Topoisomerase 1-dependent R-loop deficiency drives accelerated replication and genomic instability.

DNA replication is a complex process tightly regulated to ensure faithful genome duplication, and its perturbation leads to DNA damage and genomic instability. Replication stress is commonly associated with slow and stalled replication forks. Recently, accelerated replication has emerged as a non-canonical form of replication stress. However, the molecular basis underlying fork acceleration is largely unknown. Here, we show that mutated HRAS activation leads to increased topoisomerase 1 (TOP1) expression, causing aberrant replication fork acceleration and DNA damage by decreasing RNA-DNA hybrids or R-loops. In these cells, restoration of TOP1 expression or mild replication inhibition rescues the perturbed replication and reduces DNA damage. Furthermore, TOP1 or RNaseH1 overexpression induces accelerated replication and DNA damage, highlighting the importance of TOP1 equilibrium in regulating R-loop homeostasis to ensure faithful DNA replication and genome integrity. Altogether, our results dissect a mechanism of oncogene-induced DNA damage by aberrant replication fork acceleration.

MutSß regulates G4-associated telomeric R-loops to maintain telomere integrity in ALT cancer cells.

Up to 15% of human cancers maintain their telomeres through a telomerase-independent mechanism, termed "alternative lengthening of telomeres" (ALT) that relies on homologous recombination between telomeric sequences. Emerging evidence suggests that the recombinogenic nature of ALT telomeres results from the formation of RNA:DNA hybrids (R-loops) between telomeric DNA and the long-noncoding telomeric repeat-containing RNA (TERRA). Here, we show that the mismatch repair protein MutSß, a heterodimer of MSH2 and MSH3 subunits, is enriched at telomeres in ALT cancer cells, where it prevents the accumulation of telomeric G-quadruplex (G4) structures and R-loops. Cells depleted of MSH3 display increased incidence of R-loop-dependent telomere fragility and accumulation of telomeric C-circles. We also demonstrate that purified MutSß recognizes and destabilizes G4 structures in vitro. These data suggest that MutSß destabilizes G4 structures in ALT telomeres to regulate TERRA R-loops, which is a prerequisite for maintenance of telomere integrity during ALT.

Determinants of early antibody responses to COVID-19 mRNA vaccines in a cohort of exposed and naïve healthcare workers.

BACKGROUND: Two doses of mRNA vaccination have shown >94% efficacy at preventing COVID-19 mostly in naïve adults, but it is not clear if the second dose is needed to maximize effectiveness in those previously exposed to SARS-CoV-2 and what other factors affect responsiveness. METHODS: We measured IgA, IgG and IgM levels against SARS-CoV-2 spike (S) and nucleocapsid (N) antigens from the wild-type and S from the Alpha, Beta and Gamma variants of concern, after BNT162b2 (Pfizer/BioNTech) or mRNA-1273 (Moderna) vaccination in a cohort of health care workers (N=578). Neutralizing capacity and antibody avidity were evaluated. Data were analyzed in relation to COVID-19 history, comorbidities, vaccine doses, brand and adverse events. FINDINGS: Vaccination induced robust IgA and IgG levels against all S antigens. Neutralization capacity and S IgA and IgG levels were higher in mRNA-1273 vaccinees, previously SARS-CoV-2 exposed, particularly if symptomatic, and in those experiencing systemic adverse effects (p<0·05). A second dose in pre-exposed did not increase antibody levels. Smoking and comorbidities were associated with 43% (95% CI, 19-59) and 45% (95% CI, 63-18) lower neutralization, respectively, and 35% (95% CI, 3-57%) and 55% (95% CI, 33-70%) lower antibody levels, respectively. Among fully vaccinated, 6·3% breakthroughs were detected up to 189 days post-vaccination. Among pre-exposed non-vaccinated, 90% were IgG seropositive more than 300 days post-infection. INTERPRETATION: Our data support administering a single-dose in pre-exposed healthy individuals as primary vaccination. However, heterogeneity of responses suggests that personalized recommendations may be necessary depending on COVID-19 history and life-style. Higher mRNA-1273 immunogenicity would be beneficial for those expected to respond worse to vaccination and in face of variants that escape immunity such as Omicron. Persistence of antibody levels in pre-exposed unvaccinated indicates maintenance of immunity up to one year. FUNDING: This work was supported by Institut de Salut Global de Barcelona (ISGlobal) internal funds, in-kind contributions from Hospital Clínic de Barcelona, the Fundació Privada Daniel Bravo Andreu, and European Institute of Innovation and Technology (EIT) Health (grant number 20877), supported by the European Institute of Innovation and Technology, a body of the European Union receiving support from the H2020 Research and Innovation Programme. We acknowledge support from the Spanish Ministry of Science and Innovation and State Research Agency through the "Centro de Excelencia Severo Ochoa 2019-2023" Program (CEX2018-000806-S), and support from the Generalitat de Catalunya through the CERCA Program. L. I. work was supported by PID2019-110810RB-I00 grant from the Spanish Ministry of Science & Innovation. Development of SARS-CoV-2 reagents was partially supported by the National Institute of Allergy and Infectious Diseases Centers of Excellence for Influenza Research and Surveillance (contract number HHSN272201400008C). The funders had no role in study design, data collection and analysis, the decision to publish, or the preparation of the manuscript.

