Fragile Sites Research Articles

Disrupted cellulose aggregation leads to the reduced mechanical performance of wood–adhesive interphase during freeze–thaw cycles

The mechanical performance of engineered wooden products facing the freeze–thaw cycles (FTCs) arises as an attention-worthy issue since the application of timber architectures in cold climates spreads. Here, we reported an investigation to reveal the losses of the mechanical performance of the wood-phenol formaldehyde (PF) adhesive interphase after the FTCs. Results revealed that PF adhesive was barely affected by the FTCs due to the low moisture content and rigid networks, whereas the mechanical properties of the cell wall in wood-PF interphase reduced significantly (more than 30%) after 5 FTCs at – 40 °C. Cracks were observed in the cell wall and compound middle lamella after FTCs. Further investigation into the crystal structure of the cell wall in the wood-PF interphase demonstrated that the FTCs disrupt the aggregations of cellulose macromolecules. The stresses caused by the phase transition of free water and the external hydrogen bonds formed between water and cellulose disrupted hydrogen bond networks in the cell wall. A plausible mechanism for the FTCs reducing the mechanical properties of the wood-PF bonds can be concluded as the cracks and weakened cell walls crippled the structural integrity of the wood-PF interphase, making it a fragile and stress-concentrated site when subjected to load.

Plasma metabolites-based design of long-acting peptides and their anticancer evaluation

Antimicrobial peptides (AMPs) are generally small cationic amphipathic peptides, which are thought to be ideal antineoplastic agents, owing to their favorable selectivity to cancer cells and the ability to overcome drug-resistance. In this study, an anticancer AMP (Mastoparan (INLKALAALAKKIL-NH2)) was selected as the lead compound and a series of Mastoparan derivatives were designed. Preliminary studies verified that an analogue of Mastoparan, KM8 (KLLKINLKALAALAKKIL-NH2), exhibited prominent selective antitumor effects. Instead, it presents a significant defect of metabolic instability, with a half-life in plasma of only about 0.5 h. Metabolite profiling of KM8 was performed and indicated the structure 9AL10 in peptide sequence could be the fragile site for KM8. Thus, the Aib (unnatural amnio acid) was employed to substitute the 9Ala residue in KM8, and generating a long-acting KM8 derivative, namely KM8-Aib. Further investigations revealed KM8-Aib possessed higher metabolic stability, more potent anticancer activity in vitro &in vivo, and lower toxicity. Therefore, KM8-Aib is suggested be a potential antimalignant agent that worthy of more in-depth study.

Abstract A020: Structural variants in the pathogenesis of colorectal cancer: The elephant in the room

Abstract Background: Cancer is caused by somatic DNA alterations, comprising single/small nucleotide variants (SNVs), somatic copy number alterations (SCNAs) and chromosomal rearrangement structural variants (SVs). We previously demonstrated that SVs are recurrently identified in hundreds of genes and are highly prevalent in common fragile site genes, e.g., in MACROD2 in >40% of colorectal cancers (CRCs). However, computational methods that discriminate SV-driver from SV-passenger events are lacking and laboratory methods to detect SVs at nucleotide resolution from routinely obtained formalin-fixed paraffin-embedded (FFPE) tumor tissue material are underdeveloped. Therefore, despite the abundant presence of SVs, knowledge about their biological and clinical impact is limited. Aim: The aim of our studies is to identify genes of which the function is frequently affected by SV, to understand how these genes contribute to CRC pathogenesis, and to translate these SVs into clinically relevant biomarkers. Methods: We made use of publicly available deep whole genome DNA sequencing data and tumor-matched RNA sequencing data from the Hartwig Medical Foundation to develop the algorithm ‘CoBRA’: Computation of Biologically Relevant Alterations. Adenoma-derived organoids were used for CRISPR/Cas9-mediated gene modulation for functional analysis of SV-driver events. Cergentis’ targeted locus capture (FFPE-TLC) technology was used to detect SVs at nucleotide resolution from FFPE material, which were translated into droplet digital PCR (ddPCR) assays for the detection of SVs in cell-free circulating tumor DNA (ctDNA) in liquid biopsies. Results: The CoBRA algorithm associated the presence of SV-events in frequently affected genes to the extent in which genome-wide RNA sequencing data were altered. In this way, CoBRA ranked SV-events in genes according to their putative impact on tumor biology. SVs in MACROD2 ranked among those with the highest impact on tumor biology. Therefore, we generated focal deletions in MACROD2 in adenoma-derived organoids for functional analyses. Moreover, using FFPE tumor tissue material we detected SVs at nucleotide resolution in MACROD2 and three other genes in 21 out of 29 patients. SVs were verified by PCR on tumor tissue and subsequently translated into ddPCR biomarker assays for detection of SVs in ctDNA in blood from the same patients. Conclusions: We developed the computational method CoBRA and succeeded to detect SVs with high impact on tumor biology. These SVs are prioritized for functional analysis in pre-malignant adenoma-derived organoids; for targeted detection in routinely obtained FFPE tumor tissue material; and for translation into liquid biopsy ctDNA assays. Proof of concept was delivered for MACROD2. Our novel computational and laboratory methodologies provide valuable tools to effectively explore the biological and clinical impact of SVs, which will contribute to our understanding of these common recurrent somatic alterations in CRC and their translation into clinically relevant biomarker applications. Citation Format: Elise van Bree, Carmen Rubio Alarcón, Soufyan Lakbir, Ellen Stelloo, Caterina Buranelli, Amber Hondema, Iris van 't Erve, Daan Vessies, Pien Delis-van Diemen, Marianne Tijssen, Anne Bolijn, Mirthe Lanfermeijer, Dorothe Linders, Joost Swennenhuis, Daan van den Broek, Jaap Heringa, Gerrit Meijer, Beatriz Carvalho, Harma Feitsma, Sanne Abeln, Remond J. A. Fijneman. Structural variants in the pathogenesis of colorectal cancer: The elephant in the room [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr A020.

