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Disease diagnostics and surveillance increasingly highlight the importance of portable, cost-effective, and sensitive point-of-care (POC) detection of nucleic acids. Here, we report a CRISPR/Cas13a-responsive and RNA-bridged DNA hydrogel capillary sensor for the direct and visual detection of specific RNA with high sensitivity. The capillary sensor was simply prepared by loading RNA-cross-linking DNA hydrogel film (∼0.2 mm ± 0.02 mm) at the end of a capillary. When CRISPR/Cas13a specifically recognizes the target RNA, the RNA bridge in the hydrogel film is cleaved by the trans -cleavage activity of CRISPR/Cas13a, increasing the permeability of the hydrogel film. Different concentrations of target RNA activate different amounts of Cas13a, cleaving different amounts of the RNA bridge in the hydrogel and causing corresponding changes in the permeability of the hydrogel. Therefore, samples containing different amounts of the target RNA travel to different distances in the capillary. Visual reading of the distance provides quantitative detection of the RNA target without the need for any nucleic acid amplification or auxiliary equipment. The technique was successfully used for the determination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in clinical nasopharyngeal (NP) swab and saliva samples. Easily quantifiable distance using a ruler eliminates the need for any optical or electrochemical detection equipment, making this assay potentially useful for POC and on-site applications.

DNA-protein crosslinks (DPCs) are a frequent form of DNA lesion and are strongly inhibitive in diverse DNA transactions. Despite recent developments, the biochemical detection of DPCs remains a limiting factor for the in-depth mechanistic understanding of DPC repair. Here, we develop a sensitive and versatile assay, designated ARK, for the quantitative analysis of DPCs in cells. ARK uses sequential chaotropic and detergent-based isolation of DPCs and substantially enhances sample purity, resulting in a 5-fold increase in detection sensitivity and a 10-fold reduction in background reading. We validate the ARK assay with genetic mutants with established deficiencies in DPC repair and demonstrate its robustness by using common DPC-inducing reagents, including formaldehyde, camptothecin, and etoposide. In addition, we show that the Fanconi anemia pathway contributes to the repair of DPCs. Thus, ARK is expected to facilitate various studies aimed at understanding both fundamental biology and translational applications of DNA-protein crosslink repair., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Developing improved cationic polymer-DNA polyplexes for gene delivery requires improved understanding of DNA transport from endosomes into the nucleus. Using a FRET-capable oligonucleotide molecular beacon (OMB), we monitored the transport of intact DNA to cell organelles. We observed that for effective (jetPEI) and ineffective (G5 PAMAM) vectors, the fraction of cells displaying intact OMB in the cytosol (jetPEI ≫ G5 PAMAM) quantitatively predicted the fraction expressing transgene (jetPEI ≫ G5 PAMAM). Intact OMB delivered with PAMAM and confined to endosomes could be released to the cytosol by the subsequent addition of L-PEI, with a corresponding 10-fold increase in transgene expression. These results suggest that future vector development should optimize vectors for intercalation into, and destabilization of, the endosomal membrane. Finally, the study highlights a two-step strategy in which the pDNA is loaded in cells using one vector and endosomal release is mediated by a second agent.

We identified ubiquitin-like with PHD and RING finger domain 1 (UHRF1) as a binding factor for DNA interstrand crosslink (ICL) lesions through affinity purification of ICL-recognition activities. UHRF1 is recruited to DNA lesions in vivo and binds directly to ICL-containing DNA. UHRF1-deficient cells display increased sensitivity to a variety of DNA damages. We found that loss of UHRF1 led to retarded lesion processing and reduced recruitment of ICL repair nucleases to the site of DNA damage. UHRF1 interacts physically with both ERCC1 and MUS81, two nucleases involved in the repair of ICL lesions. Depletion of both UHRF1 and components of the Fanconi anemia (FA) pathway resulted in increased DNA damage sensitivity compared to defect of each mechanism alone. These results suggest that UHRF1 promotes recruitment of lesion-processing activities via its affinity to recognize DNA damage and functions as a nuclease recruitment scaffold in parallel to the FA pathway., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