ADAR-mediated RNA editing of DNA:RNA hybrids is required for DNA double strand break repair.

The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.

A transcription-based mechanism for oncogenic ß-catenin-induced lethality in BRCA1/2-deficient cells.

BRCA1 or BRCA2 germline mutations predispose to breast, ovarian and other cancers. High-throughput sequencing of tumour genomes revealed that oncogene amplification and BRCA1/2 mutations are mutually exclusive in cancer, however the molecular mechanism underlying this incompatibility remains unknown. Here, we report that activation of ß-catenin, an oncogene of the WNT signalling pathway, inhibits proliferation of BRCA1/2-deficient cells. RNA-seq analyses revealed ß-catenin-induced discrete transcriptome alterations in BRCA2-deficient cells, including suppression of CDKN1A gene encoding the CDK inhibitor p21. This accelerates G1/S transition, triggering illegitimate origin firing and DNA damage. In addition, ß-catenin activation accelerates replication fork progression in BRCA2-deficient cells, which is critically dependent on p21 downregulation. Importantly, we find that upregulated p21 expression is essential for the survival of BRCA2-deficient cells and tumours. Thus, our work demonstrates that ß-catenin toxicity in cancer cells with compromised BRCA1/2 function is driven by transcriptional alterations that cause aberrant replication and inflict DNA damage.

Seasonal Evaluation of Phlorotannin-Enriched Extracts from Brown Macroalgae Fucus spiralis.

Fucus spiralis that was collected in the four seasons was submitted to an extraction with ethanol:water (crude extracts Et80), followed by a liquid-liquid fractionation with organic solvents (fraction He from n-hexane; aqueous fractions AQ1, AQ2, AQ3 and AQ4; ethyl acetate fraction EA), with the aim of obtaining phlorotannin-enriched extracts. All the extracts (Et80, He, AQ1, AQ2, AQ3, AQ4 and EA) that were obtained for the F. spiralis of the four seasons were evaluated for their antioxidant capacity and total phenolic compounds. The summer extracts presented the highest contents in polyphenols (TPC), as well as the highest ferric reducing antioxidant power (FRAP), when compared to the samples from the other seasons. The reductive percentage of the DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) compound was similar between the seasons. For all the seasons, the EA extract showed the highest polyphenol content (TPC), and the highest antioxidant capacity (highest ferric reducing power (FRAP) and lowest concentration needed to reduce 50% of the DPPH compound), which is in agreement with a phlorotannin-enriched fraction. This study revealed that the polyphenol content and antioxidant power of the F. spiralis extracts are influenced by the time of harvest, as well as by the solvents used for their extraction.