Epigenomic signatures associated with spontaneous and replication stress-induced DNA double strand breaks.

Common fragile sites (CFSs) are specific regions of all individuals' genome that are predisposed to DNA double strand breaks (DSBs) and undergo subsequent rearrangements. CFS formation can be induced in vitro by mild level of DNA replication stress, such as DNA polymerase inhibition or nucleotide pool disturbance. The mechanisms of CFS formation have been linked to DNA replication timing control, transcription activities, as well as chromatin organization. However, it is unclear what specific cis- or trans-factors regulate the interplay between replication and transcription that determine CFS formation. We recently reported genome-wide mapping of DNA DSBs under replication stress induced by aphidicolin in human lymphoblastoids for the first time. Here, we systematically compared these DSBs with regards to nearby epigenomic features mapped in the same cell line from published studies. We demonstrate that aphidicolin-induced DSBs are strongly correlated with histone 3 lysine 36 trimethylation, a marker for active transcription. We further demonstrate that this DSB signature is a composite effect by the dual treatment of aphidicolin and its solvent, dimethylsulfoxide, the latter of which potently induces transcription on its own. We also present complementing evidence for the association between DSBs and 3D chromosome architectural domains with high density gene cluster and active transcription. Additionally, we show that while DSBs were detected at all but one of the fourteen finely mapped CFSs, they were not enriched in the CFS core sequences and rather demarcated the CFS core region. Related to this point, DSB density was not higher in large genes of greater than 300kb, contrary to reported enrichment of CFS sites at these large genes. Finally, replication timing analyses demonstrate that the CFS core region contain initiation events, suggesting that altered replication dynamics are responsible for CFS formation in relatively higher level of replication stress.

Global landscape of replicative DNA polymerase usage in the human genome

The division of labour among DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequencing (Pu-seq) in HCT116 cells and mapped Polε and Polα usage genome wide. The polymerase usage profiles show Polε synthesises the leading strand and Polα contributes mainly to lagging strand synthesis. Combining the Polε and Polα profiles, we accurately predict the genome-wide pattern of fork directionality plus zones of replication initiation and termination. We confirm that transcriptional activity contributes to the pattern of initiation and termination and, by separately analysing the effect of transcription on co-directional and converging forks, demonstrate that coupled DNA synthesis of leading and lagging strands is compromised by transcription in both co-directional and convergent forks. Polymerase uncoupling is particularly evident in the vicinity of large genes, including the two most unstable common fragile sites, FRA3B and FRA3D, thus linking transcription-induced polymerase uncoupling to chromosomal instability. Together, our result demonstrated that Pu-seq in human cells provides a powerful and straightforward methodology to explore DNA polymerase usage and replication fork dynamics.

Fragile sites, chromosomal lesions, tandem repeats, and disease.

