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The decentralised railway signalling systems have a potential to increase capacity, availability and reduce maintenance costs of railway networks. However, given the safety-critical nature of railway signalling and the complexity of novel distributed signalling solutions, their safety should be guaranteed by using thorough system validation methods. To achieve such a high-level of safety assurance of these complex signalling systems, scenario-based testing methods are far from being sufficient despite that they are still widely used in the industry. Formal verification is an alternative approach which provides a rigorous approach to verifying complex systems and has been successfully used in the railway domain. Despite the successes, little work has been done in applying formal methods for distributed railway systems. In our research we are working towards a multifaceted formal development methodology of complex railway signalling systems. The methodology is based on the Event-B modelling language which provides an expressive modelling language, a stepwise development and a proof-based model verification. In this paper, we present the application of the methodology for the development and verification of a distributed protocol for reservation of railway sections. The main challenge of this work is developing a distributed protocol which ensures safety and liveness of the distributed railway system when message delays are allowed in the model.

Telomere binding protein Stn1 forms the CST (Cdc13/CTC1-STN1-TEN1) complex in budding yeast and mammals. Likewise, fission yeast Stn1 and Ten1 form a complex indispensable for telomere protection. We have previously reported that stn1-1, a high-temperature sensitive mutant, rapidly loses telomere DNA at the restrictive temperature due to frequent failure of replication fork progression at telomeres and subtelomeres, both containing repetitive sequences. It is unclear, however, whether Stn1 is required for maintaining other repetitive DNAs such as ribosomal DNA. In this study, we have demonstrated that stn1-1 cells, even when grown at the permissive temperature, exhibited dynamic rearrangements in the telomere-proximal regions of subtelomere and ribosomal DNA repeats. Furthermore, Rad52 and γH2A accumulation was observed at ribosomal DNA repeats in the stn1-1 mutant. The phenotypes exhibited by the stn1-1 allele were largely suppressed in the absence of Reb1, a replication fork barrier-forming protein, suggesting that Stn1 is involved in the maintenance of the arrested replication forks. Collectively, we propose that Stn1 maintains the stability of repetitive DNAs at subtelomeres and rDNA regions.

We introduce a goal-aware extension of responsibility-sensitive safety (RSS), a recent methodology for rule-based safety guarantee for automated driving systems (ADS). Making RSS rules guarantee goal achievement -- in addition to collision avoidance as in the original RSS -- requires complex planning over long sequences of manoeuvres. To deal with the complexity, we introduce a compositional reasoning framework based on program logic, in which one can systematically develop RSS rules for smaller subscenarios and combine them to obtain RSS rules for bigger scenarios. As the basis of the framework, we introduce a program logic dFHL that accommodates continuous dynamics and safety conditions. Our framework presents a dFHL-based workflow for deriving goal-aware RSS rules; we discuss its software support, too. We conducted experimental evaluation using RSS rules in a safety architecture. Its results show that goal-aware RSS is indeed effective in realising both collision avoidance and goal achievement., 33 pages, 18 figures, 1 table. Accepted for publication in IEEE Transactions on Intelligent Vehicles

Significant effort is being put into developing industrial applications for artificial intelligence (AI), especially those using machine learning (ML) techniques. Despite the intensive support for building ML applications, there are still challenges when it comes to evaluating, assuring, and improving the quality or dependability. The difficulty stems from the unique nature of ML, namely, system behavior is derived from training data not from logical design by human engineers. This leads to black-box and intrinsically imperfect implementations that invalidate many principles and techniques in traditional software engineering. In light of this situation, the Japanese industry has jointly worked on a set of guidelines for the quality assurance of AI systems (in the Consortium of Quality Assurance for AI-based Products and Services) from the viewpoint of traditional quality-assurance engineers and test engineers. We report on the second version of these guidelines, which cover a list of quality evaluation aspects, catalogue of current state-of-the-art techniques, and domain-specific discussions in five representative domains. The guidelines provide significant insights for engineers in terms of methodologies and designs for tests driven by application-specific requirements.

