systems based on distributed services. A prototype implementation of a service-based eHealth system for the remote monitoring of patients is described; it validates the enhanced capacity of ICE to support dynamic reconfiguration of the application services. Results show that the temporal cost of the on line reconfiguration of the eHealth application is bounded, achieving a low overhead resulting from the addition of ICE compliance. Medical devices operate with strict safety and security standards in a rather closed manner, providing very restrictive interfaces. The reasons for this are, among others, their safety and security requirements, besides the fact that they are typically patented devices. As dictated by the non-stop technological evolution, medical systems have followed the path towards easing interoperability across vendors to facilitate the development of more powerful functions derived from the integration of heterogeneous services and equipment .Traditionally, clinicians would continuously monitor and operate a number of devices separately. Nevertheless, for developing modern and future medical systems, it is essential to achieve interoperability among medical devices and software services. The evolution of software paradigms and implementations, and their integration with most hardware platforms, has boosted the possibilities of medical systems. Still, the medical devices that are closest to patient monitoring tend to be based on special purpose hardware, being mostly closed systems; but this trend has started to be progressively abandoned towards more interoperable standardized open approaches.Given the critical nature of medical systems and applications, industry and suppliers have gathered around medical specifications and design approaches for realizing the interoperability goal . One of The full realization of ICE needs to consider specific middleware technologies at different levels to enable interoperation and distribution. Precisely, the required functions are data processing and exchange formats’ definition, distribution infrastructure, flexible functional composition and transport and network protocols. The realization of the ICE framework must include middleware technologies at the levels of the bare infrastructure middleware and the enhanced middleware. Infrastructure middleware provides the basic communication paradigm for distributed communication among functional units that reside in different nodes . Enhanced middleware provides additional logic tied to a specific application domain related with either its business logic or with the behavioral requirements of the application domain. Essentially, ICE is a standard for safety interoperability across nodes, defining the roles of the different participant devices and how these should interact. Therefore, this standard must be naturally supported by  an underlying communication bus  that provides communication functions among the participant devices; the used middleware must adjust to the non-functional properties required by the medical domains and in compliance with ICE safety requirements. This paper details the integration of ICE with a specific middleware that enables flexible reconfiguration capabilities inside ICE.https://sourceforge.net/projects/iland-project/) [Not all middleware technologies are suitable as communication backbones in healthcare systems. For instance, in a centralized patient monitoring system that receives and displays the information about patients in their rooms/apartments, a bad decision on the selection of a middleware technology may yield latencies of over tens of seconds; also, the wrong selection may result in lack of support for flexible on line service integration. This limits the potential of on line health monitoring systems. There are different middleware possibilities to take the strong position as the communication backbone for ICE. This is the case of DDS   that hasroject/)  is a comTherefore, integrating ICE and iLand allows on line orchestration of service-based medical applications. As an example, let us imagine a remote elderly patient monitoring scenario such as described in ; there, This paper describes the integration of on line composition capacities provided by iLand middleware into the ICE framework to comply with medical application requirements. The goal is to support on line flexible addition and/or removal of services to customize medical applications on-the-fly for specific patients.For this purpose, the paper presents the characteristics of the open source middleware technology named iLand  that is The paper is structured as follows. Current and future medical systems are progressively interconnected in order to flexibly exchange data among devices, departments and, most importantly, between patients and medical staff. Middleware is a fundamental software layer that enables this, and it is practically at the heart of almost every single distributed application nowadays. Middleware is characterized by abstracting the low level details of the communication protocols and the hardware characteristics of devices to programmers. This way, programmers can focus solely on the functional aspects of the application; this has a positive impact on their productivity and the application maturity level.Among the first middleware technologies one finds RPC . With the years, this interaction scheme evolved towards the component-based distribution paradigm of Corba  and objeIt acts as an interoperability provider, allowing information (patient records) to be communicated/transferred and formatted to the specific needs of physicians.It provides major flexibility across departments, as they may be interested in different patient data, which can be extracted as required from a global repository.It eases evolution to richer functionality in a short time such as hospitals and care centers in general, as new functions can be developed as individually-developed decoupled services.It allows the connection of mobile devices to the hospital network cloud, making data available to professionals anytime, anywhere.Patient engagement in their care could be increased as middleware can display their historic records and current monitored data in an understandable way so that they can participate in their healing process. The engagement of patients enabled by middleware requires that the middleware provide them with answers in an efficient, timely and robust manner.Middleware is also key to healthcare systems for a number of reasons; just a few of them are:One of the most frequent uses of middleware in medical systems is the automatic handling of patient records. Current practice often uses Health Information Systems (HIS) and Electronic Health Records (EHR) in an informal manner with ad hoc protocols and interoperability solutions in order to develop clinical systems. Typically, attention in these systems is paid at the pure application level where healthcare enterprise systems are put together to deliver specific clinical solutions.Aspects such as security and safety are essential in current solutions, especially when envisioning medical cyber-physical systems, as a number of subsystems will coexist and interact through a safety/security framework. Such a framework must provide rigorous design including on line verification such as exposed in  and trusThe ICE approach defines important elements, such as the protocol stack for medical device interoperability . The lowBelow this DPWS, there is the indication of a specific technology, apart from the typical de facto usage of HTTP/TCP or UDP. However, no reference to the basic communication middleware technologies  is in operation, ICE is silent on how to achieve time-bounded reconfigurations of the application due to, e.g., a change in a service or the appearance of a new service that has to be connected to the application.ICE is a framework for medical device interoperability; as stated in , it is: At the heart of the ICE idea is that medical devices must provide a compatible model aiming at interoperability  that is exposed through a specific interface, namely the ICE equipment interface. The device model is a representation of the capabilities of ICE compatible equipment that includes the information needed to qualitatively and quantitatively describe, control and monitor its operation. This includes the information exposed by the manufacturer through the equipment interface, as well as the non-functional requirements according to ASTM.The ICE network controller provides communication among ICE compatible equipment and the rest of ICE using the device model. It is in charge of ensuring that the functional capabilities, in accordance with the non-functional requirements in the device model, can be reliably delivered to the supervisor. Furthermore, it must generate alarms if the required performance cannot be delivered. An ICE network controller block manages a set of (or aggregation of) medical devices and side equipment that must provide a specified functionality meeting certain non-functional requirements. This block contains the necessary business logic according to the device model to provide the specified functional capabilities, and detecting the possible generated alarms. The ICE supervisor is a platform (both an equipment and the associated software) that provides a specific application logic  and an operator interface. It ensures that the specified functional capabilities are provided according to the ICE network controller, also handling the alarms.The ICE equipment interface is a part of an ICE-compatible equipment that provides the interface to the ICE network controller. It is typically an interface between software processes; it is not intended to be an interface between the operator and the compatible equipment.The mission of the data logger function block is to support forensic data storage and logging that enables the detection of incidents and abnormal situations in order to initiate mitigation actions; meaningful events  are recorded and stored and can later be analyzed to identify abnormal situations such as erroneous usage of the ICE-compatible equipment , failure of the devices and/or of the supervisor or Ice network controller failure.These functional blocks can be either physically separate or they can be integrated into one or more physical entities . The network controller is the interface where plug and play occurs. The external interface is the interface of the ICE outside of the clinical environment. According to the standard, this could be the facility backbone, public switched network or the Internet. The data logger is used to record everything within the ICE for future forensic activities, i.e., incident investigations and/or training scenarios.Overall, ICE provides a high-level architecture mostly containing functional blocks that need to be realized by specific proposals. Such proposals must be concrete technological options containing the distribution middleware software as a basic communication backbone connecting the different medical services, with both basic communication services, as well as other enhanced logic. Most of the ICE functional blocks are wrappers that refine the basic information model provided by the device model of each equipment or device to be integrated. One of the key blocks of ICE as concerns the interaction among medical devices and services is the network controller. This element is a point of communication of a specific service or device to other services and/or devices in the medical system; it is also a point of connection to the supervisor, which contains the application logic. The following section describes a proposal of how to integrate a specific middleware technology with ICE to support both basic decoupled communication services and enhanced logic for on-the-fly service composition.This section presents an overview of iLand, a communication middleware that is enhanced with additional intelligence to enable the time-bounded reconfiguration of distributed real-time systems based on services. This section introduces the main