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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

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.

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.

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.

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.

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.

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.

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.

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.

Cellular senescence is a tumor-suppressing mechanism that is accompanied by characteristic chromatin condensation called senescence-associated heterochromatic foci (SAHFs). We found that individual SAHFs originate from individual chromosomes. SAHFs do not show alterations of posttranslational modifications of core histones that mark condensed chromatin in mitotic chromosomes, apoptotic chromatin, or transcriptionally inactive heterochromatin. Remarkably, SAHF-positive senescent cells lose linker histone H1 and exhibit increased levels of chromatin-bound high mobility group A2 (HMGA2). The expression of N-terminally enhanced green fluorescent protein (EGFP)–tagged histone H1 induces premature senescence phenotypes, including increased levels of phosphorylated p53, p21, and hypophosphorylated Rb, and a decrease in the chromatin-bound endogenous histone H1 level but not in p16 level accumulation or SAHF formation. However, the simultaneous ectopic expression of hemagglutinin-tagged HMGA2 and N-terminally EGFP-tagged histone H1 leads to significant SAHF formation (P < 0.001). It is known that histone H1 and HMG proteins compete for a common binding site, the linker DNA. These results suggest that SAHFs are a novel type of chromatin condensation involving alterations in linker DNA–binding proteins.

Telomeric and subtelomeric regions of human chromosomes largely consist of highly repetitive and redundant DNA sequences, resulting in a paucity of unique DNA sequences specific to individual telomeres. Accordingly, it is difficult to analyze telomere metabolism on a single-telomere basis. To circumvent this problem, we have exploited a human artificial chromosome (HAC#21) derived from human chromosome 21 (hChr21). HAC#21 was generated through truncation of the long arm of native hChr21 by the targeted telomere seeding technique. The newly established telomere of HAC#21 lacks canonical subtelomere structures but possesses unique sequences derived from the target vector backbone and the internal region of hChr21 used for telomere targeting, which enabled us to molecularly characterize the single HAC telomere. We established HeLa and NIH-3T3 sub-lines containing a single copy of HAC#21, where it was robustly maintained. The seeded telomere is associated with telomeric proteins over a length similar to that reported in native telomeres, and is faithfully replicated in mid-S phase in HeLa cells. We found that the seeded telomere on HAC#21 is transcribed from the newly juxtaposed site. The transcript, HAC-telRNA, shares several features with TERRA (telomeric repeat-containing RNA): it is a short-lived RNA polymerase II transcript, rarely contains a poly(A) tail, and associates with chromatin. Interestingly, HAC-telRNA undergoes splicing. These results suggest that transcription into TERRA is locally influenced by the subtelomeric context. Taken together, we have established human and mouse cell lines that will be useful for analyzing the behavior of a uniquely identifiable, functional telomere.

peer-reviewed Globally distributed software development teams face problems with software development life cycle phases, as the distributed nature of each of these phases make it even more challenging to communicate between the stakeholders. Global distance can give rise to incomplete requirements handovers which make the situation more difficult. It is important to address this issue as the end product is likely to deliver less business value when such problems arise. In this research, we propose a process to facilitate non-verbal communication among globally distributed requirements engineering teams. The focus of this research is the situation that occurs after requirements are handed to another site. Our proposed process endeavors to ensure that incomplete and conflicting requirements are identified and mitigated.

Induced pluripotent stem cell (iPSC) technology can be used to model human disorders, create cell-based models of human diseases, including neurodegenerative diseases, and in establishing therapeutic strategies. To detect subtle cellular abnormalities associated with common late-onset disease in iPSCs, valid control iPSCs derived from healthy donors free of serious late-onset diseases are necessary. Here, we report the generation of iPSCs from fibroblasts obtained immediately postmortem from centenarian donors (106- and 109-years-old) who were extremely healthy until an advanced age. The iPSCs were generated using a conventional method involving OCT4, SOX2, KLF4, and c-MYC, and then differentiated into neuronal cells using a neurosphere method. The expression of molecules that play critical roles in late-onset neurodegenerative diseases by neurons differentiated from the centenarian-iPSCs was compared to that of neurons differentiated from iPSCs derived from familial Alzheimer's disease and familial Parkinson's disease (PARK4: triplication of the α synuclein gene) patients. The results indicated that our series of iPSCs would be useful in neurodegeneration research. The iPSCs we describe, which were derived from donors with exceptional longevity who were presumed to have no serious disease risk factors, would be useful in longevity research and as valid super-controls for use in studies of various late-onset diseases.