The human SSB homologue 1 (hSSB1) has been shown to facilitate homologous recombination and double-strand break signalling in human cells. Here, we compare the DNA-binding properties of the SOSS1 complex, containing SSB1, with Replication Protein A (RPA), the primary single-strand DNA (ssDNA) binding complex in eukaryotes. Ensemble and single-molecule approaches show that SOSS1 binds ssDNA with lower affinity compared to RPA, and exhibits less stable interactions with DNA substrates. Nevertheless, the SOSS1 complex is uniquely capable of promoting interaction of human Exo1 with double-strand DNA ends and stimulates its activity independently of the MRN complex in vitro. Both MRN and SOSS1 also act to mitigate the inhibitory action of the Ku70/80 heterodimer on Exo1 activity in vitro. These results may explain why SOSS complexes do not localize with RPA to replication sites in human cells, yet have a strong effect on double-strand break resection and homologous recombination.

Fanconi anemia (FA) is caused by mutations in 13 Fanc genes and renders cells hypersensitive to DNA interstrand cross-linking (ICL) agents. A central event in the FA pathway is mono-ubiquitylation of the FANCI-FANCD2 (ID) protein complex. Here, we characterize a previously unrecognized nuclease, Fanconi anemia-associated nuclease 1 (FAN1), that promotes ICL repair in a manner strictly dependent on its ability to accumulate at or near sites of DNA damage and that relies on mono-ubiquitylation of the ID complex. Thus, the mono-ubiquitylated ID complex recruits the downstream repair protein FAN1 and facilitates the repair of DNA interstrand cross-links.

DNA double-strand breaks (DSBs) represent one of the most lethal types of DNA damage cells encounter. CtIP (also known as RBBP8) acts together with the MRN (MRE11-RAD50-NBS1) complex to promote DNA end resection and the generation of single-stranded DNA, which is critically important for homologous recombination repair. However, it is not yet clear exactly how CtIP participates in this process. Here, we demonstrate that besides the known conserved C terminus, the N terminus of CtIP protein is also required in DSB end resection and DNA damage-induced G(2)/M checkpoint control. We further show that both termini of CtIP can interact with the MRN complex and that the N terminus of CtIP, especially residues 22-45, binds to MRN and plays a critical role in targeting CtIP to sites of DNA breaks. Collectively, our results highlight the importance of the N terminus of CtIP in directing its localization and function in DSB repair.

Mutations in breast cancer susceptibility gene 1 and 2 (BRCA1 and BRCA2) predispose individuals to breast and ovarian cancer development. We previously reported an in vivo interaction between BRCA1 and BRCA2. However, the biological significance of their association is thus far undefined. Here, we report that PALB2, the partner and localizer of BRCA2, binds directly to BRCA1, and serves as the molecular scaffold in the formation of the BRCA1-PALB2-BRCA2 complex. The association between BRCA1 and PALB2 is primarily mediated via apolar bonding between their respective coiled-coil domains. More importantly, BRCA1 mutations identified in cancer patients disrupted the specific interaction between BRCA1 and PALB2. Consistent with the converging functions of the BRCA proteins in DNA repair, cells harboring mutations with abrogated BRCA1-PALB2 interaction resulted in defective homologous recombination (HR) repair. We propose that, via its direct interaction with PALB2, BRCA1 fine-tunes recombinational repair partly through its modulatory role in the PALB2-dependent loading of BRCA2-RAD51 repair machinery at DNA breaks. Our findings uncover PALB2 as the molecular adaptor between the BRCA proteins, and suggest that impaired HR repair is one of the fundamental causes for genomic instability and tumorigenesis observed in patients carrying BRCA1, BRCA2, or PALB2 mutations.