terminology and concepts of iLand, presenting the essential structure of the overall reconfiguration process.https://sourceforge.net/projects/iland-project/) of iLand uses a DDS backbone. iLand includes a number of enhanced functions to support dynamically reconfigurable applications based on services: light-weight services in the real-time version and web services in the soft and best effort version with QoS guarantees.iLand  is an opThe middleware provides time-deterministic reconfiguration mechanisms  and Service: A self-contained piece of code with well-defined interfaces that communicates with other services via message exchanges, as indicated in the Service-oriented Paradigm (SOA).Service implementation: It is a particular realization of a service. A given service can have different implementations that differ from one another due to their particular coding of the service interface. Two different implementations will require distinct computational resources, depending on the quality of the produced results.Service composition: A process by which different services are combined and orchestrated to merge as an application with global requirements such as the end to end response time. An example of this could be the connection of an oximeter service with a blood pressure service that might yield a basic daily health application for an in-house patient.Configuration: It is the set of software functions  that execute at a particular instant. A configuration is achieved by a specific service composition process. A configuration is a given set of service implementations.Reconfiguration: A transition from the current configuration to a new  configuration. This reflects a change in the application functions. An example might be the addition of a third temperature sensing service to the former basic daily health application.Time bounded reconfiguration: It is a reconfiguration process performed within a guaranteed maximum amount of time, i.e., deadline. It includes a time-bounded service composition algorithm .Real-time pruning and low-complexity reconfiguration: A reconfiguration process may involve the exploration of all possible combinations of configurations that could yield a high-complexity process. iLand designed a low-complexity reconfiguration process ,35 whereThe following is the basic key terminology of iLand:Off line exploration of the space of solutions: Prior to execution, the system design is fine-tuned for efficiency and timeliness. Two tools, generalized BTEA for timely service composition  and RealReliable communications: The communication time between distributed services assumes that the underlying communication transport is reliable and there is a real-time network with guaranteed message scheduling.Centralized verification of the configurations: The iLand reference implementation includes a single verification entity that has the view of the whole system see . The verCentralized reconfiguration coordination: There is a single entity that coordinates the reconfiguration process, mandating over the execution of the reconfiguration phases that are required for the transition between the current and the target configurations.Emergency configuration: As a mechanism to achieve fault tolerance, iLand has a back-up configuration that satisfies the system specification at all times. In the event that the reconfiguration process does not find a target configuration that satisfies the system specification, the emergency configuration is applied.Distributed real-time systems can be reconfigured with iLand middleware ensuring the timeliness of the transition from the current configuration to a new one. The reconfiguration logic is contained inside the control manager component. Configurations are expressed as service graphs with end to end timing properties, and a set of restrictions are imposed to achieve time-bounded transitions on line:The interface between the application level (including an operator) and iLAND is at the level of the application/service manager and communication manager components. Applications or an operator can register services and configure specific applications with the App/Service manager; the communication among services is supported by the functions of the communication manager component.The experiences gained with iLand have shown that the middleware is easy to deploy and also facilitates the design and execution of applications. For applications designed as decoupled stateless services, the middleware provides a plug and play approach for application reconfiguration and service orchestration, i.e., new services may be added, and running services can be deleted or replaced. The design of the application is indicated in the form of a configuration file or using a front-end tool.https://sourceforge.net/projects/iland-project/). In the interface, a service specifies a functional part and the non-functional part [The initial step is to model the application as a Service-Oriented Architecture (SOA). Services must provide the interface specified by iLand API , planet (for nodes without the reconfiguration coordination modules) and satellite  and forward them to a planet or star configuration and also to receive commands from a planet node).The star configuration supports the design of the specific application SOA in both a configuration file or with a model-driven tool front-end. Furthermore, it allows launching the services. Once services are in execution, these are automatically connected as specified.A list of reconfiguration events can also be specified in the configuration file. Apart from these, the logic of the services also allows detecting reconfiguration triggers .Following, the path to having an application up and running on iLand middleware is explained:Once in execution (and upon the triggering of the reconfiguration events), iLand automatically handles the system structure and makes timely transitions from the current configuration to the new one. iLand implements a constant monitoring activity to detect situations requiring a reconfiguration of the system. This logic has proven to be efficient with little overhead, as well as the reconfiguration logic that performs a previous selection of the target system configuration.Based on the previously presented ICE and iLand architecture, this section presents their integration. As a result of the integration, ICE embeds additional logic to: (1) support flexible communication among the software services and the medical devices and to (2) provide reconfiguration capabilities to applications for on line integration  of services.ICE is simply a generic architecture to design medical devices and services that can be easily interconnected. However, it only defines the general view of the basic components to interconnect devices among themselves and with a clinician or operator. Consequently, ICE does require the integration of specific software technology designs to: (1) enable flexible communications among devices; and (2) additional logic for improved behavior, such as the on line composition of services.By integrating iLand into ICE, the capacities of the latter are increased by offering flexible on line service plug and play; as iLand supports on line application definition based on service graphs with end-to-end time requirements. This is a clear improvement for the ICE architecture as applications can be defined and modified on line by operators according to the specific changing patient needs.The iLand architecture is based on the classical definition of middleware architecture , later rDeviceDeviceDeviceFollowing, it is indicated how the interaction between ICE and iLand occurs for the on line service registry and application reconfiguration. In this case, there is an application running made of two devices or services that are connected as a directed graph of the form new_service_register) can then be granted. However, requests to run a new application (or to reconfigure an existing one) have to be checked for feasibility and correctness. The verification logic of iLand is run to make sure that the new application execution will comply with the requirements expressed in the invocation to new_app_run and that the rest of the already running services will not suffer interference. If the requested new application is feasible, then the control manager component of iLand will execute the reconfiguration protocol and ensure a timely transition to the new configuration.There are two ways to perform this integration. A first approach is to include specific iLand components that provide on line service composition as functional blocks. This option is, however, invasive with respect to the ICE architecture, and it would require modifications to the standard high-level definition of ICE. A second approach is to preserve the independence of ICE functional blocks, including only an additional block that contains the minimum iLand services that guarantee the time-bounded service communication, composition and reconfiguration. This second approach is adopted as it minimizes invasion and coupling among ICE and iLand while providing the benefits of both. The integration of the reconfiguration logic does not affect the medical devices’ operation. Medical devices are connected to the distributed medical system through a gateway: the ICE equipment interface. This interface interacts with the ICE network controller that is the entry point to the ICE manager part, in the sphere of: the supervisor, the external interface and the data logger. iLand is located in the ICE manager part, and it only interacts with the ICE supervisor and with the external interface.Communication Manager (Comm. Mngr): It includes the main infrastructure middleware functions that provides real-time communications (through a custom protocol stack) or QoS communication (through standard transport protocols). If the underlying communication backbone supports QoS parameters , iLand offers the possibility of setting such parameters.One or more medical device can be managed under the same ICE supervisor to provide a specific functionality. These medical devices can be provided to other applications as services, e.g., oximeter data reads provided as a software service to a nurse control center set. Therefore, the ICE supervisor needs to integrate some additional logic to keep the structure of applications and services interconnection; this is enabled by the application/service manager component. In iLand CFL, there are two separate components for managing applications and services. The Service Manager (SM) allows registering and eliminating individual services in the system, specifying their functional and non-functional properties and interfaces. The Application Manager (AM) component supports the definition of applications that are made of aggregations of services and that have global functional and non-functional properties; these are specified as service graphs. For simplicity, the interfaces to these two components are provided in a unified way in this integration of iLand-ICE.For supporting on line composition of the services, ICE needs to integrate both the service composition logic (composition logic component) to provide on line medical services integration, as well as the logic that coordinates the actual selection of services ((Reconfig.) control manager component).Vendors guarantee device interoperability by providing a standard interface for each device: the ICE equipment interface. Compatibility across the functions of different devices is provided at the application level, within the ICE supervisor that is in charge of collecting, analyzing and displaying the data from devices either individually or in an integrated way; this will depend on the specific logic of the application that is running, e.g., the ICE supervisor may be executing an application that simply displays the different data collected by the medical devices that monitor the patient vital conditions; or it may also display inferred data derived from the cross-processing of the monitored data.This section presents a monitoring system