Systematic techniques to improve quality of deep neural networks (DNNs) are critical given the increasing demand for practical applications including safety-critical ones. The key challenge comes from the little controllability in updating DNNs. Retraining to fix some behavior often has a destructive impact on other behavior, causing regressions, i.e., the updated DNN fails with inputs correctly handled by the original one. This problem is crucial when engineers are required to investigate failures in intensive assurance activities for safety or trust. Search-based repair techniques for DNNs have potentials to tackle this challenge by enabling localized updates only on "responsible parameters" inside the DNN. However, the potentials have not been explored to realize sufficient controllability to suppress regressions in DNN repair tasks. In this paper, we propose a novel DNN repair method that makes use of the training history for judging which DNN parameters should be changed or not to suppress regressions. We implemented the method into a tool called NeuRecover and evaluated it with three datasets. Our method outperformed the existing method by achieving often less than a quarter, even a tenth in some cases, number of regressions. Our method is especially effective when the repair requirements are tight to fix specific failure types. In such cases, our method showed stably low rates (

Additional training of a deep learning model can cause negative effects on the results, turning an initially positive sample into a negative one (degradation). Such degradation is possible in real-world use cases due to the diversity of sample characteristics. That is, a set of samples is a mixture of critical ones which should not be missed and less important ones. Therefore, we cannot understand the performance by accuracy alone. While existing research aims to prevent a model degradation, insights into the related methods are needed to grasp their benefits and limitations. In this talk, we will present implications derived from a comparison of methods for reducing degradation. Especially, we formulated use cases for industrial settings in terms of arrangements of a data set. The results imply that a practitioner should care about better method continuously considering dataset availability and life cycle of an AI system because of a trade-off between accuracy and preventing degradation.

Although automated machine learning (AutoML) is receiving attention in the field of data science, most AutoML open source software focuses on supervised learning tasks, and little attention has been given to unsupervised learning tasks. Moreover, AutoML has a disadvantage in that it tends to deprive users of a chance to acquire knowledge about machine learning and data science because it usually works as a black box. The purpose of this study is to help inexperienced data scientists and machine learning engineers conduct clustering data analysis, which is an unsupervised learning task, while simultaneously enabling them to acquire knowledge about clustering data analysis by extending our existing AutoML software, the machine learning support system (MALSS). The MALSS helps with clustering data analysis by automatically determining the optimal number of clusters, which is one of the main purposes of clustering data analysis. Furthermore, the MALSS helps users to acquire knowledge about clustering data analysis by generating a report after automated clustering data analysis is conducted. We validated the effectiveness of our approach by using open datasets and by running an experiment on a crowdsourcing platform.

Autonomous Driving Systems (ADSs) are complex critical systems that must be thoroughly tested. Still, assessing the strength of tests for ADSs is an open and complex problem. Weight Coverage is a test criterion targeting ADSs which are based on a weighted cost function. It measures how much each weight, related to different aspects of the ADS's decision process, is involved in the decisions taken in a test scenario. All weights/aspects should be involved for a strong test suite. Although weight coverage can measure the quality of a test suite, it does not provide clear guidance for test generation. This work proposes weight coverage-driven search-based test generation for ADSs. It describes and compares three designs of the search process: a single-objective search aiming at generating a test covering a single weight; a multi-objective search where each objective targets a different weight; and a single-objective search where the fitness function is an aggregate function representing the coverage over multiple weights. Experiments using an ADS system provided by our industry partner show the validity of the method and provide insights into the benefits of each search design. This Hot-off-the-Press paper summarises the paper [2]: T. Laurent, P. Arcaini, F. Ishikawa and A. Ventresque, "Achieving Weight Coverage for an Autonomous Driving System with Search-based Test Generation", 25th International Conference on Engineering of Complex Computer Systems (ICECCS 2020).

A common approach in testing automated and autonomous driving systems (ADS) consists in running the ADS in a simulator where driving and environmental conditions are specified in terms of scenarios. An important aspect in ADS testing is to cover different driving situations in which the autonomous car must perform different types of maneuvers. In this paper, we consider the path planner of our industry partner; the path planner is responsible for deciding the path that must be followed by the autonomous car. A path is characterized by the driving characteristics (as forward acceleration, lateral acceleration, curvature, and so on) that are needed, at each time point, to implement it. For different driving characteristics, a good test suite should contain a scenario for which the path planner chooses a path that requires the application of the selected driving characteristics for a non-negligible period of time: this means that the characteristics are relevant in that path. With such a test suite, engineers can observe the different types of decision taken by the path planner, and so possibly better assess its correctness. In the paper, we introduce the notion of patterns of driving characteristics, to characterize their interaction (i.e., simultaneous or not) and measure their duration. Exploiting this definition, we propose two search-based approaches (for single and pairs of driving characteristics) to find scenarios in which such patterns occur and their duration is maximized. Experimental results show that the approaches are effective in finding scenarios for which the path planner generates paths where the different driving characteristics occur in terms of the specified pattern.

We introduce a new logic named Quantitative Confidence Logic (QCL) that quantifies the level of confidence one has in the conclusion of a proof. By translating a fault tree representing a system's architecture to a proof, we show how to use QCL to give a solution to the test resource allocation problem that takes the given architecture into account. We implemented a tool called Astrahl and compared our results to other testing resource allocation strategies.