Expanded tandem repeat DNAs are associated with various unusual chromosomal lesions, despiralizations, multi-branched inter-chromosomal associations, and fragile sites. Fragile sites cytogenetically manifest as localized gaps or discontinuities in chromosome structure and are an important genetic, biological, and health-related phenomena. Common fragile sites (∼230), present in most individuals, are induced by aphidicolin and can be associated with cancer; of the 27 molecularly-mapped common sites, none are associated with a particular DNA sequence motif. Rare fragile sites ( 40 known), 5% of the population (may be as few as a single individual), can be associated with neurodevelopmental disease. All 10 molecularly-mapped folate-sensitive fragile sites, the largest category of rare fragile sites, are caused by gene-specific CGG/CCG tandem repeat expansions that are aberrantly CpG methylated and include FRAXA, FRAXE, FRAXF, FRA2A, FRA7A, FRA10A, FRA11A, FRA11B, FRA12A, and FRA16A. The minisatellite-associated rare fragile sites, FRA10B, FRA16B, can be induced by AT-rich DNA-ligands or nucleotide analogs. Despiralized lesions and multi-branched inter-chromosomal associations at the heterochromatic satellite repeats of chromosomes 1, 9, 16 are inducible by de-methylating agents like 5-azadeoxycytidine and can spontaneously arise in patients with ICF syndrome (Immunodeficiency Centromeric instability and Facial anomalies) with mutations in genes regulating DNA methylation. ICF individuals have hypomethylated satellites I-III, alpha-satellites, and subtelomeric repeats. Ribosomal repeats and subtelomeric D4Z4 megasatellites/macrosatellites, are associated with chromosome location, fragility, and disease. Telomere repeats can also assume fragile sites. Dietary deficiencies of folate or vitamin B12, or drug insults are associated with megaloblastic and/or pernicious anemia, that display chromosomes with fragile sites. The recent discovery of many new tandem repeat expansion loci, with varied repeat motifs, where motif lengths can range from mono-nucleotides to megabase units, could be the molecular cause of new fragile sites, or other chromosomal lesions. This review focuses on repeat-associated fragility, covering their induction, cytogenetics, epigenetics, cell type specificity, genetic instability (repeat instability, micronuclei, deletions/rearrangements, and sister chromatid exchange), unusual heritability, disease association, and penetrance. Understanding tandem repeat-associated chromosomal fragile sites provides insight to chromosome structure, genome packaging, genetic instability, and disease.

Sister chromatid exchanges induced by perturbed replication can form independently of BRCA1, BRCA2 and RAD51

Sister chromatid exchanges (SCEs) are products of joint DNA molecule resolution, and are considered to form through homologous recombination (HR). Indeed, SCE induction upon irradiation requires the canonical HR factors BRCA1, BRCA2 and RAD51. In contrast, replication-blocking agents, including PARP inhibitors, induce SCEs independently of BRCA1, BRCA2 and RAD51. PARP inhibitor-induced SCEs are enriched at difficult-to-replicate genomic regions, including common fragile sites (CFSs). PARP inhibitor-induced replication lesions are transmitted into mitosis, suggesting that SCEs can originate from mitotic processing of under-replicated DNA. Proteomics analysis reveals mitotic recruitment of DNA polymerase theta (POLQ) to synthetic DNA ends. POLQ inactivation results in reduced SCE numbers and severe chromosome fragmentation upon PARP inhibition in HR-deficient cells. Accordingly, analysis of CFSs in cancer genomes reveals frequent allelic deletions, flanked by signatures of POLQ-mediated repair. Combined, we show PARP inhibition generates under-replicated DNA, which is processed into SCEs during mitosis, independently of canonical HR factors.

CmirC: an integrated database of clustered miRNAs co-localized with copy number variations in cancer

Genomic rearrangements and copy number variations (CNVs) are the major regulators of clustered microRNAs (miRNAs) expression. Several clustered miRNAs are harbored in and around chromosome fragile sites (CFSs) and cancer-associated genomic hotspots. Aberrant expression of such clusters can lead to oncogenic or tumor suppressor activities. Here, we developed CmirC (Clustered miRNAs co-localized with CNVs), a comprehensive database of clustered miRNAs co-localized with CNV regions. The database consists of 481 clustered miRNAs co-localized with CNVs and their expression patterns in 35 cancer types of the TCGA. The portal also provides information on CFSs, miRNA cluster candidates, genomic coordinates, target gene networks, and gene functionality. The web portal is integrated with advanced tools such as JBrowse, NCBI-BLAST, GeneSCF, visNetwork, and NetworkD3 to help the researchers in data analysis, visualization, and browsing. This portal provides a promising avenue for integrated data analytics and offers additional evidence for the complex regulation of clustered miRNAs in cancer. The web portal is freely accessible at http://slsdb.manipal.edu/cmirclust to explore clinically significant miRNAs.