for an elderly house that is based on an initial implementation on iLand middleware. In this section, it is explained how it has been adapted to the Integrated Clinical Environment (ICE) to enhance its interoperability with devices following the ICE architecture.In this specific example, patients may be in severe physical conditions; therefore, they need to be monitored in real time. They are located in a centralized building (elderly house), and each patient has an individual room specially configured according to the patient’s health requirements. This means that the devices and sensors across rooms are different and, also for a specific patient, these devices can vary over time according to the person’s needs. Each room has a service patient manager (or room manager) and a set of medical sensors to monitor the patient health conditions. The room manager is a front-end monitoring and decision making system that performs basic analysis of the patient’s monitored data. The medical equipment subsystem is simulated, and the gathered data are synthetically generated to test monitoring algorithms with physical parameters analysis. The room manager is an embedded system with a basic interface, integrated with the room. The clinician supervisor server (or control center) is equipped with iLand middleware and a database connection.Time requirements are specified in this system as there are different criticality levels, some being highly critical. The periodicity of the medical patient monitoring samples range from 1 s to 1 min, according to the patient health conditions. Data logs to the control center are stored in 1-s periods. For any detected alarm, the patient manager must trigger the clinical supervisor within a specified deadline in the range of 500 ms to 1 s.Different reconfiguration scenarios are possible. Reconfigurations are triggered by the occurrence of some specific events (reconfiguration triggers), and they start a process to perform a new service composition in the system. The occurrence of a reconfiguration trigger may be the arrival of a new patient; room adaptation according to patient health conditions; and alarm event detection . Therefore, it is evidenced that a reconfiguration trigger requires a modification in the set of services that are active in the system at the moment that it occurs.data collection services  datdata collection services obtain the data sampled by the medical sensors; each sensor has an individual service that collects its sampled data;data analysis fuses the data and reasons about the profile of the patient in order to determine the current health conditions of the patient and commits the result to the local storage;data logs format the sampled data and the obtained health status of the patient and send them to the clinical supervisor;transfer logs execute at the clinical supervisor side by retrieving the logs sent by the room managers and forwards them to a storage server;store logs store the monitored data of the patients in persistent storage.The set of services of the patient monitorization application see  are: (i)An additional SOA performs log data display to medical staff in the control center. The log data display SOA has two services: read DB (for interfacing to the database) and display for handling data formatting and display.This design allows handling each room individually while preserving continuous operation through a single end point for control. As an example, upon arrival of a new patient, it is possible to instantiate the two SOAs needed for data collection and supervision/monitoring in this room to configure them without affecting the rest of the system. Likewise, reconfiguration only concerns the data collection SOA of a single room and not the whole system. A similar scenario to the one proposed above was originally implemented with bare iLand . Here, iPreserving the service-based design, as it supports decoupled application development and enhances system flexibility; for this purpose, all services must be registered in the iLand component that will keep the overall view of the medical application .Mapping all functional blocks of ICE to the specific services that are present in the example; the service-based design and iLand low-complexity architecture provides a natural way to perform this mapping.Each sensor in the example is a medical device that runs an ICE equipment interface according to its specified device model. These devices perform autonomous sampling of patient data that is restricted to data sensing and reading simulations that provide the monitored data at a predefined frequency.Data collection services are the software interface of the sensors. Measure data services obtain the patient monitored data by the sensors via the ICE equipment interface. Sensors are exported as services in the iLand application; this way, each sensor can be accessed individually and connected on line to build a specific application. Therefore, iLand services are simply wrappers to the sensors’ functionality.Collect log and retrieve DB logs are services that correspond to the ICE data logger functional block; again, they only require providing wrappers to the data logger functionality. The services data are exported to the data logger via an interface to guarantee abstraction independence between the services implementation and the functional ICE blocks. In this way, the iLand application structure information, the reconfiguration logic and registry/deletion of services is kept independent from the ICE functional blocks.Services read new room configuration and device control control each of the services implemented for sampling data . Every time a new device or sensor is installed, these services are uploaded to make it usable in the iLand system.The room manager module contains a number of services that are mapped to the ICE supervisor. This service is, precisely, the local monitoring. Again, this iLand service is exported to the ICE supervisor via an interface to guarantee abstraction independence between the services implementation and the functional ICE blocks.Send log service is mapped to the ICE external interface that provides communication to other devices that are, in this case, used for sending the patient data to another system, such as a server, etc.Following, the specific mappings between the elderly house application design based on services and ICE architecture are presented:This section presents the practical implementation of this example. Initially, the implementation over iLand is presented; later, the example is ported to comply with the ICE architectural framework. This section shows the feasibility of integrating an iLand-enabled distributed service-oriented medical application that supports reconfiguration into an ICE architecture, providing experimental results that show the reconfiguration time achieved with iLand for the timely delivery of alarms.Firstly, a real-time situation is presented with low latency requirements. It is considered that during the normal operation of the system , an alarm may occur. The reasons for this may be the detection of abnormal sensed values that would require immediate signaling.iLand supports a number of underlying communication backbones using both Internet transports and/or custom real-time network protocols. In this situation, the underlying communication protocol needs to be highly reliable and timely; data cannot be lost nor suffer unbounded delays. For this system, iLand middleware was adapted to time-triggered communication, which enables real-time schedulability analysis in the verification component. The time-triggered protocol  was analThe goal of this initial experiment is to measure the reconfiguration time as the time to forward an alarm to the room manager and to the clinician terminal when an alarm has been detected during the normal operation of the system. Results are shown in In the following experiment, a reconfiguration use case is provided as the result of a new service being registered in the system , to obtain patient data logs  and remote video surveillance of the patient . The reconfiguration is triggered when an alarm is detected. The figure shows the time to start the video surveillance service that is physically linked to an IP camera connected to its controlling node via an Ethernet interface. A UDP transport is used for iLand over DDS. It is shown that the worst case reconfiguration time never exceeds This paper has presented a contribution for supporting dynamic reconfiguration in ICE. The approach is based on the usage of iLand middleware that provides time-bounded reconfiguration of distributed applications based on the service-oriented paradigm. This approach is supported by the proven flexibility of iLand in different real application domains, including medical environments.The paper has presented the characteristics of the Integrated Clinical Environment, indicating its gaps with reference to the specific technologies and middleware for enabling decoupled communication and on line flexibility for service deployment. iLand middleware has been presented as a choice for the flexible development of distributed systems based on medical services. This integration of iLand in ICE has been done for two fundamental reasons, which are explained as follows.On the one hand, the current trend to provide technologies that enable flexible and interoperable services requires that contributions meet the safety guarantees that are fundamental in the medical domains. The latter is the main purpose and goal of the Integrated Clinical Environment.On the other hand, ICE is an abstract architecture that defines general functional blocks to define safety-compliant architectures for medical systems. However, the initial definition of ICE focuses on medical device interoperability, and it is silent about functional reconfigurations. The latter is precisely covered by iLand middleware. The interaction paradigm of iLand is a decoupled one, therefore supporting decoupled integration of services that can be either local in the same node or distributed among different nodes in different network segments. iLand middleware has a modular architecture enabling easy porting to different underlying communication middleware technologies; these range from the most conventional and de facto standards, such as DDS, Corba, RMI, Ada DSA, or lower level networking protocols operating at the medium access layer such as time-triggered ones. The reconfiguration logic of iLand is based, on the one hand, on graph algorithms for representing applications and the relation among their constituent services; on the other hand, it is based on search techniques that target timely decision making and a set of steps to guarantee the reconfiguration of a whole system based on services.Therefore, there is a benefit in integrating both worlds so that service-oriented applications with real-time guarantees can be provided in a way that is compliant with ICE.The paper has provided the mappings between the services of a medical application based on iLand and the ICE architecture. The fact that iLand uses a decoupled service-oriented interaction scheme is an advantage for its integration within ICE. The paper has shown the mapping of the different services that are registered in the iLand middleware to the different functional blocks of ICE. A specific example application has been implemented for the remote monitoring of elderly patients, showing the timeliness of the middleware, and the bounded reconfiguration times achieved in two different reconfiguration situations: on line addition of new sensors and on line activation of existing services.Reconfigurations are guaranteed to be completed in bounded time: the modification of a service (replacing a specific implementation) or an SOA (replacement of the service graph) is achieved within the specified deadline, showing the time measurements performed for the elderly house case; time measures confirm the real-time operation of the system and the stability of the reconfiguration logic within an ICE-enabled framework.