In male germ cells the repair of DNA double strand breaks (DSBs) differs from that described for somatic cell lines. Irradiation induced immunofluorescent foci (IRIF's) signifying a double strand DNA breaks, were followed in spermatogenic cells up to 16 h after the insult. Foci were characterised for Mdc1, 53BP1 and Rad51 that always were expressed in conjecture with gamma-H2AX. Subsequent spermatogenic cell types were found to have different repair proteins. In early germ cells up to the start of meiotic prophase, i.e. in spermatogonia and preleptotene spermatocytes, 53BP1 and Rad51 are available but no Mdc1 is expressed in these cells before and after irradiation. The latter might explain the radiosensitivity of spermatogonia. Spermatocytes from shortly after premeiotic S-phase till pachytene in epithelial stage IV/V express Mdc1 and Rad51 but no 53BP1 which has no role in recombination involved repair during the early meiotic prophase. Mdc1 is required during this period as in Mdc1 deficient mice all spermatocytes enter apoptosis in epithelial stage IV when they should start mid-pachytene phase of the meiotic prophase. From stage IV mid pachytene spermatocytes to round spermatids, Mdc1 and 53BP1 are expressed while Rad51 is no longer expressed in the haploid round spermatids. Quantifying foci numbers of gamma-H2AX, Mdc1 and 53BP1 at various time points after irradiation revealed a 70% reduction after 16 h in pachytene and diplotene spermatocytes and round spermatids. Although the DSB repair efficiency is higher then in spermatogonia where only a 40% reduction was found, it still does not compare to somatic cell lines where a 70% reduction occurs in 2 h. Taken together, DNA DSBs repair proteins differ for the various types of spermatogenic cells, no germ cell type possessing the complete set. This likely leads to a compromised efficiency relative to somatic cell lines. From the evolutionary point of view it may be an advantage when germ cells die from DNA damage rather than risk the acquisition of transmittable errors made during the repair process.

Mutations in the breast cancer susceptibility gene 1 (BRCA1) are associated with an increased risk of breast and ovarian cancers. BRCA1 participates in the cellular DNA damage response. We report the identification of receptor-associated protein 80 (RAP80) as a BRCA1-interacting protein in humans. RAP80 contains a tandem ubiquitin-interacting motif domain, which is required for its binding with ubiquitin in vitro and its damage-induced foci formation in vivo. Moreover, RAP80 specifically recruits BRCA1 to DNA damage sites and functions with BRCA1 in G2/M checkpoint control. Together, these results suggest the existence of a ubiquitination-dependent signaling pathway involved in the DNA damage response.

The tumor suppressor BRCA1 has an important function in the maintenance of genomic stability. Increasing evidence suggests that BRCA1 regulates cell cycle checkpoints and DNA repair after DNA damage. However, little is known about its normal function in the absence of DNA damage. Here we show that BRCA1 interacts and colocalizes with topoisomerase IIalpha in S phase cells. Similar to cells treated with the topoisomerase IIalpha inhibitor ICRF-193, BRCA1-deficient cells show lagging chromosomes, indicating a defect in DNA decatenation and chromosome segregation. More directly, BRCA1 deficiency results in defective DNA decatenation in vitro. Finally, topoisomerase IIalpha is ubiquitinated in a BRCA1-dependent manner, and topoisomerase IIalpha ubiquitination correlates with higher DNA decatenation activity. Together these results suggest an important role of BRCA1 in DNA decatenation and reveal a previously unknown function of BRCA1 in the maintenance of genomic stability.

53BP1 participates early in the DNA damage response and is involved in cell cycle checkpoint control. Moreover, the phenotype of mice and cells deficient in 53BP1 suggests a defect in DNA repair (Ward et al., 2003b). Therefore, we asked whether or not 53BP1 would be required for the efficient repair of DNA double strand breaks. Our data indicate that homologous recombination by gene conversion does not depend on 53BP1. Moreover, 53BP1-deficient mice support normal V(D)J recombination, indicating that 53BP1 is not required for "classic" nonhomologous end joining. However, class switch recombination is severely impaired in the absence of 53BP1, suggesting that 53BP1 facilitates DNA end joining in a way that is not required or redundant for the efficient closing of RAG-induced strand breaks. These findings are similar to those observed in mice or cells deficient in the tumor suppressors ATM and H2AX, further suggesting that the functions of ATM, H2AX, and 53BP1 are closely linked.