Replication stress generates distinctive landscapes of DNA copy number alterations and chromosome scale losses

BackgroundA major driver of cancer chromosomal instability is replication stress, the slowing or stalling of DNA replication. How replication stress and genomic instability are connected is not known. Aphidicolin-induced replication stress induces breakages at common fragile sites, but the exact causes of fragility are debated, and acute genomic consequences of replication stress are not fully explored.ResultsWe characterize DNA copy number alterations (CNAs) in single, diploid non-transformed cells, caused by one cell cycle in the presence of either aphidicolin or hydroxyurea. Multiple types of CNAs are generated, associated with different genomic regions and features, and observed copy number landscapes are distinct between aphidicolin and hydroxyurea-induced replication stress. Coupling cell type-specific analysis of CNAs to gene expression and single-cell replication timing analyses pinpointed the causative large genes of the most recurrent chromosome-scale CNAs in aphidicolin. These are clustered on chromosome 7 in RPE1 epithelial cells but chromosome 1 in BJ fibroblasts. Chromosome arm level CNAs also generate acentric lagging chromatin and micronuclei containing these chromosomes.ConclusionsChromosomal instability driven by replication stress occurs via focal CNAs and chromosome arm scale changes, with the latter confined to a very small subset of chromosome regions, potentially heavily skewing cancer genome evolution. Different inducers of replication stress lead to distinctive CNA landscapes providing the opportunity to derive copy number signatures of specific replication stress mechanisms. Single-cell CNA analysis thus reveals the impact of replication stress on the genome, providing insights into the molecular mechanisms which fuel chromosomal instability in cancer.

Judgment, Doubt, and Self-Doubt: A Reflexive Turn from the Borderland of Lampedusa

Abstract As borders kill under the premises of saving lives and distributing order, border regimes emerge in everyday life as sites of uncertainty, fragility, and doubt. The paper explores the spaces of uncertainty that pervade everyday life at the borderlands of Europe with a particular focus on the island of Lampedusa (Italy), and it deploys doubt as an analytical tool to produce a reflexive turn in border studies. From an autoethnographic perspective, it calls for a re-evaluation of the directions we take as scholars when we think and write about borders. By exploring the potential of selfreflection as a path for knowledge of the "other", the paper suggests a shift in gaze which builds on judgment and self-dialogue as forms of responsibility.

Nonamer dependent RAG cleavage at CpGs can explain mechanism of chromosomal translocations associated to lymphoid cancers.

Chromosomal translocations are considered as one of the major causes of lymphoid cancers. RAG complex, which is responsible for V(D)J recombination, can also cleave non-B DNA structures and cryptic RSSs in the genome leading to chromosomal translocations. The mechanism and factors regulating the illegitimate function of RAGs resulting in oncogenesis are largely unknown. Upon in silico analysis of 3760 chromosomal translocations from lymphoid cancer patients, we find that 93% of the translocation breakpoints possess adjacent cryptic nonamers (RAG binding sequences), of which 77% had CpGs in proximity. As a proof of principle, we show that RAGs can efficiently bind to cryptic nonamers present at multiple fragile regions and cleave at adjacent mismatches generated to mimic the deamination of CpGs. ChIP studies reveal that RAGs can indeed recognize these fragile sites on a chromatin context inside the cell. Finally, we show that AID, the cytidine deaminase, plays a significant role during the generation of mismatches at CpGs and reconstitute the process of RAG-dependent generation of DNA breaks both in vitro and inside the cells. Thus, we propose a novel mechanism for generation of chromosomal translocation, where RAGs bind to the cryptic nonamer sequences and direct cleavage at adjacent mismatch generated due to deamination of meCpGs or cytosines.