Alaugc drop off in biochemical assays of successive incorporations. Here we predict the function of this tRNA 2′OH in the ribosomal A, P and E sites using recent co-crystal structures of ribosomes and tRNA substrates and test these structure-function models by systematic kinetics analyses. Unexpectedly, the C75 2′H did not affect A- to P-site translocation nor peptidyl donor activity of tRNAAlaugc. Rather, the peptidyl acceptor activity of the A-site Ala-tRNAAlaugc and the translocation of the P-site deacylated tRNAAlaugc to the E site were impeded. Delivery by EF-Tu was not significantly affected. This broadens our view of the roles of 2′OH groups in tRNAs in translation.Aminoacyl-tRNAs containing a deoxy substitution in the penultimate nucleotide (C75 2′OH → 2′H) have been widely used in translation for incorporation of unnatural amino acids (AAs). However, this supposedly innocuous modification surprisingly increased peptidyl-tRNA Importantly, the 2′OH can be involved in intra- and inter-molecular hydrogen bonding interactions to contribute to the structure and function of RNA.The 2′-hydroxyl group (2′OH) of the ribose in the backbone of ribonucleic acids (RNAs) is the main difference between RNAs and deoxyribonucleic acids (DNAs). Because of the presence of the 2′OH in RNAs, the sugar pucker is predominantly in the C3′-endo conformation in contrast to the C2′-endo conformation that predominates in DNAs2. The ribosome catalyzes the polymerization of amino acids (AAs) in the sequence encoded by the messenger RNA (mRNA); the site in the ribosome where peptide bond formation occurs, the peptidyl transferase center (PTC), is composed entirely of RNA; AAs are linked to transfer RNAs (tRNAs) via ester bonds with the terminal A76 of the tRNAs before they can serve as the substrates of ribosomes2.RNAs play crucial roles in protein synthesis where the macromolecular translation machinery, the ribosome, is an RNA-protein complex3. Pleiss and Uhlenbeck found that five distinct 2′OH groups in unmodified yeast tRNAPhe were critical for the binding affinity of the AA-tRNA to elongation factor Tu (EF-Tu)4, noting that prior co-crystallographic data showed that four of these 2′OH groups formed inter-molecular hydrogen bonds with EF-Tu and the fifth formed an intra-molecular hydrogen bond. Interestingly, though EF-Tu recognizes the AA and A76 terminal 2′OH of the AA-tRNA, this 2′OH and the neighboring C75 penultimate 2′OH were shown to be unimportant for EF-Tu binding4. However, another study showed that replacing the A76 terminal 2′OH with 2′H abolished binding between a different AA-tRNA and EF-Tu5. This latter study also demonstrated important roles of this terminal 2′OH in peptidyl transfer and peptide release reactions, presumably by orienting the substrate for catalysis and/or facilitating proton transfer in the catalysis.The roles of the 2′OH groups in tRNA bodies have been studied in different translation steps. For example, Feinberg and Joseph identified two 2′OH groups, at positions 71 and 76 of tRNA, that were essential for translocation of the P-site tRNA to the E site, and these two 2′OH groups were shown to contact the backbone of 23S rRNA in the E site in the crystal structure of the ribosome8. The lack of modifications and the C75 2′H in the tRNA body showed negligible effect on single incorporation yield of an AA into polypeptide10, and therefore such changes were believed to play only minor roles in translation. Surprisingly, when incorporating multiple AAs consecutively with the unmodified AA-tRNAAlaugc bearing the 2′H in C75, we found that these supposedly benign changes caused a big reduction in full-length peptide yields10 by allowing more time for peptidyl-tRNA drop off11. This reduction was not due to the lack of tRNA modifications as the kinetics and yields with unmodified C75 2′ OH transcript were indistinguishable from the native modified isoacceptor11, despite studies showing critical roles of tRNA modifications in some other tRNAs17. Here we use recent ribosome-substrate crystal structures to predict the function of the 2′OH in C75 of the A-, P- and E-site tRNAAlaugc and test these structure-function models by fast kinetics.The 2′OH in the penultimate C75 of tRNAs, however, was believed to be unimportant in translation. AA-tRNAs containing a 2′H in C75, created by ligation of the 3′CA-truncated unmodified tRNAs to pdCpA-amino acids, had been widely used in translation for incorporation of unnatural AAsAlaugc, we examined recent co-crystal structures of the 70S ribosome bound to substrate analogs18. These structures show substrates in A-, P- and E-sites  are affected by the dC75 atomic mutation in tRNAtes Fig.  and are tes Fig.  to show tes Fig. .Figure 1Given that the C75 2′OH forms H bonds at the A, P and E sites, it is easy to rationalize our overall inhibition of translation observed by replacement with a 2′H in these 3 positions on the ribosome. The loss of the A-site H bond to the O4 of the A76 sugar, which in turn is attached to the AA nucleophile, may be predicted to affect correct positioning for nucleophilic attack. The loss of the P-site H bonds with W2 and with the 2′OH of ribose C2073 of 23S rRNA may be predicted to affect correct positioning of the electrophile and also translocation of the A site peptidyl-tRNA to the P site (because the A site tRNA would not form the H-bond with the P site rRNA). The loss of the E-site intramolecular H-bond Fig.  might beAla substrates with either C75 2′OH , the subsequent time for fMet-Ala (fMA) dipeptide bond formation (to assay peptidyl acceptor activity of Ala-tRNAAla), A to P site translocation of fMA-tRNAAla, fMet-Ala-Phe (fMAF) tripeptide formation from EF-Tu:GTP:Phe-tRNAPhe and fMA-tRNAAla (to assay peptidyl donor activity of P site fMA-tRNAAla), and P to E site translocation of tRNAAla.To test our predictions based on crystal structures, we prepared Ala-tRNAmentclass2pt{minimAla ternary complex concentrations of the two kinds of substrates . This indicated that one or more steps up to the peptidyl transfer reaction were impeded by replacing the C75 2′OH with the 2′H. Next, we measured the mean times for GTP hydrolysis on EF-Tu (τGTP) and dipeptide formation (τdip) simultaneously ; (ii) fMAF tripeptide formation from fMet-tRNAfMet, Ala-tRNAAla and Phe-tRNAPhe (tot); and (iii) fMAF tripeptide formation from translocated fMA-tRNAAla and Phe-tRNAPhe (step3). The mean time for translocation from A site to P site could then be calculated as τstep2 = τtot − τstep1 − τstep3 , as expected. Surprisingly, the τstep3 for the two cases had no significant difference  fMAAla. This step occurred 3 times during our incorporation of 5 consecutive Ala-tRNAAla substrates11. In order to isolate the effect of the C75 2′H on a single translocation step, we used a simpler translocation assay here: monitoring the mRNA movement with a 3′ pyrene-labeled mMA2FT mRNA28. A post-translocation complex programmed with this fluorescent mRNA and fMA2-tRNAAla in the P site  was rapidly mixed with EF-Tu:GTP:Phe-tRNAPhe ternary complex together with EF-G:GTP on a stopped-flow apparatus . The over-expressed E. coli tRNAfMet and tRNAPhe were prepared respectively as described33. All other reagents were prepared as described34.in vitro translation reactions were at 37 °C in pH 7.5 polymix buffer supplemented with 1 mM ATP, 1 mM GTP, 10 mM PEP, 1 μg/mL pyruvate kinase and 0.1 μg/mL myokinase for energy regeneration34. Two mixtures were prepared on ice in the polymix buffer. A ribosomal mix contained MRE600 E. coli 70 S ribosomes (1 μM), IF1 (1.5 μM), IF2 (0.5 μM), IF3 (1.5 μM), [3H]fMet-tRNAfMet (2 μM), and mMAF (4 μM). For the case of Ala-3H] peptides and the unreacted [3H]fMet from each time point was analyzed by C18 reverse phase HPLC coupled with a β-RAM model 3 radioactivity detector (IN/US Systems). The separation of fMet and fMA dipeptide was done by elution with 0% methanol/99.9% H2O/0.1% trifluoroacetic acid for 20 min at 1 mL/min followed by another 40 min at 0.45 mL/min. After quantifying the [3H] fMA