The human nucleoporin Tpr protects cells from RNA-mediated replication stress.

Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery.

The SWI/SNF chromatin remodeling complex helps resolve R-loop-mediated transcription-replication conflicts.

ATP-dependent chromatin remodelers are commonly mutated in human cancer. Mammalian SWI/SNF complexes comprise three conserved multisubunit chromatin remodelers (cBAF, ncBAF and PBAF) that share the BRG1 (also known as SMARCA4) subunit responsible for the main ATPase activity. BRG1 is the most frequently mutated Snf2-like ATPase in cancer. In the present study, we have investigated the role of SWI/SNF in genome instability, a hallmark of cancer cells, given its role in transcription, DNA replication and DNA-damage repair. We show that depletion of BRG1 increases R-loops and R-loop-dependent DNA breaks, as well as transcription-replication (T-R) conflicts. BRG1 colocalizes with R-loops and replication fork blocks, as determined by FANCD2 foci, with BRG1 depletion being epistatic to FANCD2 silencing. Our study, extended to other components of SWI/SNF, uncovers a key role of the SWI/SNF complex, in particular cBAF, in helping resolve R-loop-mediated T-R conflicts, thus, unveiling a new mechanism by which chromatin remodeling protects genome integrity.

BRCA2 promotes DNA-RNA hybrid resolution by DDX5 helicase at DNA breaks to facilitate their repair‡.

The BRCA2 tumor suppressor is a DNA double-strand break (DSB) repair factor essential for maintaining genome integrity. BRCA2-deficient cells spontaneously accumulate DNA-RNA hybrids, a known source of genome instability. However, the specific role of BRCA2 on these structures remains poorly understood. Here we identified the DEAD-box RNA helicase DDX5 as a BRCA2-interacting protein. DDX5 associates with DNA-RNA hybrids that form in the vicinity of DSBs, and this association is enhanced by BRCA2. Notably, BRCA2 stimulates the DNA-RNA hybrid-unwinding activity of DDX5 helicase. An impaired BRCA2-DDX5 interaction, as observed in cells expressing the breast cancer variant BRCA2-T207A, reduces the association of DDX5 with DNA-RNA hybrids, decreases the number of RPA foci, and alters the kinetics of appearance of RAD51 foci upon irradiation. Our findings are consistent with DNA-RNA hybrids constituting an impediment for the repair of DSBs by homologous recombination and reveal BRCA2 and DDX5 as active players in their removal.

Detection of DNA Double-Strand Breaks by γ-H2AX Immunodetection.

DNA double-strand breaks (DSBs) are the most deleterious type of DNA damage and a cause of genetic instability as they can lead to mutations, genome rearrangements, or loss of genetic material when not properly repaired. Eukaryotes from budding yeast to mammalian cells respond to the formation of DSBs with the immediate phosphorylation of a histone H2A isoform. The modified histone, phosphorylated in serine 139 in mammals (S129 in yeast), is named γ-H2AX. Detection of DSBs is of high relevance in research on DNA repair, aging, tumorigenesis, and cancer drug development, given the tight association of DSBs with different diseases and its potential to kill cells. DSB levels can be obtained by measuring levels of γ-H2AX in extracts of cell populations or by counting foci in individual nuclei. In this chapter some techniques to detect γ-H2AX are described.

Exploring the cytotoxic activity of new phenanthroline salicylaldimine Zn(II) complexes.