Formal reasoning on the safety of controller systems interacting with plants is complex because developers need to specify behavior while taking into account perceptual uncertainty. To address this, we propose an automated workflow that takes an Event-B model of an uncertainty-unaware controller and a specification of uncertainty as input. First, our workflow automatically injects the uncertainty into the original model to obtain an uncertainty-aware but potentially unsafe controller. Then, it automatically robustifies the controller so that it satisfies safety even under the uncertainty. The case study shows how our workflow helps developers to explore multiple levels of perceptual uncertainty. We conclude that our workflow makes design and analysis of uncertainty-aware controller systems easier and more systematic.

To date, although machine learning has been successful in various practical applications, generic methods of testing machine learning code have not been established yet. Here we present a new approach to test machine learning code using the possible input region obtained as a polyhedron. If an ML system generates different output for multiple input in the polyhedron, it is ensured that there exists a bug in the code. This property is known as one of theoretical fundamentals in statistical inference, for example, sparse regression models such as the lasso, and a wide range of machine learning algorithms satisfy this polyhedral condition, to which our testing procedure can be applied. We empirically show that the existence of bugs in lasso code can be effectively detected by our method in the mutation testing framework.

The decentralisation of railway signalling systems has the potential to increase railway network capacity, availability and reduce maintenance costs. Given the safety-critical nature of railway signalling and the complexity of novel distributed signalling solutions, their safety should be guaranteed by using thorough system validation methods. In this paper, we present a rigorous formal development and verification of a distributed protocol for reservation of railway sections, which we believe could deliver benefits of a decentralised signalling while ensuring safety and liveness properties. For the formal distributed protocol development and verification, we devised a multifaceted framework, which aims to reduce modelling and verification effort, while still providing complementary techniques to study protocol from all relevant perspectives.

Autonomous cars are subjected to several different kind of inputs (other cars, road structure, etc.) and, therefore, testing the car under all possible conditions is impossible. To tackle this problem, scenario-based testing for automated driving defines categories of different scenarios that should be covered. Although this kind of coverage is a necessary condition, it still does not guarantee that any possible behaviour of the autonomous car is tested. In this paper, we consider the path planner of an autonomous car that decides, at each timestep, the short-term path to follow in the next few seconds; such decision is done by using a weighted cost function that considers different aspects (safety, comfort, etc.). In order to assess whether all the possible decisions that can be taken by the path planner are covered by a given test suite T, we propose a mutation-based approach that mutates the weights of the cost function and then checks if at least one scenario of T kills the mutant. Preliminary experiments on a manually designed test suite show that some weights are easier to cover as they consider aspects that more likely occur in a scenario, and that more complicated scenarios (that generate more complex paths) are those that allow to cover more weights., Comment: Preprint version of paper accepted at the 26th Asia-Pacific Software Engineering Conference (APSEC 2019)

Recent years have witnessed substantial progress in understanding tumor heterogeneity and the process of tumor progression; however, the entire process of the transition of tumors from a benign to metastatic state remains poorly understood. In the present study, we performed a prospective cancer genome-sequencing analysis by employing an experimental carcinogenesis mouse model of squamous cell carcinoma to systematically understand the evolutionary process of tumors. We surgically collected a part of a lesion of each tumor and followed the progression of these tumors in vivo over time. Comparative time-series analysis of the genomes of tumors with different fates, i.e., those that eventually metastasized and regressed, suggested that these tumors acquired and inherited different mutations. These findings suggest that despite the occurrence of an intra-tumor selection event for malignant alteration during the transformation from early- to late-stage papilloma, the fate determination of tumors might be determined at an even earlier stage.

Nowadays it is becoming more and more attractive to execute workflow applications in the cloud because it enables workflow applications to use computing resources on demand. Meanwhile, it also challenges traditional workflow scheduling algorithms that only concentrate on optimizing the execution time. This paper investigates how to minimize execution cost of a workflow in clouds under a deadline constraint and proposes a metaheuristic algorithm L-ACO as well as a simple heuristic ProLiS. ProLiS distributes the deadline to each task, proportionally to a novel definition of probabilistic upward rank, and follows a two-step list scheduling methodology: rank tasks and sequentially allocates each task a service which meets the sub-deadline and minimizes the cost. L-ACO employs ant colony optimization to carry out deadline-constrained cost optimization: the ant constructs an ordered task list according to the pheromone trail and probabilistic upward rank, and uses the same deadline distribution and service selection methods as ProLiS to build solutions. Moreover, the deadline is relaxed to guide the search of L-ACO towards constrained optimization. Experimental results show that compared with traditional algorithms, the performance of ProLiS is very competitive and L-ACO performs the best in terms of execution costs and success ratios of meeting deadlines.