fraction from the total [3H] signal at each time point, the data were analyzed by the nonlinear regression program Origin 7.5 with a 2-step kinetic model31 and dipeptide formation reaction mean times, τdip, at each ternary complex concentration were obtained. The inverses of the mean times were then plotted against ternary complex concentrations and fitted to the hyperbolic Michaelis-Menten equation to deduce the maximal overall rate constant for dipeptide formation, kcat, as well as KM values34.To compare the A site acceptor reactivity of Ala-τGTP) and dipeptide formation (τdip), a ribosomal mix was prepared similarly to that described above, except that the 70S ribosome was added to 2 μM and [3H]fMet-tRNAfMet to 2.5 μM. In the ternary complex mix, unlabeled GTP was omitted and ATP was added to 2 mM. The ternary complex mix contained 1 μM [3H]GTP, 1 μM EF-Tu and 1.2 μM Ala-31. The overall mean time, τacc,pep, for all the steps that lead to peptide bond formation after GTP hydrolysis on EF-Tu was obtained by subtracting the mean time for GTP hydrolysis on EF-Tu from the mean time for dipeptide formation.To simultaneously measure the mean times for GTP hydrolysis on EF-Tu (27. The first reaction was fMAF tripeptide synthesis starting from 70S initiation complex such that the mean times for fMA dipeptide formation (τstep1) and fMAF tripeptide formation (τtot) could be deduced. In this reaction, the ribosomal mix contained 0.6 μM 70S ribosome, 0.9 μM IF1, 0.3 μM IF2, 0.9 μM IF3, 1 μM mMAF and 0.5 μM [3H]fMet-tRNAfMet. The ternary complex mix contained 20 μM EF-Tu, 2 μM EF-Ts, 22 μM EF-G, 0.4 mM phenylalanine, 0.4 unit/μL PheRS, 8 μM tRNAPhe and 6 μM Ala-τstep3, could be obtained. In this reaction, three mixtures were prepared. The ribosomal mix had doubled concentrations of the components as in the ribosomal mix in the first reaction. An elongation mix, E1, contained 8 μM EF-Tu, 1 μM EF-Ts, 4 μM EF-G and 12 μM Ala-E2, contained 16 μM EF-Tu, 1.5 μM EF-Ts, 20 μM EF-G, 0.4 mM phenylalanine, 0.4 unit/μL PheRS and 8 μM tRNAPhe. After preincubation for 15 min at 37 °C for the three mixtures, equal volumes of the ribosomal mix and E1 mix were rapidly mixed in the reaction tube for 5 sec before incubation on ice for 10 min. The resulting mixture and E2 mix were applied to the quench-flow apparatus for kinetic experiments. The reaction and sample treatment were performed in the same way as described above. Dipeptide, tripeptide and unreacted fMet was separated on C18 reverse phase HPLC by eluting with 0% methanol/99.9% H2O/0.1% trifluoroacetic acid for 20 min at 1 mL/min and another 10 min at 0.45 mL/min, followed by 30 min elution with 58% methanol/42% H2O/0.1% trifluoroacetic acid at 0.45 mL/min. Data analysis was done as described26 and the complete translocation time, τstep2, was calculated as τstep2 = τtot − τstep1 − τstep3.To measure the rate of the translocation step between the formation of fMA dipeptide and the formation of fMet-Ala-Phe (fMAF) tripeptide, two reactions were done in parallel2FT mRNA was chemically synthesized by IBA GmbH (Göttingen), kindly provided M. Holm and S. Sanyal, and had the sequence 5′ uaacaauaaggagguauuaaauggcagcauuuacg 3′. Three mixtures were prepared for this measurement similar to the three mixtures in the A- to P- site translocation assay. A ribosomal mix contained 0.8 μM 70S ribosome, 1.2 μM IF1, 0.4 μM IF2, 1.2 μM IF3, 1 μM [3H]fMet-tRNAfMet and 1.5 μM pyrene-labeled mRNA. An elongation mix, E1, contained 2 μM EF-Tu, 0.25 μM EF-Ts, 4 μM EF-G and 2 μM Ala-E2, contained 10 μM EF-Tu, 0.5 μM EF-Ts, 38 μM EF-G, 0.4 mM phenylalanine, 0.4 unit/μL PheRS and 6 μM tRNAPhe. After preincubation of the three mixtures for 15 min at 37 °C, equal volumes of the ribosomal mix and E1 mix were rapidly mixed in the reaction tube for 10 sec before incubation on ice for 10 min. The resulting mixture and E2 mix were loaded onto a stopped flow instrument (Applied Photophysics SX20). Pyrene fluorescence was excited at 343 nm and the change in fluorescence after mixing was recorded using a 360 nm long pass filter (Newport 10CGA-360). Data analysis was done as described27.The 3′ pyrene-labeled mMAAll data generated or analyzed during this study are included in this published article (and its Supplementary Information files).Supplementary Information


Autophagy is a bulk degradation pathway for long-lived proteins, protein aggregates, and damaged organelles. ULK1 protein kinase and Vps34 lipid kinase are two key autophagy regulators that are critical for autophagosome biogenesis. However, it isn’t fully understood how ULK1 regulates Vps34, especially in the context of disease. Polyglutamine expansion in huntingtin (Htt) causes aberrant accumulation of the aggregated protein and disrupts various cellular pathways including autophagy, a lysosomal degradation pathway, underlying the pathogenesis of Huntington’s disease (HD). Although autophagic clearance of Htt aggregates is under investigation as therapeutic strategy for HD, the precise mechanism of autophagy impairment remains poorly understood. Moreover, in-vivo assays of autophagy have been particularly challenging due to lack of reliable and robust molecular biomarkers.We generated anti-phosphorylated ATG14 antibody to determine ATG14-mediated autophagy regulation; we employed Huntington’s disease (HD) genetic cell models and animal models as well as autophagy reporter animal model to understand autophagy signaling and regulation in vivo. We applied biochemical analysis and molecular biology approaches to dissect the alteration of autophagy kinase activity and regulation.Here, we demonstrate that ULK1 phosphorylates ATG14 at serine 29 in an mTOR-dependent manner. This phosphorylation critically regulates ATG14-Vps34 lipid kinase activity to control autophagy level. We also show that ATG14-associated Vps34 activity and ULK1-mediated phosphorylation of ATG14 and Beclin 1 are compromised in the Q175 mouse model of Huntington’s disease. Finally, we show that ATG14 phosphorylation is decreased during general proteotoxic stress caused by proteasomal inhibition. This reduction of the specific phosphorylation of ATG14 and Beclin 1 is mediated, in part, by p62-induced sequestration of ULK1 to an insoluble cellular fraction. We show that increased ULK1 levels and phosphor-mimetic mutant ATG14 facilitate the clearance of polyQ mutant in cells.Our study identifies a new regulatory mechanism for ATG14-Vps34 kinase activity by ULK1, which can be used as valuable molecular markers for in-vivo autophagic activity as well as potential therapeutic target for the clearance of polyglutamine disease protein.The online version of this article (doi:10.1186/s13024-016-0141-0) contains supplementary material, which is available to authorized users. Macroautophagy (simply referred to as autophagy) is a lysosome degradation pathway involving the synthesis, trafficking, and degradation of autophagic vacuoles, or autophagosomes. Basal autophagy is responsible for the turnover of long-lived proteins, protein aggregates, and damaged organelles, but can also be upregulated to cope with various cellular stressors. In fact, autophagy disregulation has been implicated in many disease states. Several studies of postmortem Huntington’s disease (HD) brains and animal models have indicated altered autophagic activity –4. As a Autophagy is tightly regulated by multiple signaling pathways related to nutrient sensing and cellular stress. ULK1 is a serine/threonine kinase that initiates the autophagy cascade . ULK1 isHD is a fatal neurodegenerative disease caused by mutations in the Htt gene that code for expanded polyglutamine tracts (polyQ) in the first exon, which causes protein aggregation and neuronal loss throughout the brain, most notably in the striatum and cortex. The precise nature of autophagy alterations in HD is not completely understood. However, increasing autophagy has been shown to facilitate the clearance of mutant Htt aggregates , 13. TheRecent work has aimed at understanding the autophagy pathway in finer