Zinc(II) complexes bearing N-salicylideneglycinate (Sal-Gly) and 1,10-phenanthroline (phen) or phenanthroline derivatives [NN = 5-chloro-1,10-phenanthroline, 5-amine-1,10-phenanthroline (amphen), 4,7-diphenyl-1,10-phenanthroline (Bphen) and 5,6-epoxy-5,6-dihydro-1,10-phenanthroline] are synthesized. Complexes formulated as [Zn(NN)2(H2O)2]2+(NN = phen and amphen), are also prepared. The cytotoxicity of the compounds is evaluated towards a panel of human cancer cells: ovarian (A2780), breast (MCF7) and cervical (HeLa), as well as non-tumoral V79 fibroblasts. All compounds display higher cytotoxicity than cisplatin (IC50 = 22.5 ±â€¯5.0 µM) towards ovarian cells, showing IC50values in the low micromolar range. Overall, all compounds show higher selectivity for the A2780 cells than for the non-tumoral cells and higher selectivity indexes (IC50(V79)/IC50(A2780) than cisplatin. [Zn(Sal-Gly)(NN)(H2O)] complexes induce caspase-dependent apoptosis in A2780 cells, except [Zn(Sal-Gly)(Bphen)(H2O)], one of the most cytotoxic of the series. The cellular uptake in the ovarian cells analyzed by Inductively Coupled Plasma mass spectrometry indicates different Zn distribution profiles. Transmission electronic microscopy shows mitochondria alterations and apoptotic features consistent with caspase activation; cells incubated with [Zn(Sal-Gly)(amphen)(H2O)] present additional nuclear membrane alterations in agreement with significant association with the nucleus. The increase of reactive oxygen species and lipid peroxidation forms could be related to apoptosis induction. [Zn(NN)2(H2O)2]2+complexes have high ability to bind DNA through intercalation/groove binding, and circular dichroism data suggests that the main type of species that interact with DNA is [Zn(NN)]2+. Studies varying the % of fetal bovine serum (1-15%) in cell media show that albumin binding decreases the complex activity, indicating that distinct speciation of Zn- and phen-containing species in cell media may affect the cytotoxicity.

The Antitumor Drugs Trabectedin and Lurbinectedin Induce Transcription-Dependent Replication Stress and Genome Instability.

R-loops are a major source of replication stress, DNA damage, and genome instability, which are major hallmarks of cancer cells. Accordingly, growing evidence suggests that R-loops may also be related to cancer. Here we show that R-loops play an important role in the cellular response to trabectedin (ET743), an anticancer drug from marine origin and its derivative lurbinectedin (PM01183). Trabectedin and lurbinectedin induced RNA-DNA hybrid-dependent DNA damage in HeLa cells, causing replication impairment and genome instability. We also show that high levels of R-loops increase cell sensitivity to trabectedin. In addition, trabectedin led to transcription-dependent FANCD2 foci accumulation, which was suppressed by RNase H1 overexpression. In yeast, trabectedin and lurbinectedin increased the presence of Rad52 foci, a marker of DNA damage, in an R-loop-dependent manner. In addition to providing new insights into the mechanisms of action of these drugs, our study reveals that R-loops could be targeted by anticancer agents. Given the increasing evidence that R-loops occur all over the genome, the ability of lurbinectedin and trabectedin to act on them may contribute to enhance their efficacy, opening the possibility that R-loops might be a feature shared by specific cancers. IMPLICATIONS: The data presented in this study provide the new concept that R-loops are important cellular factors that contribute to trabectedin and lurbinectedin anticancer activity.

Epigenetic features of human telomeres.

Although subtelomeric regions in humans are heterochromatic, the epigenetic nature of human telomeres remains controversial. This controversy might have been influenced by the confounding effect of subtelomeric regions and interstitial telomeric sequences (ITSs) on telomeric chromatin structure analyses. In addition, different human cell lines might carry diverse epigenetic marks at telomeres. We have developed a reliable procedure to study the chromatin structure of human telomeres independently of subtelomeres and ITSs. This procedure is based on the statistical analysis of multiple ChIP-seq experiments. We have found that human telomeres are not enriched in the heterochromatic H3K9me3 mark in most of the common laboratory cell lines, including embryonic stem cells. Instead, they are labeled with H4K20me1 and H3K27ac, which might be established by p300. These results together with previously published data argue that subtelomeric heterochromatin might control human telomere functions. Interestingly, U2OS cells that exhibit alternative lengthening of telomeres have heterochromatic levels of H3K9me3 in their telomeres.

Detection of DNA-RNA Hybrids In Vivo.