53BP1 participates in the cellular response to DNA damage. Like many proteins involved in the DNA damage response, 53BP1 becomes hyperphosphorylated after radiation and colocalizes with phosphorylated H2AX in megabase regions surrounding the sites of DNA strand breaks. However, it is not yet clear whether the phosphorylation status of 53BP1 determines its localization or vice versa. In this study we mapped a region upstream of the 53BP1 C terminus that is required and sufficient for the recruitment of 53BP1 to these DNA break areas. In vitro assays revealed that this region binds to phosphorylated but not unphosphorylated H2AX. Moreover, using H2AX-deficient cells reconstituted with wild-type or a phosphorylation-deficient mutant of H2AX, we have shown that phosphorylation of H2AX at serine 140 is critical for efficient 53BP1 foci formation, implying that a direct interaction between 53BP1 and phosphorylated H2AX is required for the accumulation of 53BP1 at DNA break sites. On the other hand, radiation-induced phosphorylation of the 53BP1 N terminus by the ATM (ataxia-telangiectasia mutated) kinase is not essential for 53BP1 foci formation and takes place independently of 53BP1 redistribution. Thus, these two damage-induced events, hyperphosphorylation and relocalization of 53BP1, occur independently in the cell.

We present novoBreak, a genome-wide local assembly algorithm that discovers somatic and germline structural variation breakpoints in whole-genome sequencing data. novoBreak consistently outperformed existing algorithms on real cancer genome data and on synthetic tumors in the ICGC-TCGA DREAM 8.5 Somatic Mutation Calling Challenge primarily because it more effectively utilized reads spanning breakpoints. novoBreak also demonstrated great sensitivity in identifying short insertions and deletions.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.tibs.2009.09.001 Byline: Michael S.Y. Huen (1)(2), Junjie Chen (3) Abstract: The remarkably coordinated nature of the DNA damage response pathway relies on numerous mechanisms that facilitate the assembly of checkpoint and repair factors at DNA breaks. Post-translational modifications on and around chromatin have critical roles in allowing the timely and sequential assembly of DNA damage responsive elements at the vicinity of DNA breaks. Notably, recent advances in forward genetics and proteomics-based approaches have enabled the identification of novel components within the DNA damage response pathway, providing a more comprehensive picture of the molecular network that assists in the detection and propagation of DNA damage signals. Author Affiliation: (1) Department of Anatomy (2) Centre for Cancer Research, University of Hong Kong, L1-59, Laboratory Block, 21 Sassoon Road, Hong Kong S.A.R. (3) Department of Experimental Radiation Oncology, University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030 USA

The~Fanconi anemia (FA) pathway participates in interstrand crosslink (ICL) repair and the maintenance of genomic stability. The FA core complex consists of eight FA proteins and two Fanconi anemiaassociated proteins (FAAPZ4 and FAAP100). The FA core complex has ubiquitin ligase activity responsible for monoubiquitination of the FANCI-FANCD2 (ID) complex, which in turn initiates a cascade of biochemical events that allow processing and removal of crosslinked DNA and thereby promotes cell survival following DNA damage. Here, we report the identification of a unique component of the FA core complex, namely, FAAP2O, which contains a RAD18like ubiquitin-binding zinc-finger domain. Our data suggest that FAAP2O promotes the functional integrity of the FA core complex via its direct interaction with the FA gene product, FANCA. Indeed, somatic knockout cells devoid of FAAP2O displayed the hallmarks of FA cells, including hypersensitivity to DNA cross-linking agents, chromosome aberrations, and reduced FANCD2 monoubiquitination. Taking these data together, our study indicates that FAAP2O is an important player involved in the FA pathway. [ABSTRACT FROM AUTHOR]

Cell cycle checkpoints are essential for maintaining genomic integrity. Human topoisomerase II binding protein 1 (TopBP1) shares sequence similarity with budding yeast Dpb11, fission yeast Rad4/Cut5, and Xenopus Cut5, all of which are required for DNA replication and cell cycle checkpoints. Indeed, we have shown that human TopBP1 participates in the activation of replication checkpoint and DNA damage checkpoints, following hydroxyurea treatment and ionizing radiation. In this study, we address the physiological function of TopBP1 in S phase by using small interfering RNA. In the absence of exogenous DNA damage, TopBP1 is recruited to replicating chromatin. However, TopBP1 does not appear to be essential for DNA replication. TopBP1-deficient ceils have increased H2AX phosphorylation and ATM-Chk 2 activation, suggesting the accumulation of DNA double-strand breaks in the absence of TopBP1. This leads to formation of gaps and breaks at fragile sites, 4N accumulation, and aberrant cell division. We propose that the cellular function of TopBP1 is to monitor ongoing DNA replication. By ensuring proper DNA replication, TopBP1 plays a critical role in the maintenance of genomic stability during normal S phase as well as following genotoxic stress. [ABSTRACT FROM AUTHOR]

Presents a study conducted on the effect of deoxyribonucleic acid (DNA) damage on BRCA1 gene subnuclear location and phosphorylation state during S phase. Phosphorylation of BRCA1 after DNA damage in S phase without arrest of scheduled DNA synthesis; Results of the study.