Telomere length and chromosomal fragility increase in car painters exposed to organic solvents

ObjectiveTo comparing the chromosome fragility ratio and relative telomere length, of a group of car painters occupationally exposure to organic solvents with a group of non-exposed individuals in the city of Bogotá (Colombia). MethodsThis was a retrospective study of two cohorts (33 men each), matched by age (±2 years) at 1:1 ratio. The differences in the fragility ratio and relative telomere length distributions were determined for both groups. Case group was composed for adult males working in artisanal shops located in Bogotá (Colombia), who were occupationally exposed to organic solvents through automotive painting for a minimum of two years. Blood samples were analyzed. The chromosomal fragile sites were detected using cytogenetics techniques and DNA was extracted to determine the length of telomeres using qPCR (Cawthon’s method). Chromosomal fragility ratio and relative telomeric length were compared between car painters exposed to organic solvents and no-exposed individuals. ResultsStatistically significant differences in Chromosomal fragility ratio were found between exposed (0.645 ± 0.440, med = 0.520) and non-exposed (0.414 ± 0.217, med = 0.400) (p = 0.037, Wilcoxon one-tailed test). Also, in relative telomere length, in exposed (2.728 ± 5.581, med = 1.668) and non-exposed (1.835 ± 4.727, med = 0.732) individuals (p = 0.002, Wilcoxon one-tailed test). Low degree correlation for the two parameters, using Spearman's Rho, was found (0.21, p = 0.419). ConclusionsExposure to organic solvents increase both the telomere length and the rate of chromosomal sites fragile in the exposed group compared to the unexposed group. This study is a pioneer in the evaluation of the genotoxicity of exposure to organic solvents obtained the chromosomal fragility ratio and measuring the relative length of telomeres. We suggest applying this methodology to workers in other types of industries, for example carpentry shops, paint factories and stores where organic solvents are mixed and there is occupational exposure due to the inadequate use of protection measures or space conditioning.

Chromosomal Diversification in Pseudacanthicus Species (Loricariidae, Hypostominae) Revealed by Comparative Mapping of Repetitive Sequences.

Simple SummaryThe fishes of the Loricariidae family have a huge genetic diversity, mainly involving variations in the number and shape of chromosomes. The recognition of the species genus Pseudacanthicus is complex due to the large diversity of forms and limited knowledge of their genetic diversity. In this study, the karyotypes of three Pseudacanthicus species were comparatively analyzed using classical and molecular methods. They presented the same diploid number, but with different compositions of repetitive DNA sequences. Such information can be useful for the recognition of distinct species, in addition to providing important insights into the real biodiversity of this important group of Neotropical fish.Pseudacanthicus is a genus of Neotropical fish with eight valid species, in addition to numerous lineages not formally identified. It occurs along the Amazon and Tocantins River basins, in Suriname and in the Guiana shield. There are no karyotypic data in the literature for species of this genus. Here, the karyotypes of three Pseudacanthicus species (P. spinosus, P. leopardus and Pseudacanthicus sp.) were comparatively analyzed by classical cytogenetics and fluorescence in situ hybridization using 18S and 5S rDNA probes, U2 snDNA and telomeric sequences. The analyzed species presented 52 chromosomes and KF = 18 m + 34 sm. Constitutive heterochromatin occurred in blocks on a few chromosomes. The 18S rDNA occurred in a single pair; interestingly, P. leopardus presented only one locus of this sequence in its diploid genome. The 5S rDNA sequence occurred in only one pair in P. leopardus, and in multiple sites in Pseudacanthicus sp. and P. spinosus. The snDNA U2 occurred in only one pair in all analyzed species. Telomeric sequences did not show interstitial sites. Although Pseudacanthicus species share the same 2n and KF, repetitive sequence analysis revealed karyotypic diversity among these species. The occurrence of DNA double-strand breaks related to fragile sites, unequal crossing over and transpositions is proposed as the mechanism of karyotypic diversification, suggesting that the conservation of the karyotypic macrostructure is only apparent in this group of fish.