DNA-RNA hybrids form naturally during essential cellular functions such as transcription and replication. However, they may be an important source of genome instability, a hallmark of cancer and genetic diseases. Detection of DNA-RNA hybrids in cells is becoming crucial to understand an increasing number of molecular biology processes in genome dynamics and function and to identify new factors and mechanisms responsible for disease in biomedical research. Here, we describe two different procedures for the reliable detection of DNA-RNA hybrids in the yeast Saccharomyces cerevisiae and in human cells: DNA-RNA Immunoprecipitation (DRIP) and Immunofluorescence.

Roles of human POLD1 and POLD3 in genome stability.

DNA replication is essential for cellular proliferation. If improperly controlled it can constitute a major source of genome instability, frequently associated with cancer and aging. POLD1 is the catalytic subunit and POLD3 is an accessory subunit of the replicative Pol δ polymerase, which also functions in DNA repair, as well as the translesion synthesis polymerase Pol ζ, whose catalytic subunit is REV3L. In cells depleted of POLD1 or POLD3 we found a differential but general increase in genome instability as manifested by DNA breaks, S-phase progression impairment and chromosome abnormalities. Importantly, we showed that both proteins are needed to maintain the proper amount of active replication origins and that POLD3-depletion causes anaphase bridges accumulation. In addition, POLD3-associated DNA damage showed to be dependent on RNA-DNA hybrids pointing toward an additional and specific role of this subunit in genome stability. Interestingly, a similar increase in RNA-DNA hybrids-dependent genome instability was observed in REV3L-depleted cells. Our findings demonstrate a key role of POLD1 and POLD3 in genome stability and S-phase progression revealing RNA-DNA hybrids-dependent effects for POLD3 that might be partly due to its Pol ζ interaction.

Chelating bis-N-heterocyclic carbene complexes of iron(ii) containing bipyridyl ligands as catalyst precursors for oxidation of alcohols.

Chelating bis-N-heterocyclic carbene (bis-NHC) complexes of iron(ii) containing pyridyl ligands have been prepared by the reaction of [FeCl2L] [L = bipy (1), phen (2)] with [LiN(SiMe3)2] and a bis(imidazolium) salt. The [Fe(bis-NHC)L(I)2] complexes were active pre-catalysts in the oxidation of 1-phenylethanol with tert-butyl hydroperoxide in neat conditions, affording a quantitative yield of acetophenone in 4.5 h. The catalyst could be reused up to six cycles giving a turnover number (TON) of 1500. Various secondary alcohols, both aromatic and aliphatic were selectivity oxidised to the corresponding ketones in excellent yields. Compound 1 is stable in acetonitrile solution for ca. 4 h, although after 16 h, it evolves to a mixture of [Fe(bis-NHC)(bipy)2]I2 (3), [Fe(bipy)3](2+) and bis-imidazolium salt. The molecular structure of 3 has been determined by X-ray diffraction studies.

The Fanconi Anemia Pathway Protects Genome Integrity from R-loops.

Co-transcriptional RNA-DNA hybrids (R loops) cause genome instability. To prevent harmful R loop accumulation, cells have evolved specific eukaryotic factors, one being the BRCA2 double-strand break repair protein. As BRCA2 also protects stalled replication forks and is the FANCD1 member of the Fanconi Anemia (FA) pathway, we investigated the FA role in R loop-dependent genome instability. Using human and murine cells defective in FANCD2 or FANCA and primary bone marrow cells from FANCD2 deficient mice, we show that the FA pathway removes R loops, and that many DNA breaks accumulated in FA cells are R loop-dependent. Importantly, FANCD2 foci in untreated and MMC-treated cells are largely R loop dependent, suggesting that the FA functions at R loop-containing sites. We conclude that co-transcriptional R loops and R loop-mediated DNA damage greatly contribute to genome instability and that one major function of the FA pathway is to protect cells from R loops.

Synthesis, cytotoxic and hydrolytic studies of titanium complexes anchored by a tripodal diamine bis(phenolate) ligand.