Summary The molecular mechanism of memory formation remains a mystery. Here, we show that TERT, the catalytic subunit of telomerase, gene knockout (Tert−/−) causes extremely poor ability in spatial memory formation. Knockdown of TERT in the dentate gyrus of adult hippocampus impairs spatial memory processes, while overexpression facilitates it. We find that TERT plays a critical role in neural development including dendritic development and neuritogenesis of hippocampal newborn neurons. A monosynaptic pseudotyped rabies virus retrograde tracing method shows that TERT is required for neural circuit integration of hippocampal newborn neurons. Interestingly, TERT regulated neural development and spatial memory formation in a reverse transcription activity-independent manner. Using X-ray irradiation, we find that hippocampal newborn neurons mediate the modulation of spatial memory processes by TERT. These observations reveal an important function of TERT through a non-canonical pathway and encourage the development of a TERT-based strategy to treat neurological disease-associated memory impairment., Highlights • Tert gene knockout causes extremely poor ability in spatial memory formation • Dendritic development and neuritogenesis are impaired in Tert−/− mice • TERT is required for neural circuit integration of hippocampal newborn neurons • TERT regulates spatial memory formation in an activity-independent manner, In this article, Qi-Gang Zhou and colleagues show that spatial memory formation, neural development including dendritic development and neuritogenesis, and neural circuit integration are impaired in Tert gene knockout mice. Hippocampal TERT accounts for these phenotypes in a reverse transcription activity-independent manner.

The CST complex is a phylogenetically conserved protein complex consisting of CTC1/Cdc13, Stn1 and Ten1 that protects telomeres on linear chromosomes. Deletion of the fission yeast homologs stn1 and ten1 results in complete telomere loss; however, the precise function of Stn1 is still largely unknown. Here, we have isolated a high-temperature sensitive stn1 allele (termed stn1-1). stn1-1 cells abruptly lost telomeric sequence almost completely at the restrictive temperature. The loss of chromosomal DNA happened without gradual telomere shortening, and extended to 30 kb from the ends of chromosomes. We found transient and modest single-stranded G-strand exposure, but did not find any evidence of checkpoint activation in stn1-1 at the restrictive temperature. When we probed neutral-neutral 2D gels for subtelomere regions, we found no Y-arc-shaped replication intermediates in cycling cells. We conclude that the loss of telomere and subtelomere DNAs in stn1-1 cells at the restrictive temperature is caused by very frequent replication fork collapses specifically in subtelomere regions. Furthermore, we identified two independent suppressor mutants of the high-temperature sensitivity of stn1-1: a multi-copy form of pmt3 and a deletion of rif1. Collectively, we propose that fission yeast Stn1 primarily safeguards the semi-conservative DNA replication at telomeres and subtelomeres.

Quality of service (QoS)-aware optimal service composition aims to maximize the overall QoS value of the resulting composite service instance while meeting user-specified global QoS constraints. Traditional methods only consider as candidates service instances that implement one abstract service in the composite service and neglect those that could perform multiple abstract services. To overcome this shortcoming, this paper proposes the concept of generalized component services (GCSs), which is defined in a semantic manner, to expand the selection scope so as to achieve a better solution. A QoS-aware multigranularity service composition model is formulated and how to identify all the GCSs for a composite service is elaborated. A backtracking-based algorithm and an extended genetic algorithm are proposed to optimize the resulting composite service instance. Lastly, evaluation results of these algorithms are described.