Peroxymonosulfate activation by Mg-introduced Fe-N carbon nanotubes to accelerate sulfamethoxazole degradation: Singlet oxygen-dominated nonradical pathway

Here, Mg-introduced Fe-N carbon nanotube catalysts (FeMg@NCNTs) were prepared for peroxymonosulfate (PMS). Experiments suggested that the introduction of Mg improved the removal of sulfamethoxazole (SMX) and found a transformation from the original radical pathway to a nonradical pathway dominated by singlet oxygen (1O2). XPS characterization demonstrated an enhanced pyridinic N content with the introduction of Mg. SMX degradation was not inhibited by pH, coexisting ions, or humic acid (HA). The free radical scavengers and electron paramagnetic resonance (EPR) indicate that the primary reactive substance was 1O2. Experimentally, 1O2 was proven to have a predominant effect on the degradation of SMX. The concentration of 1O2 increased 6.37 times after the introduction of Mg. Four decomposition pathways and three fragile sites for SMX were proposed by DFT calculations and HPLC-MS. The toxicity estimation software tool (T.E.S.T) analyzed the intermediates with lower ecotoxicity than SMX. The different contaminants (CIP, BPA, TBBPA, TCS, TC and Rh B) had a removal rate of more than 99% at 60 min except TC. The removal rates of SMX in tap water, river water and lake water were 100%, 96.6% and 93.1% in 40 min, respectively. The SMX degradation efficiency remained good after 5 cycles of the experiment. FeMg@NCNTs hydrogel catalyst continuous flow device exceeded 90% within 15 h for SMX removal efficiency and the Rh B is always running at 99% removal rate. In conclusion, this work provides a singlet oxygen-dominated non-radical pathway activating material and provides a new option for antibiotic wastewater degradation and practical applications.

Error-prone repair of stalled replication forks drives mutagenesis and loss of heterozygosity in haploinsufficient BRCA1 cells

Germline mutations in the BRCA genes are associated with a higher risk of carcinogenesis, which is linked to an increased mutation rate and loss of the second unaffected BRCA allele (loss of heterozygosity, LOH). However, the mechanisms triggering mutagenesis are not clearly understood. The BRCA genes contain high numbers of repetitive DNA sequences. We detected replication forks stalling, DNA breaks, and deletions at these sites in haploinsufficient BRCA cells, thus identifying the BRCA genes as fragile sites. Next, we found that stalled forks are repaired by error-prone pathways, such as microhomology-mediated break-induced replication (MMBIR) in haploinsufficient BRCA1 breast epithelial cells. We detected MMBIR mutations in BRCA1 tumor cells and noticed deletions-insertions (>50bp) at the BRCA1 genes in BRCA1 patients. Altogether, these results suggest that under stress, error-prone repair of stalled forks is upregulated and induces mutations, including complex genomic rearrangements at the BRCA genes (LOH), in haploinsufficient BRCA1 cells.

Different classes of genomic inserts contribute to human antibody diversity

Recombination of antibody genes in B cells can involve distant genomic loci and contribute a foreign antigen-binding element to form hybrid antibodies with broad reactivity for Plasmodium falciparum. So far, antibodies containing the extracellular domain of the LAIR1 and LILRB1 receptors represent unique examples of cross-chromosomal antibody diversification. Here, we devise a technique to profile non-VDJ elements from distant genes in antibody transcripts. Independent of the preexposure of donors to malaria parasites, non-VDJ inserts were detected in 80% of individuals at frequencies of 1 in 104 to 105 B cells. We detected insertions in heavy, but not in light chain or T cell receptor transcripts. We classify the insertions into four types depending on the insert origin and destination: 1) mitochondrial and 2) nuclear DNA inserts integrated at VDJ junctions; 3) inserts originating from telomere proximal genes; and 4) fragile sites incorporated between J-to-constant junctions. The latter class of inserts was exclusively found in memory and in invitro activated B cells, while all other classes were already detected in naïve B cells. More than 10% of inserts preserved the reading frame, including transcripts with signs of antigen-driven affinity maturation. Collectively, our study unravels a mechanism of antibody diversification that is layered on the classical V(D)J and switch recombination.

Mitotic DNA synthesis is caused by transcription-replication conflicts in BRCA2-deficient cells

Aberrant replication causes cells lacking BRCA2 to enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here, we identify genome-wide the sites where MiDAS reactions occur when BRCA2 is abrogated. High-resolution profiling revealed that these sites are different from MiDAS at aphidicolin-induced common fragile sites in that they map to genomic regions replicating in the early S-phase, which are close to early-firing replication origins, are highly transcribed, and display R-loop-forming potential. Both transcription inhibition in early S-phase and RNaseH1 overexpression reduced MiDAS in BRCA2-deficient cells, indicating that transcription-replication conflicts (TRCs) and R-loops are the source of MiDAS. Importantly, the MiDAS sites identified in BRCA2-deficient cells also represent hotspots for genomic rearrangements in BRCA2-mutated breast tumors. Thus, our work provides a mechanism for how tumor-predisposing BRCA2 inactivation links transcription-induced DNA damage with mitotic DNA repair to fuel the genomic instability characteristic of cancer cells.