Summary: Dynamic changes of chromatin structure facilitate diverse biological events, including DNA replication, repair, recombination, and gene transcription. Recent evidence revealed that DNA damage elicits alterations to the chromatin to facilitate proper checkpoint activation and DNA repair. Here we report the identification of the PWWP domain-containing protein EXPAND1/MUM1 as an architectural component of the chromatin, which in response to DNA damage serves as an accessory factor to promote cell survival. Depletion of EXPAND1/MUM1 or inactivation of its PWWP domain resulted in chromatin compaction. Upon DNA damage, EXPAND1/MUM1 rapidly concentrates at the vicinity of DNA damage sites via its direct interaction with 53BP1. Ablation of this interaction impaired damage-induced chromatin decondensation, which is accompanied by sustained DNA damage and hypersensitivity to genotoxic stress. Collectively, our study uncovers a chromatin-bound factor that serves an accessory role in coupling damage signaling with chromatin changes in response to DNA damage. [Copyright &y& Elsevier]

Summary: Human TopBP1 plays a critical role in the control of DNA replication checkpoint. In this study, we report a specific interaction between TopBP1 and BACH1/FANCJ, a DNA helicase involved in the repair of DNA crosslinks. The TopBP1/BACH1 interaction is mediated by the very C-terminal tandem BRCT domains of TopBP1 and S phase-specific phosphorylation of BACH1 at Thr 1133 site. Interestingly, we demonstrate that depletion of TopBP1 or BACH1 attenuates the loading of RPA on chromatin. Moreover, both TopBP1 and BACH1 are required for ATR-dependent phosphorylation events in response to replication stress. Taken together, our data suggest that BACH1 has an unexpected early role in replication checkpoint control. A specific interaction between TopBP1 and BACH1 is likely to be required for the extension of single-stranded DNA regions and RPA loading following replication stress, which is a prerequisite for the subsequent activation of replication checkpoint. [Copyright &y& Elsevier]

Summary: The molecular pathways leading from genomic instability to cellular senescence and/or cell death remain incompletely characterized. Using mouse embryonic fibroblasts with constitutively increased DNA damage due to the absence of the full-length form of the tumor suppressor Brca1 (Brca1 Δ11/Δ11), we show that deletion of p53 binding protein 1 (53BP1) selectivity abrogates senescence and cell death stimulated by reduced Brca1 activity. Furthermore, the embryonic lethality induced by Brca1 mutation can be alleviated by 53BP1 deletion. Adult Brca1 Δ11/Δ11 53BP1 −/− manifest constitutively high levels of genomic instability, yet age relatively normally, with a surprisingly low incidence of overall tumor formation. Together, these in vitro and in vivo data suggest that 53BP1 is specifically required for the development of premature senescence and apoptosis induced by Brca1 deficiency. These observations may have important implications for Brca1-mediated tumor formation as well as for the molecular pathway leading from genomic instability to organismal aging. [Copyright &y& Elsevier]

Summary: Phosphorylated histone H2AX (“γ-H2AX”) recruits MDC1, 53BP1, and BRCA1 to chromatin near a double-strand break (DSB) and facilitates efficient repair of the break. It is unclear to what extent γ-H2AX-associated proteins act in concert and to what extent their functions within γ-H2AX chromatin are distinct. We addressed this question by comparing the mechanisms of action of MDC1 and 53BP1 in DSB repair (DSBR). We find that MDC1 functions primarily in homologous recombination/sister chromatid recombination, in a manner strictly dependent upon its ability to interact with γ-H2AX but, unexpectedly, not requiring recruitment of 53BP1 or BRCA1 to γ-H2AX chromatin. In contrast, 53BP1 functions in XRCC4-dependent nonhomologous end-joining, likely mediated by its interaction with dimethylated lysine 20 of histone H4 but, surprisingly, independent of H2AX. These results suggest a specialized adaptation of the “histone code” in which distinct histone tail-protein interactions promote engagement of distinct DSBR pathways. [Copyright &y& Elsevier]