Genomic rearrangements (gross chromosomal rearrangements, GCRs) threatens genome integrity and cause cell death or tumor formation. At the terminus of linear chromosomes, a telomere-binding protein complex, called shelterin, ensures chromosome stability by preventing chromosome end-to-end fusions and regulating telomere length homeostasis. As such, shelterin-mediated telomere functions play a pivotal role in suppressing GCR formation. However, it remains unclear whether the shelterin proteins play any direct role in inhibiting GCR at non-telomeric regions. Here, we have established a GCR assay for the first time in fission yeast and measured GCR rates in various mutants. We found that fission yeast cells lacking shelterin components Taz1 or Rap1 (mammalian TRF1/2 or RAP1 homologues, respectively) showed higher GCR rates compared to wild-type, accumulating large chromosome deletions. Genetic dissection of Rap1 revealed that Rap1 contributes to inhibiting GCRs via two independent pathways. The N-terminal BRCT-domain promotes faithful DSB repair, as determined by I-SceI-mediated DSB-induction experiments; moreover, association with Poz1 mediated by the central Poz1-binding domain regulates telomerase accessibility to DSBs, leading to suppression of de novo telomere additions. Our data highlight unappreciated functions of the shelterin components Taz1 and Rap1 in maintaining genome stability, specifically by preventing non-telomeric GCRs., Author summary Tips of chromosomes, telomeres, are bound and protected by a telomere-binding protein complex called shelterin. Most previous studies focused on shelterin’s telomere-specific role, and its general role in genome maintenance has not been explored extensively. In this study, we first set up an assay measuring the spontaneous formation rate per cell division of gross chromosomal rearrangements (GCRs) in fission yeast. We found that the rate of GCRs is elevated in mutants defective for shelterin components Taz1 or Rap1. Detailed genetic experiments revealed unexpectedly that Taz1 and Rap1 have a novel role in repairing DNA double-strand breaks (DSBs) and suppressing GCRs at non-telomeric regions. Given that shelterin components are conserved between fission yeast and humans, future studies are warranted to test whether shelterin dysfunction leads to genome-wide GCRs, which are frequently observed in cancers.

More and more software practitioners are tackling towards industrial applications of artificial intelligence (AI) systems, especially those based on machine learning (ML). However, many of existing principles and approaches to traditional systems do not work effectively for the system behavior obtained by training not by logical design. In addition, unique kinds of requirements are emerging such as fairness and explainability. To provide clear guidance to understand and tackle these difficulties, we present an analysis on what quality concepts we should evaluate for AI systems. We base our discussion on ISO/IEC 25000 series, known as SQuaRE, and identify how it should be adapted for the unique nature of ML and $\textit{Ethics guidelines for trustworthy AI}$ from European Commission. We thus provide holistic insights for quality of AI systems by incorporating the ML nature and AI ethics to the traditional software quality concepts.

Cellular senescence, a stress‐induced irreversible cell cycle arrest, has been defined for mitotic cells and is implicated in aging of replicative tissues. Age‐related functional decline in the brain is often attributed to a failure of protein homeostasis (proteostasis), largely in postmitotic neurons, which accordingly is a process distinct by definition from senescence. It is nevertheless possible that proteostasis failure and cellular senescence have overlapping molecular mechanisms. Here, we identify postmitotic cellular senescence as an adaptive stress response to proteostasis failure. Primary rat hippocampal neurons in long‐term cultures show molecular changes indicative of both senescence (senescence‐associated β‐galactosidase, p16, and loss of lamin B1) and proteostasis failure relevant to Alzheimer's disease. In addition, we demonstrate that the senescent neurons exhibit resistance to stress. Importantly, treatment of the cultures with an mTOR antagonist, protein synthesis inhibitor, or chemical compound that reduces the amount of protein aggregates relieved the proteotoxic stresses as well as the appearance of senescence markers. Our data propose mechanistic insights into the pathophysiological brain aging by establishing senescence as a primary cell‐autonomous neuroprotective response., Loss of protein homeostasis (proteostasis) is a hallmark of brain aging, yet the adaptive mechanism that contributes to life‐long neuronal preservation is poorly understood. Long‐term cultures of primary post‐mitotic neurons increase a proteotoxic burden and establish cellular senescence, which is alleviated by prolonged treatment of neurons with rapamycin. Post‐mitotic cell senescence is accompanied by stress resilience, suggesting an intrinsic neuroprotective role of senescence.

Chromosome fusion is deleterious among oncogenic chromosome rearrangements, and has been proposed to cause multiple tumor-driving abnormalities. Conventional methodologies, however, lack the strictness of the experimental controls on the fusion such as the exact timing, the number and the types of fusion in a given cell. Here, we developed a human cell-based sister chromatid fusion visualization system (FuVis), in which a single defined sister chromatid fusion is induced by CRISPR/Cas9 concomitantly with mCitrine expression. Fused chromosome developed numerical and structural abnormalities, including chromosome fragmentation, an indicative of eventual chromothripsis. Live cell imaging and hierarchical Bayesian modeling indicated that micronucleus (MN) is generated in the first few cell cycle, and that cells with MN tend to possess cell cycle abnormalities. These results demonstrate that, although most cells can tolerate a single fusion, even a single sister chromatid fusion destabilizes cell cycle through MN formation.

Safety analyses in the automotive domain (in particular automated driving) present unprecedented challenges due to its complexity and tight integration with the physical environment. Given the diversity in the types of cars, potentially unlimited number of possible environmental and driving conditions, it is crucial to devise a systematic way of managing variability in hazards, driving and environmental conditions in individual cars, families of cars, and families of families of cars to facilitate analyses efficiently. To this end, we present our ongoing work in a research project that focuses on devising a model-based reasoning framework for systematically managing hazards in the automotive domain and supporting safety analyses (e.g., falsification).