detail, during the HD pathogenesis. As part of this, a link between the ULK1 kinase and the autophagy receptor, p62/SQSTM1, has been identified. ULK1 phosphorylates p62 to promote selective autophagy in response to proteotoxic stress . ExpressHerein, we report a mechanism whereby ATG14-Vps34 activity is regulated by ULK1-mediated phosphorylation of ATG14. This phosphorylation occurs in an mTOR-dependent fashion. In contrast to our previous report of increased ULK1-mediated p62 phosphorylation in animal and cellular HD models ATP (PerkinElmer), Calf intestinal alkaline phosphatase (New England Biolabs), and TALON metal affinity resin (Clontech) were purchased from the companies listed.Vps34 (#4263), Myc (#2276), Actin (#3700), and Beclin 1 pS14 (#84966) were purchased from Cell Signaling Technologies . Vps15 (#A302-571A), and NRBF2 (#A301-852A) were purchased from Bethyl Laboratories. ATG14 , polyQ , huntingtin aggregates  Beclin 1 , LC3 , Flag-M2 , p62 , p62 pS409 (in house), ULK1 , GAPDH , and GFP  were purchased from indicated company. The ATG14 antibody used to detect the purified fragment (a.a. 20–73) was made in house [Myc-ULK1 wildtype and kinase inhibited (K46I) mutant were provided by Dr. Sharon Tooze (London Research Institute). Beclin 1-AsRed, ATG14-GFP, FLAG-ATG14, and myc-Vps34-his-Vps15 plasmids were generated as previously described . ATG14 aMEF, HEK293T, HCT116, and HeLa cells were maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum (Invitrogen) and 50 μg/mL penicillin and streptomycin (Invitrogen). Wild type and p62 KO MEF cells were provided by Dr. Masaaki Komatsu (Niigata University)  and wereAll IP’s were performed using 10 mM Tris–HCl pH 7.5, 2 mM EDTA, 100 mM NaCl, 1% NP-40, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche). All other lysates were prepared using 50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, an protease/phosphatase inhibitors (Roche). Supernatants were collected after centrifugation at 13,000 g for 15 min at 4 °C. Supernatants were subjected to BCA assay and then resolved by SDS-PAGE.°C. Samples were incubated with 30 μl Dynabeads (Life Technologies) for 1– 2 h at 4 °C and then washed three times with lysis buffer. Two thirds of the beads were reserved for western blotting, while the remaining third was used for the kinase assay. Beads for the kinase assay were washed once with wash buffer  and then resuspended to a final volume of 50 μl with reaction buffer . 10 nM wortmannin was included in the indicated controls. The reaction was started with the addition of γ-32P-ATP (5 mCi) and the samples were shaken at 37 °C for 30 min. Reactions were stopped with 120 μl stop buffer (CHCl3/CH3OH/HCl at a 10:20:0.2 volume ratio) and then shaken for another 10 min at room temperature. Samples were centrifuged for 5 min at 1000 g. 15 μl of the lower, organic phase was resolved on silica coated TLC plate (Millipore) using CHCl3/CH3OH/NH4OH/H2O (86:76:10:14 volume ratio) and visualized with the Typhoon 9400 Variable Imager .Immunoprecipitation was performed with 500 μg protein from post-nuclear supernatant, using antibodies against ATG14 (MBL) overnight at 4 2, 5 mM EGTA, 0.25 mM DTT, 100 μM cold ATP, protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail (Roche)) and then resuspended to a final volume of 50 μl with reaction buffer including 5 μg of substrate (ATG14 a.a. 20–73 or MBP) and 5 μCi 32P-ATP. Reaction was incubated at 37 °C for 30 min. Samples were subject to gel electrophoresis in a 4–12% bis-tris gel. The gel was visualized with the Typhoon 9400 Variable Imager .Myc-ULK1 WT, myc-ULK1 kinase inhibited, or myc protein was immunopurified from HEK cells using anti-myc antibodies. IPs were washed once with kinase buffer  cells by growing at room temperature for 16 h with 0.05 mM of IPTG. Bacterial cell lysis and protein purification was done using the TALON metal affinity resin kit in strict accordance with the recommended manufacturers protocol (Clontech).Expression of His-ATG14 a.a. 20–73 was induced in °C, Triton X-100 insoluble fractions were collected. Pellets were washed 4x in lysis buffer. Pellets were further solubilized in PBS containing 1% SDS, 1% Triton X-100, and phosphatase/protease inhibitors at 60 °C for 1 h. Triton X-100 insoluble fractions were collected after centrifugation at 15,000 g for 30 min at 4 °C. A BCA assay was done on the lysates to ensure equal protein concentrations for western blot. This procedure was described elsewhere [Samples were lysed in PBS containing 1% Triton X-100 and phosphatase/protease inhibitors on ice for 30 min. After centrifugation at 15,000 g for 30 min at 4 lsewhere .Mice were transcardially perfused with ice cold PBS to remove excess blood, then perfused with cold 4% paraformaldehyde. Brain sections were blocked in PBS containing 3% BSA and 0.1% Triton X-100 for 1 h at room temperature. Sections were then incubated with primary antibodies in blocking buffer overnight at 4 °C. After washing 3 times in PBS sections were incubated with Alexa-conjugated secondary antibody for 1 h at room temperature. Secondary antibody was goat anti-mouse Alexa Fluor 555. After 3 more washes with PBS, sections were mounted with mounting medium . Cells were examined under Carl Zeiss upright or invert confocal microscopes (LSM780 system). Images were taken with 63X oil immersion objective lens at room temperature and image acquisition was performed by Zen2012 software.All animal studies were performed in compliance with IACUC  at Icahn School of Medicine at Mount Sinai. Heterozygous z_Q175 and littermate controls were obtained from the CHDI colony at the Jackson Laboratories. For imaging analysis, GFP-LC3 expressing mice  were croP values <0.05 were considered as statistically significant.Data are presented as mean ± SEM from at least three independent experiments. Statistical analysis was performed with GraphPad Prism v5.0 (Graphpad Software). For data normalized with control group , one sample t-tests against a hypothetical value of 1 were used to compare the difference between the other group and control group. For other non-normalized values, unpaired Student’s t-tests were used. For multifactorial analysis, a two-way ANOVA was used. Bonferroni’s correction was used for multiple comparisons. 32P labeled ATP. The ATG14 fragment showed incorporation of 32P when incubated with ULK1 WT but not with mutant KI  MEF cells with Torin 1. Torin 1-induced ATG14 phosphorylation was detected in wildtype MEF cells but not in ULK1/2 DKO MEF cells Fig. . KnockinTo examine the role for pS29 of ATG14 in Vps34 kinase activity, we generated ATG14 phosphorylation mutants either to mimic phosphorylation (S/E) or suppress phosphorylation (S/A). FLAG-ATG14 S29E-associated Vps34 showed a significant increase in lipid kinase activity compared to that of FLAG-ATG14 S29A Fig. . PreviouRecent structural analysis of the Vps34 complex reveals that the N-termini of ATG14 and Beclin 1 are in close proximity , 27. BecIn an earlier study, we identified an ULK1-mediated phosphorylation site on p62 . During We also examined ATG14 phosphorylation an HD cell model, which expresses the N-terminus of Htt following 103 polyQ repeats . Cells eTo understand autophagic activity in HD, we examined autophagosome formation in the Q175 mice, which expressed green fluorescence protein fused with LC3 (GFP-LC3) as reporter  after crProteotoxic stress induced by proteasomal inhibition also promotes ULK1-mediated phosphorylation of p62/SQSTM1, an autophagy receptor in selective autophagy . To undeGiven that ULK1 phosphorylates both p62 and ATG14, it is surprising that these signaling events appear to be differentially regulated. Moreover, we showed previously that p62-ULK1 binding is increased in the presence of mutant Htt or proteotoxic stress [ATG13 and FIP200 are components of ULK1 complex and important for ULK1 kinase regulation. We then tested whether MG132 can alter the binding of ATG13 and FIP200 with ULK1 and localization of ATG13 and FIP200, which may account for the decrease of ATG14 phosphorylation. We