Summary: DNA-damage signaling utilizes a multitude of posttranslational modifiers as molecular switches to regulate cell-cycle checkpoints, DNA repair, cellular senescence, and apoptosis. Here we show that RNF8, a FHA/RING domain-containing protein, plays a critical role in the early DNA-damage response. We have solved the X-ray crystal structure of the FHA domain structure at 1.35 Å. We have shown that RNF8 facilitates the accumulation of checkpoint mediator proteins BRCA1 and 53BP1 to the damaged chromatin, on one hand through the phospho-dependent FHA domain-mediated binding of RNF8 to MDC1, on the other hand via its role in ubiquitylating H2AX and possibly other substrates at damage sites. Moreover, RNF8-depleted cells displayed a defective G2/M checkpoint and increased IR sensitivity. Together, our study implicates RNF8 as a novel DNA-damage-responsive protein that integrates protein phosphorylation and ubiquitylation signaling and plays a critical role in the cellular response to genotoxic stress. [Copyright &y& Elsevier]

Summary: Histone H2AX promotes DNA double-strand break (DSB) repair and immunoglobulin heavy chain (IgH) class switch recombination (CSR) in B-lymphocytes. CSR requires activation-induced cytidine deaminase (AID) and involves joining of DSB intermediates by end joining. We find that AID-dependent IgH locus chromosome breaks occur at high frequency in primary H2AX-deficient B cells activated for CSR and that a substantial proportion of these breaks participate in chromosomal translocations. Moreover, activated B cells deficient for ATM, 53BP1, or MDC1, which interact with H2AX during the DSB response, show similarly increased IgH locus breaks and translocations. Thus, our findings implicate a general role for these factors in promoting end joining and thereby preventing DSBs from progressing into chromosomal breaks and translocations. As cellular p53 status does not markedly influence the frequency of such events, our results also have implications for how p53 and the DSB response machinery cooperate to suppress generation of lymphomas with oncogenic translocations. [Copyright &y& Elsevier]

Summary: MDC1 functions in checkpoint activation and DNA repair following DNA damage. To address the physiological role of MDC1, we disrupted the MDC1 gene in mice. MDC1−/− mice recapitulated many phenotypes of H2AX−/− mice, including growth retardation, male infertility, immune defects, chromosome instability, DNA repair defects, and radiation sensitivity. At the molecular level, H2AX, MDC1, and ATM form a positive feedback loop, with MDC1 directly mediating the interaction between H2AX and ATM. MDC1 binds phosphorylated H2AX through its BRCT domain and ATM through its FHA domain. Through these interactions, MDC1 accumulates activated ATM flanking the sites of DNA damage, facilitating further ATM-dependent phosphorylation of H2AX and the amplification of DNA damage signals. In the absence of MDC1, many downstream ATM signaling events are defective. These results suggest that MDC1, as a signal amplifier of the ATM pathway, is vital in controlling proper DNA damage response and maintaining genomic stability. [Copyright &y& Elsevier]

BRCT tandem domains, found in many proteins involved in DNA damage checkpoint and DNA repair pathways, were recently shown to be phosphopeptide binding motifs. Using solution nuclear magnetic resonance (NMR) spectroscopy and mutational analysis, we have characterized the interaction of BRCA1-BRCT domains with a phosphoserine-containing peptide derived from the DNA repair helicase BACH1. We show that a phenylalanine in the +3 position from the phosphoserine of BACH1 is bound to a conserved hydrophobic pocket formed between the two BRCT domains and that recognition of the phosphate group is mediated by lysine and serine side chains from the amino-terminal BRCT domain. Mutations that prevent phosphopeptide binding abolish BRCA1 function in DNA damage-induced checkpoint control. Our NMR data also reveal a dynamic interaction between BRCA1-BRCT and BACH1, where the bound phosphopeptide exists as an equilibrium of two conformations and where BRCA1-BRCT undergoes a transition to a more rigid conformation upon peptide binding. [Copyright &y& Elsevier]