International audience; From smart buildings to medical devices to smart nations, software systems increasingly integrate computation, networking, and interaction with the physical environment. These systems are known as Cyber-Physical Systems (CPS). While these systems open new opportunities to deliver improved quality of life for people and reinvigorate computing, their engineering is a difficult problem given the level of heterogeneity and dynamism they exhibit. While progress has been made, we argue that complexity is now at a level such that existing approaches need a major rethink to define principles and associated techniques for CPS. In this paper, we identify research challenges when modelling, analysing and engineering CPS. We focus on three key topics: theoretical foundations of CPS, self-adaptation methods for CPS, and exemplars of CPS serving as a research vehicle shared by a larger community. For each topic, we present an overview and suggest future research directions, thereby focusing on selected challenges. This paper is one of the results of the Shonan Seminar 118 on Modelling and Analysing Resilient Cyber-Physical Systems, which took place in December 2018.

Newly developed Fusion Visualization (FuVis) system reveals a single Xp sister chromatid fusion induces micronuclei formation, acentric chromosome fragments, and cell cycle instabilities., Chromosome fusion is a frequent intermediate in oncogenic chromosome rearrangements and has been proposed to cause multiple tumor-driving abnormalities. In conventional experimental systems, however, these abnormalities were often induced by randomly induced chromosome fusions involving multiple different chromosomes. It was therefore not well understood whether a single defined type of chromosome fusion, which is reminiscent of a sporadic fusion in tumor cells, has the potential to cause chromosome instabilities. Here, we developed a human cell-based sister chromatid fusion visualization system (FuVis), in which a single defined sister chromatid fusion is induced by CRISPR/Cas9 concomitantly with mCitrine expression. The fused chromosome subsequently developed extra-acentric chromosomes, including chromosome scattering, indicative of chromothripsis. Live-cell imaging and statistical modeling indicated that sister chromatid fusion generated micronuclei (MN) in the first few cell cycles and that cells with MN tend to display cell cycle abnormalities. The powerful FuVis system thus demonstrates that even a single sporadic sister chromatid fusion can induce chromosome instability and destabilize the cell cycle through MN formation.

Shelterin component TPP1 plays critical roles in chromosome end protection and telomere length regulation. Specifically, TPP1 contains an OB-fold domain that provides an interface to recruit telomerase. However, it remains largely unknown how telomerase recruitment is regulated by cell cycle regulators. We show that TPP1 interacts with the cell cycle regulator kinase NEK6 in human cells. We found that NEK6-mediated phosphorylation of TPP1 Ser255 in G2/M phase regulates the association between telomerase activity and TPP1. Furthermore, we found evidence that POT1 negatively regulates TPP1 phosphorylation because the level of Ser255 phosphorylation was elevated when telomeres were elongated by a POT1 mutant lacking its OB-fold domains. Ser255 is located in the intervening region between the telomerase-recruiting OB-fold and the POT1 recruitment domains. Ser255 and the surrounding amino acids are conserved among vertebrates. These observations suggest that a region adjacent to the OB-fold domain of TPP1 is involved in telomere length regulation via telomerase recruitment.

Telomeres maintain the integrity of chromosome ends and telomere length is an important marker of aging. The epidemiological studies suggested that many types of stress including psychosocial stress decrease telomere length. However, it remains unknown how various stresses induce telomere shortening. Here, we report that the stress-responsive transcription factor ATF7 mediates TNF-α–induced telomere shortening. ATF7 and telomerase, an enzyme that elongates telomeres, are localized on telomeres via interactions with the Ku complex. In response to TNF-α, which is induced by various stresses including psychological stress, ATF7 was phosphorylated by p38, leading to the release of ATF7 and telomerase from telomeres. Thus, a decrease of ATF7 and telomerase on telomeres in response to stress causes telomere shortening, as observed in ATF7-deficient mice. These findings give credence to the idea that various types of stress might shorten telomere.

Mobile applications (apps) are software developed for use on mobile devices and made available through app stores. App stores are highly competitive markets where developers need to cater to a large number of users spanning multiple countries. This work hypothesizes that there exist country differences in mobile app user behavior and conducts one of the largest surveys to date of app users across the world, in order to identify the precise nature of those differences. The survey investigated user adoption of the app store concept, app needs, and rationale for selecting or abandoning an app. We collected data from more than 15 countries, including USA, China, Japan, Germany, France, Brazil, United Kingdom, Italy, Russia, India, Canada, Spain, Australia, Mexico, and South Korea. Analysis of data provided by 4,824 participants showed significant differences in app user behaviors across countries, for example users from USA are more likely to download medical apps, users from the United Kingdom and Canada are more likely to be influenced by price, users from Japan and Australia are less likely to rate apps. Analysis of the results revealed new challenges to market-driven software engineering related to packaging requirements, feature space, quality expectations, app store dependency, price sensitivity, and ecosystem effect.