found that MG132 treatment does not alter the interaction between ULK1 and ATG13 or FIP200 as assayed by co-IP; however, MG132 treatment causes FIP200 and ATG13 redistribution into insoluble fraction  starvation, which in turn up-regulates Vps34 kinase activity. Importantly, we find that the specific ATG14 phosphorylation and ATG14-associated VSP34 activity, which is autophagy-specific, are impaired in HD cell and animal models. Interestingly, our current assays suggest no obvious impairment of autophagic activity in the brain of Q175 mice. Thus the 35% decrease of ATG14-linked Vps34 activity does not corroborate a significance reduction of basal autophagy in the HD animal models. Note that our autophagy assays are based on GFP-LC3 reporter and protein levels of several autophagy markers including p62 [GFP-LC3 mice is consistent with the idea that basal autophagosome formation is extremely rare in wildtype neurons as described by Mizushima’s group [Interestingly, a previous study in HdhQ200 knockin model, which manifests an accelerated and robust phenotypes than Q175 mice, has perinulear mHtt foci accompanied by colocalized LC3 and p62 aggregates. The HdhQ200 mice have increased levels of LC3II and p62 levels in the striatal neurons [ULK1-mediated phosphorylation of ATG14 may represent an important signaling event for autophagy induction that coordinates the action of the two key components of autophagy machinery, the ULK1 kinase complex and ATG14-Vps34 complex (autophagy specific). During the preparation of this manuscript, Park, et al. published their independent discovery of ATG14 phosphorylation by ULK1 . Their rOur detailed study of ULK1 kinase and ATG14-Vps34 lipid kinase show reduced activity of both kinases, suggesting altered regulation of the autophagy pathway in HD models. Both phosphorylated Beclin 1 and ATG14 levels are decreased in HD brains, indicating the lower ULK1 kinase activity, which is regulated by mTOR nutrient pathway. The reduced ULK1-mediated ATG14 or Beclin 1 phosphorylation was initially surprising, given that ULK1-mediated p62 phosphorylation (mTOR-independent) increases in the same models of HD . AlternaFinally, our current work extends our previous report that ULK1-mediated p62 phosphorylation in selective autophagy helps clear polyQ protein  by showiAs the major pathway for protein aggregate clearance, autophagy has been heavily implicated in the HD pathogenesis and considered a therapeutic target. However, the exact molecular mechanisms at work remain elusive. In this work, we identify a critical process during autophagosome biogenesis that is disregulated following mutant Htt-induced proteotoxic stress. We find that in the canonical autophagy pathway, ULK1 phosphorylates ATG14, which promotes Vps34 lipid kinase activity. ATG14 phosphorylation and Vps34 activity, however, are impaired in HD models. Our study highlights ULK1 and Vps34 as potential targets for the treatment of HD.


The level of selectivity of the novel inhibitor on several major transmembrane ion currents  was also determined.In this study the effects of a new, highly selective sodium-calcium exchanger (NCX) inhibitor, ORM-10962 were investigated on cardiac NCX current, Ca2+ transients and cell shortening were measured in fluorescent dye loaded isolated dog myocytes. Antiarrhythmic effects of ORM-10962 were studied in anesthetized ouabain (10 μg/kg/min i.v.) pretreated guinea pigs and in ischemia-reperfusion models (I/R) of anesthetized coronary artery occluded rats and Langendorff perfused guinea pigs hearts.Ion currents in single dog ventricular cells , and action potentials in dog cardiac multicellular preparations were recorded utilizing the whole-cell patch clamp and standard microelectrode techniques, respectively. CaCaL in CMs, neither had any apparent influence on the inward rectifier, transient outward, the rapid and slow components of the delayed rectifier potassium currents, the late and peak sodium and Na+/K+ pump currents. NCX inhibition exerted moderate positive inotropic effect under normal condition, negative inotropy when reverse, and further positive inotropic effect when forward mode was facilitated. In dog Purkinje fibres 1 μM ORM-10962 decreased the amplitude of digoxin induced delayed afterdepolarizations (DADs). Pre-treatment with 0.3 mg/kg ORM-10962 (i.v.) 10 min before starting ouabain infusion significantly delayed the development and recurrence of ventricular extrasystoles (by about 50%) or ventricular tachycardia (by about 30%) in anesthetized guinea pigs. On the contrary, ORM-10962 pre-treatment had no apparent influence on the time of onset or the severity of I/R induced arrhythmias in anesthetized rats and in Langendorff perfused guinea-pig hearts.ORM-10962 significantly reduced the inward/outward NCX currents with estimated EC50 values of 55/67 nM, respectively. The compound, even at a high concentration of 1 μM, did not modify significantly the magnitude of Iin vivo, however, its effect on I/R induced arrhythmias is still uncertain.The present study provides strong evidence for a high efficacy and selectivity of the NCX-inhibitory effect of ORM-10962. Selective NCX inhibition can exert positive as well as negative inotropic effect depending on the actual operation mode of NCX. Selective NCX blockade may contribute to the prevention of DAD based arrhythmogenesis,  The ARRIVE guidelines were adhered to during the study, .In vivo guinea pigs were used for ouabain induced arrhythmias and rats for coronary artery occlusion induced reperfusion induced arrhythmia studies. Guinea pigs were used for zero flow ischaemia study on Langendorff apparatus.Majority of measurements were performed in dog preparations. Guinea-pig papillary muscle preparations were used for slow response experiments. Mongrel dogs of either sex weighing 8 to 16 kg, and male Sprague-Dawley CFY rats weighing 290–330 g obtained from a licensed supplier were used for the study. The experimental animals were young adults.Adult mongrel dogs exclusively bred for experimental purposes  were used for the study. The dogs were identified by a chip implanted subcutaneously in the neck area. All the dogs were under one roof and a common airspace, however, each dog had its own kennel space bordered by metal bars, so that animals could interact and see/hear each other while being safely separated. Each kennel had an “outdoor” area (about 4 x 2 m) still under a common roof of the animal house on the top level of the Department) and an indoor area (about 4 x 2 m) with central heating and a resting place, separated by a trapdoor. The animals were provided various types of chew toys for enrichment. A certified animal handler tended to the dogs every day .2. Male Sprague-Dawley CFY rats weighing 290–330 g were maintained in standard rat cages. All the animal house was kept at standard temperature, humidity and lighting. The animals’ food intake and tap water consumption were provided ad libitum. The tap water is regularly checked for any pathogens.Male guinea-pigs (weighing 250–300 g) were kept in a separate room in groups of 6–10 in areas divided by large wooden boards on the floor covered with wooden chips for bedding. Each section had a surface area of 5 mCell preparations: Ventricular myocytes were enzymatically dissociated from dog hearts by adapting a method described earlier in detail o was reduced to 70 mM in this study, the reversal potential may shifted toward more negative range enhancing the contribution of the reverse mode to establish a new equilibrium. Thus, under this condition the NCX inhibition decreases the Ca2+ transient amplitude and cell shortening presumably via the suppression of the reverse mode.In contrast with the small positive inotropic effect of ORM-10962 found in this study , in our 2+ level without major rise of the intracellular Na+ may shift NCX reversal potential toward more positive values promoting forward mode activity. In this setting the observed positive inotropy may reflect further increase of SR Ca2+ content by