FLASH has been shown to be required for S phase progression and to interact with a nuclear protein, ataxia-telangiectasia locus (NPAT), a component of Cajal bodies in the nucleus and an activator of histone transcription. We investigated the role of human FLASH by using an inducible FLASH knockdown system in the presence or absence of various mutant forms of mouse FLASH. While carboxyl-terminal deletion mutants of FLASH, which do not interact with NPAT, can support S phase progression, its amino-terminal deletion mutants, which are unable to self associate, cannot support S phase progression, replication-dependent histone transcription, or the formation of Cajal bodies. Furthermore, FLASH was shown to be associated with arsenite resistance protein 2 (ARS2) through its central region, which is composed of only 13 amino acids. The expression of ARS2 and the interaction between FLASH and ARS2 are required for S phase progression. Taking these results together, FLASH functions in S phase progression through interaction with ARS2.

A significant fraction of human cancer cells and immortalized cells maintain telomeres in a telomerase-independent manner called alternative lengthening of telomeres (ALT). It has been suggested that ALT involves homologous recombination that is expected to generate unique intermediate DNAs. However, the precise molecular mechanism of ALT is not known. To gain insight into how telomeric DNAs (T-DNAs) are maintained in ALT, we examined the physical structures of T-DNAs in ALT cells. We found abundant single-stranded regions in both G and C strands of T-DNAs. Moreover, two-dimensional gel electrophoreses and native in-gel hybridization analyses revealed novel ALT-specific single-stranded T-DNAs, in addition to previously reported t-circles. These newly identified ALT-specific T-DNAs include (i) the t-complex, which consists of highly branched T-DNAs with large numbers of internal single-stranded portions; (ii) ss-G, which consists of mostly linear single-G-strand T-DNAs; and (iii) ss-C, which consists of most likely circular single-C-strand T-DNAs. Cellular-DNA fractionation by the Hirt protocol revealed that t-circles and ss-G exist in ALT cells as extrachromosomal and chromatin-associated DNAs. We propose that such ALT-specific T-DNAs are produced by telomere metabolism specific to ALT, namely, homologous recombination and the rolling-circle replication mechanism.

The requirements of distributed software systems become increasingly complex. Among the aspects that characterize such a complexity are the inherent distribution of resources and activity - that makes global control practically infeasible and emphasizes the need for decentralized control - and the highly dynamic and changing operating conditions in which today's distributed applications operate - that calls for open and adaptive software.

INTRODUCTION. Ataxia teleangiectasia (AT) is an autosomal recessive disorder characterized by progressive neurological degeneration, teleangiectasia, high sensitivity to ionizing radiation, high incidence of cancer, and premature aging of skin and hair (1). Cells derived from AT patients exhibit a variety of abnormalities in culture, such as hypersensitivity to ionizing radiation, rapid shortening of telomere length, and premature replicative senescence. Normal diploid fibroblasts in culture have a finite proliferative lifespan and enter a nondividing state termed replicative senescence (2). Ectopic expression of the catalytic subunit of telomerase, hTERT, has been shown to restore telomerase activity, stabilize telomere length and bypass replicative senescence in telomerase-negative normal fibroblasts (3) as well as in the premature aging Werner syndrome (4). In order to address a role of ATM (AT mutated) protein in the regulation of telomere length and a relationship between telomere length and premature replicative senescence in AT cells, we have examined whether fibroblasts from AT patients can be immortalized by introduction of hTERT. METHODS. Primary dermal fibroblasts from normal and AT individuals were used. Amphotrophic retrovirus expressing hTERT was created with pMX-puro-hTERT in Phenix-A cells. Cells were infected with the hTERT retrovirus, and selected in culture containing 0.5 µg / ml puromycin. Telomere length was measured by terminal restriction fragment (TRF) assay, and telomerase activity was assessed by telomeric repeat amplification protocol (TRAP) using TRAPeze kit (Intergen, Purchase, NY). RESULTS. Expression of hTERT mRNA and telomerase activity in hTERT-infected AT as well as normal cells was confirmed by RT-PCR and TRAP assays, respectively. Telomere length of hTERT-infected AT cells was extended to approximately 9 kb, compared with short telomeric DNA of about 6kb in the parent AT cells at near mortal 1 (M1) phase. Whereas parental AT cells underwent replicative senescence at about 24 population doublings (PDs), hTERT-positive AT cells survived over 40 PDs, similar to hTERT-infected normal fibroblasts (Fig. 1).

Rat c-raf was found to be activated in a transformant obtained with DNA of a hepatocellular carcinoma induced by 2-amino-3-methylimidazo[4,5-f]quinoline. This activated c-raf was cloned in a cosmid vector and actively transforming clones were obtained. Comparison of the restriction maps of this activated c-raf and cloned normal rat c-raf revealed a recombination in the 5'-terminal region of the activated form of this gene. The recombined DNA was shown to be actively transcribed and possibly to form a fused mRNA with c-raf, which is slightly smaller than normal c-raf mRNA. Since this recombination was not detected in the original tumor by Southern blot analysis, it presumably occurred during transfection.