QoS-aware service composition aims to satisfy users’ quality of services (QoS) needs during service composition. Traditional methods simply attempt to maximize user satisfaction by provisioning the composite service instance with the best QoS. These “best-effort” methods fail to take into account that there also exist other consumers competing for the service resources and their decisions of service selection/composition can impact on QoS. Since user's QoS needs can be met once the demanded level is reached, in this paper, we propose an “on-demand” strategy for QoS-aware service composition to replace the traditional “best-effort” strategy. The service broker is introduced to facilitate implementation of this strategy: it first purchases a number of service instances for each component from providers and then provisions the composite services with different QoS classes to consumers. This paper focuses on how the broker follows the service level agreement (SLA) to provision composite services in the “on-demand” manner. This problem is formally expressed as the minimization of the QoS distance function between SLA and QoS of composite service instances, under a series of constraints. Heuristic approaches are proposed for the problem and experiments are conducted at last to verify their effectiveness and efficiency.

The proper location and timing of Dnmt1 activation are essential for DNA methylation maintenance. We demonstrate here that Dnmt1 utilizes two-mono-ubiquitylated histone H3 as a unique ubiquitin mark for its recruitment to and activation at DNA methylation sites. The crystal structure of the replication foci targeting sequence (RFTS) of Dnmt1 in complex with H3-K18Ub/23Ub reveals striking differences to the known ubiquitin-recognition structures. The two ubiquitins are simultaneously bound to the RFTS with a combination of canonical hydrophobic and atypical hydrophilic interactions. The C-lobe of RFTS, together with the K23Ub surface, also recognizes the N-terminal tail of H3. The binding of H3-K18Ub/23Ub results in spatial rearrangement of two lobes in the RFTS, suggesting the opening of its active site. Actually, incubation of Dnmt1 with H3-K18Ub/23Ub increases its catalytic activity in vitro. Our results therefore shed light on the essential role of a unique ubiquitin-binding module in DNA methylation maintenance.

Faithful propagation of DNA methylation patterns during DNA replication is critical for maintaining cellular phenotypes of individual differentiated cells. Although it is well established that Uhrf1 (ubiquitin-like with PHD and ring finger domains 1; also known as Np95 and ICBP90) specifically binds to hemi-methylated DNA through its SRA (SET and RING finger associated) domain and has an essential role in maintenance of DNA methylation by recruiting Dnmt1 to hemi-methylated DNA sites, the mechanism by which Uhrf1 coordinates the maintenance of DNA methylation and DNA replication is largely unknown. Here we show that Uhrf1-dependent histone H3 ubiquitylation has a prerequisite role in the maintenance DNA methylation. Using Xenopus egg extracts, we successfully reproduce maintenance DNA methylation in vitro. Dnmt1 depletion results in a marked accumulation of Uhrf1-dependent ubiquitylation of histone H3 at lysine 23. Dnmt1 preferentially associates with ubiquitylated H3 in vitro though a region previously identified as a replication foci targeting sequence. The RING finger mutant of Uhrf1 fails to recruit Dnmt1 to DNA replication sites and maintain DNA methylation in mammalian cultured cells. Our findings represent the first evidence, to our knowledge, of the mechanistic link between DNA methylation and DNA replication through histone H3 ubiquitylation.

Genetic instability is the driving force of the malignant progression of cancer cells. Recently, replication stress has attracted much attention as a source of genetic instability that gives rise to an accumulation of mutations during the lifespan of individuals. However, the molecular details of the process are not fully understood. Here, recent progress in understanding how genetic alterations accumulate at telomeres will be reviewed. In particular, two aspects of telomere replication will be discussed in this context, covering conventional semi-conservative replication, and DNA synthesis by telomerase plus the C-strand fill-in reactions. Although these processes are seemingly telomere-specific, I will emphasize the possibility that the molecular understanding of the telomere events may shed light on genetic instability at other genetic loci in general.