reduction of the NCX mediated Ca2+ extrusion.In parallel experiments we intended to facilitate the NCX forward mode by applying forskolin to increase the adenylate-cyclase activity. We assume that the increase of the intracellular Ca2+ load is coupled with marked increase of intracellular Na+ and, this condition may facilitate the reverse mode activity of the NCX. This hypothesis was tested in dog Purkinje fibers where NCX inhibition almost completely abolished the digoxin induced DADs  in which calcium handling and NCX function may substantially differ from their state in healthy hearts, or following acute coronary ligation in hearts of otherwise healthy animals. Furthermore, species other than rat, having differing Ca2+-handling mechanisms, may respond differently to NCX inhibition in ischemia/reperfusion induced arrhythmias. Therefore, a number of further studies are needed to explore the potential therapeutic value of NCX inhibition in cardiac arrhythmogenesis.We suggested that the antiarrhythmic action of NCX inhibition is mainly achieved by inhibition of the reverse mode and partially by suppression of the forward mode. This interpretation is based on speculations relating to our Ca results . HoweverTo our knowledge ORM-10962 is the first reported compound that can inhibit both forward and reverse NCX currents at well below submicromolar concentrations without affecting further essential sarcolemmal transport mechanisms. Selective NCX inhibition also exerts potent antiarrhythmic effect in digitalis induced arrhythmias. The previously reported antiarrhythmic effect of moderately selective compounds in ischemia/reperfusion induced arrhythmogenesis can be seriously questioned. While we suggest that selective NCX inhibition may have therapeutic implications by counteracting triggered automaticity, further detailed studies are needed before its cardioprotective role can be properly established.


This research features parameterized depth edge detection using structured light imaging that exploits a single color stripes pattern and an associated binary stripes pattern. By parameterized depth edge detection, we refer to the detection of all depth edges in a given range of distances with depth difference greater or equal to a specific value. While previous research has not properly dealt with shadow regions, which result in double edges, we effectively remove shadow regions using statistical learning through effective identification of color stripes in the structured light images. We also provide a much simpler control of involved parameters. We have compared the depth edge filtering performance of our method with that of the state-of-the-art method and depth edge detection from the Kinect depth map. Experimental results clearly show that our method finds the desired depth edges most correctly while the other methods cannot. The goal of this work, as illustrated in There has been a considerable amount of research on structured light imaging in the literature ,2,3,4,5.A while ago, in ,8, similA major drawback of the previous methods is that they did not address the issue of shadow region. Regions that the projector light cannot reach create shadow regions and result in double edges. In this work, we present an accurate control of depth edge filtering by overcoming the disadvantages of the previous works ,8. We prWe have compared the depth edge filtering performance of our method with that of  and the By parameterized structured light imaging, we refer to the technologies using structured light imaging that can control associated parameters. To the best of our knowledge, Park et al.’s work  was the  idea in  to detecThe control of parameters developed in  can be s made in  so that To better describe our method, let us revisit the key Equation (1) for the modelled imaging geometry of a camera, projector and object in ,8. This Here, r and d fixed, the relation between Since, in ,8, they L,0,0], , , and , L = 255 or 128, where L denotes lightness intensity. To create a color pattern, we exploit De Brujin sequences , L = 255 or 128. That is, we also make use of bright (L = 255) and dark (L = 128) versions for binary stipes. We have designed the stripe patterns so that in both color stripes and binary stripes, bright and dark stripes appear alternately. The color stripes are associated with the binary stripes so that bright stripes in the color pattern correspond to dark stripes in the binary pattern. Refer to Additionally, we use an associated binary stripes of which RGB values can be represented as [The most attractive advantage of employing color stripes pattern of De Bruijin sequence is that  used in ,8. Thus,L in each color channel. We exploit the associated binary image as reference to avoid decision errors. L = 256) color stripe or dark (L = 128) color stripe. Then, we recover the value of L in each color channel. The problem of recovering the original color of color stripes in the structured light image is to determine the lightness C and B, respectively. Let us denote a pixel in the color structured light image and its corresponding pixel in the binary structure light images, L. Luminance and ambient reflected light can vary in different situations. We take an adaptive thresholding scheme to make a decision. In case of a pixel in bright color stripes, Next, we decide the value of each channel, In the case of a pixel in dark color stripes, We set a bias The relationship between the original color and captured color is nonlinear. We seek to use a simple statistical method to determine parameters, N is the number of sets, ith set. In bright stripes, we already know that L = 0, 128 and 255, respectively, We initially model Lastly, we check whether the recovered color is one of the four colors we adopt to use. If not, we change it to the most probable color in four. We achieve this in two steps: (1) compare recovered color with four default colors to see how many channels match; (2) among the colors having the most matching channels, choose the color with minimum threshold difference over mismatching channels. Although empirically determined parameters are used, the whole thing works pretty well in non-shadow regions. However, recovered color is meaningless in shadow regions where stripe patterns are totally lost. We detect shadow regions and extend color stripes there that otherwise would have been projected. Details follow in the next section.In structured light imaging, shadows are created in regions that the projector light cannot reach. In shadow regions, stripe patterns are totally lost, and parameter controlled depth edge detection is not possible there. In order to prevent double edges and missing edges in shadow regions, we proactively identify shadow regions and extend color stripes that otherwise have been projected.There has been research on natural shadow removal ,13. AlthL channel beforehand saves a great deal of time on training and clustering data.We convert recovered color into Lab space and build color histogram. As provided by Guo et al. , we set Textons, a concept stated in , can helShadow is colorless. We look at each pixel in a color pattern image and its corresponding pixel in a binary pattern image in the RGB space. Let us denote them by We use color, texture, and angle features together as in We use Gabor filtering for depth edge detection as in ,8. They We have coded our method in Matlab  and the codes have not been optimized. We have used 2.9 GHz Intel Core i5 CPU, 8GB 1867 MHz DDR3 memory  and Intel Graphics . We have presented a novel method that accurately controls depth edge filtering of the input scene using a color stripes pattern. For accuracy, we employed an associated binary pattern. Our method can be used for active sensing of specific 3D information about the scene. We think that if a task is to find accurate depth edges, our method provides a better solution. Further research is in progress to use the proposed method to create a sketch of 3D reconstruction by compiling depth edges with various depth differences.