The reactivity, cytotoxic studies and hydrolytic behaviour of diamine bis(phenolate) titanium complexes are reported. The reactions of [Ti((tBu2)O2NN')Cl]2(µ-O) (1) with LiO(I)Pr or HO(I)Pr in the presence of NEt3, aiming at the synthesis of the alkoxido derivative of 1 led to no reaction or to the synthesis of the monomeric complex [Ti((tBu2)O2NN')(O(i)Pr)2] (3), respectively. A small amount of the alkoxidotitanium dimer [Ti((tBu2)O2NN')(O(i)Pr)]2(µ-O) (2) crystallized out of a solution of 3 and DFT calculations showed that the transformation of 1 into 3 is a thermodynamically favorable process in the presence of a base (NEt3) (ΔG = -14.7 kcal mol(-1)). 2 was quantitatively obtained through the direct reaction of the ligand precursor H2((tBu2)O2NN') with titanium tetra(isopropoxido). Further reaction of 2 with an excess of TMSCl was revealed to be the most suitable method for the preparation of [Ti((tBu2)O2NN')Cl2] (4). 1 and 3 disclosed cytotoxic activity towards HeLa, Fem-x, MDA-MB-361 and K562 cells and 1 exhibited moderate binding affinity to FS-DNA. (1)H NMR hydrolysis studies attested the fast decomposition of 4 in the presence of D2O. The hydrolysis of 3 is slower and proceeds through the formation of [Ti((tBu2)O2NN')(OH)]2(µ-O) (5) that was crystallographically characterized. Upon D2O addition 1 immediately forms complex new species, stable in solution for long periods (weeks).

BRCA2 prevents R-loop accumulation and associates with TREX-2 mRNA export factor PCID2.

Genome instability is central to ageing, cancer and other diseases. It is not only proteins involved in DNA replication or the DNA damage response (DDR) that are important for maintaining genome integrity: from yeast to higher eukaryotes, mutations in genes involved in pre-mRNA splicing and in the biogenesis and export of messenger ribonucleoprotein (mRNP) also induce DNA damage and genome instability. This instability is frequently mediated by R-loops formed by DNA-RNA hybrids and a displaced single-stranded DNA. Here we show that the human TREX-2 complex, which is involved in mRNP biogenesis and export, prevents genome instability as determined by the accumulation of γ-H2AX (Ser-139 phosphorylated histone H2AX) and 53BP1 foci and single-cell electrophoresis in cells depleted of the TREX-2 subunits PCID2, GANP and DSS1. We show that the BRCA2 repair factor, which binds to DSS1, also associates with PCID2 in the cell. The use of an enhanced green fluorescent protein-tagged hybrid-binding domain of RNase H1 and the S9.6 antibody did not detect R-loops in TREX-2-depleted cells, but did detect the accumulation of R-loops in BRCA2-depleted cells. The results indicate that R-loops are frequently formed in cells and that BRCA2 is required for their processing. This link between BRCA2 and RNA-mediated genome instability indicates that R-loops may be a chief source of replication stress and cancer-associated instability.

Genome instability and transcription elongation impairment in human cells depleted of THO/TREX.

THO/TREX connects transcription with genome integrity in yeast, but a role of mammalian THO in these processes is uncertain, which suggests a differential implication of mRNP biogenesis factors in genome integrity in yeast and humans. We show that human THO depletion impairs transcription elongation and mRNA export and increases instability associated with DNA breaks, leading to hyper-recombination and γH2AX and 53BP1 foci accumulation. This is accompanied by replication alteration as determined by DNA combing. Genome instability is R-loop-dependent, as deduced from the ability of the AID enzyme to increase DNA damage and of RNaseH to reduce it, or from the enhancement of R-loop-dependent class-switching caused by THOC1-depletion in CH12 murine cells. Therefore, mammalian THO prevents R-loop formation and has a role in genome dynamics and function consistent with an evolutionary conservation of the functional connection between these mRNP biogenesis factors and genome integrity that had not been anticipated.