A small number of transcription factors, including Oct-3/4 and Sox2, constitute the transcriptional network that maintains pluripotency in embryonic stem (ES) cells. Previous reports suggested that some of these factors form a complex that binds the Oct-Sox element, a composite sequence consisting of closely juxtaposed Oct-3/4 binding and Sox2 binding sites. However, little is known regarding the components of the complex. In this study we show that Sall4, a member of the Spalt-like family of proteins, directly interacts with Sox2 and Oct-3/4. Sall4 in combination with Sox2 or Oct-3/4 simultaneously occupies the Oct-Sox elements in mouse ES cells. Overexpression of Sall4 in ES cells increased reporter activities in a luciferase assay when the Pou5f1- or Nanog-derived Oct-Sox element was included in the reporter. Microarray analyses revealed that Sall4 and Sox2 bound to the same genes in ES cells significantly more frequently than expected from random coincidence. These factors appeared to bind the promoter regions of a subset of the Sall4 and Sox2 double-positive genes in precisely similar distribution patterns along the promoter regions, suggesting that Sall4 and Sox2 associate with such Sall4/Sox2-overlapping genes as a complex. Importantly, gene ontology analyses indicated that the Sall4/Sox2-overlapping gene set is enriched for genes involved in maintaining pluripotency. Sall4/Sox2/Oct-3/4 triple-positive genes identified by referring to a previous study identifying Oct-3/4-bound genes in ES cells were further enriched for pluripotency genes than Sall4/Sox2 double-positive genes. These results demonstrate that Sall4 contributes to the transcriptional network operating in pluripotent cells together with Oct-3/4 and Sox2. Background: Oct-3/4 and Sox2 form a complex to regulate gene expression and maintain pluripotency. Results: Sall4 directly interacts with Oct-3/4 or Sox2. Sall4 and Sox2 occupy the same promoter regions of genes active in ES cells. Conclusion: Sall4 is involved in transcriptional networks retaining pluripotency, in combination with Oct-3/4 or Sox2. Significance: This study provides a molecular understanding of how ES cells maintain pluripotency.

Summary Efficient chromosomal movements are important for the fidelity of chromosome segregation during mitosis; however, movements are constrained during interphase by tethering of multiple domains to the nuclear envelope (NE) [1]. Higher eukaryotes undergo open mitosis accompanied by NE breakdown, enabling chromosomes to be released from the NE, whereas lower eukaryotes undergo closed mitosis, in which NE breakdown does not occur [2]. Although the chromosomal movements in closed mitosis are thought to be restricted compared to open mitosis, the cells overcome this problem by an unknown mechanism that enables accurate chromosome segregation. Here, we report the spatiotemporal regulation of telomeres in Schizosaccharomyces pombe closed mitosis. We found that the telomeres, tethered to the NE during interphase [3], are transiently dissociated from the NE during mitosis. This dissociation from the NE is essential for accurate chromosome segregation because forced telomere tethering to the NE causes frequent chromosome loss. The phosphorylation of the telomere protein Rap1 during mitosis, primarily by Cdc2, impedes the interaction between Rap1 and Bqt4, a nuclear membrane protein, thereby inducing telomere dissociation from the NE. We propose that the telomere dissociation from the NE promoted by Rap1 phosphorylation is critical for the fidelity of chromosome segregation in closed mitosis.

Cells that have been pre-exposed to mild stress (priming stress) acquire transient resistance to subsequent severe stress even under different combinations of stresses. This phenomenon is called cross-tolerance. Although it has been reported that cross-tolerance occurs in many organisms, the molecular basis is not clear yet. Here, we identified slm9+ as a responsible gene for the cross-tolerance in the fission yeast Schizosaccharomyces pombe. Slm9 is a homolog of mammalian HIRA histone chaperone. HIRA forms a conserved complex and gene disruption of other HIRA complex components, Hip1, Hip3, and Hip4, also yielded a cross-tolerance-defective phenotype, indicating that the fission yeast HIRA is involved in the cross-tolerance as a complex. We also revealed that Slm9 was recruited to the stress-responsive gene loci upon stress treatment in an Atf1-dependent manner. The expression of stress-responsive genes under stress conditions was compromised in HIRA disruptants. Consistent with this, Pol II recruitment and nucleosome eviction at these gene loci were impaired in slm9Δ cells. Furthermore, we found that the priming stress enhanced the expression of stress-responsive genes in wild-type cells that were exposed to the severe stress. These observations suggest that HIRA functions in stress response through transcriptional regulation. Background: HIRA is a conserved histone chaperone required for regulation of chromatin structure. Results: Genes that encode HIRA proteins are responsible for cross-tolerance. Specifically, stress-responsive gene expression was most profoundly compromised in HIRA disruptants. Conclusion: HIRA is involved in cross-tolerance via regulation of stress-responsive gene expression. Significance: This study provides evidence that fission yeast HIRA functions in stress response.