074 ALK-positive desmoplastic Spitz naevus in a patient with corresponding ALK-positive anaplastic large cell lymphoma

Aim:Spitz naevus is a benign melanocytic tumour with distinct histopathological features of spindle-shaped melanocytes. An understanding of genetic aberrations can help to characterise the different subsets of Spitzoid neoplasms. Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor that was first described in the pathogenesis of anaplastic large cell lymphoma (ALCL). ALK mutations have been reported in up to 10% of Spitz tumours.This study describes the rare occurrence of an ALK mutation in two different diseases in the same patient – ALCL and Spitz naevus. Method:Presentation, management, and clinical outcomes were retrospectively reviewed for a patient with ALK-positive ALCL and ALK-positive Spitz naevus at a tertiary paediatric hospital. Result:The patient was a 14-year-old female with a 12-month history of a red-brown plaque on the right shoulder.This was on a background stage III ALCL treated with chemotherapy and stem cell transplantation. She underwent surgical excision of the skin lesion. Histopathological examination showed an ALK-positive desmoplastic Spitz naevus. A bone marrow aspirate and trephine (BMAT) of the right axillary lymph node at the time of ALCL diagnosis also revealed an ALK mutation. She is currently in remission 2 years post stem cell transplantation. Conclusion:Our case reports on a rare coincidence of a patient having the same mutation for two distinct disease processes. There has been one similarly reported case in the literature in a patient with ALK-positive cutaneous ALCL and Spitz naevi with the same fusion partner gene (TPM3). Whilst this occurrence is not fully understood, further studies may identify whether this is an independent coincidence in two different cell lineages, or a fragile site similarity which may predispose to the same translocation event.

Effects of environmental protection policies on fragile areas of a watershed occupied by agriculture in the Brazilian Cerrado

Brazil has become an antagonist in natural resource conservation policies in recent years. The Brazilian Cerrado is one of the biomes most affected by this setback due to the occupation of areas of economic interest in agriculture. Interestingly, there are projects throughout the biome that aim to encourage the preservation of natural resources, but there is uncertainty about whether these incentives have provided conservation. That is the case of the Feio River hydrographic basin, located in the Cerrado, near the city of Patrocínio, state of Minas Gerais. Indeed, the Feio River watershed has projects incentivizing the Cerrado's conservation. Thus, in this article, we aimed to explain how the advance of agriculture in fragile areas of the Feio river watershed occurs, their causes and whether protection strategies have produced satisfactory results. We used an old Environmental Fragility proposal methodwith our contributions to estimate the variations regarding nature conservation in the basin. Our findings show considerable heterogeneity in the potential fragility of the Feio's river basin. We observe that less fragile sites are used for coffee cultivation (74 and 72% of the two places), while more fragile sites are occupied by reserves (around 50% of the area) and high-impact activities such as traditional monoculture and cattle ranching (25 and 44% for the two sites). Temporally, we observed little change in use and occupation, with a slight increase in no-till farming to replace coffee and conventional cultivation. The permanent preservation areas in the basin have improved vegetation quality, while the reserve areas composed of fields show a degradation process. Considering the importance of the Feio's river basin, different incentives and inspection strategies are needed to guarantee the site's preservation and environmental restoration.

Role of transcribed ultraconserved regions in gastric cancer and therapeutic perspectives.

Gastric cancer (GC) is the fourth leading cause of cancer-related death. The occurrence and development of GC is a complex process involving multiple biological mechanisms. Although traditional regulation modulates molecular functions related to the occurrence and development of GC, the comprehensive mechanisms remain unclear. Ultraconserved region (UCR) refers to a genome sequence that is completely conserved in the homologous regions of the human, rat and mouse genomes, with 100% identity, without any insertions or deletions, and often located in fragile sites and tumour-related genes. The transcribed UCR (T-UCR) is transcribed from the UCR and is a new type of long noncoding RNA. Recent studies have found that the expression level of T-UCRs changes during the occurrence and development of GC, revealing a new mechanism underlying GC. Therefore, this article aims to review the relevant research on T-UCRs in GC, as well as the function of T-UCRs and their regulatory role in the occurrence and development of GC, to provide new strategies for GC diagnosis and treatment.