The European Green Deal outlines ambitions to build a more sustainable, climate neutral, and circular economy by 2050. To achieve this, the European Commission has published the Chemicals Strategy... Show moreThe European Green Deal outlines ambitions to build a more sustainable, climate neutral, and circular economy by 2050. To achieve this, the European Commission has published the Chemicals Strategy for Sustainability: Towards a Toxic-Free Environment, which provides targets for innovation to better protect human and environmental health, including challenges posed by hazardous chemicals and animal testing. The European project PATROLS (Physiologically Anchored Tools for Realistic nanOmateriaL hazard aSsessment) has addressed multiple aspects of the Chemicals Strategy for Sustainability by establishing a battery of new approach methodologies, including physiologically anchored human and environmental hazard assessment tools to evaluate the safety of engineered nanomaterials. PATROLS has delivered and improved innovative tools to support regulatory decision-making processes. These tools also support the need for reducing regulated vertebrate animal testing; when used at an early stage of the innovation pipeline, the PATROLS tools facilitate the safe and sustainable development of new nano-enabled products before they reach the market. Show less
Nucleotide excision repair (NER) is a DNA repair pathway present in all domains of life. In bacteria, UvrA protein localizes the DNA lesion, followed by verification by UvrB helicase and excision... Show moreNucleotide excision repair (NER) is a DNA repair pathway present in all domains of life. In bacteria, UvrA protein localizes the DNA lesion, followed by verification by UvrB helicase and excision by UvrC double nuclease. UvrA senses deformations and flexibility of the DNA duplex without precisely localizing the lesion in the damaged strand, an element essential for proper NER. Using a combination of techniques, we elucidate the mechanism of the damage verification step in bacterial NER. UvrA dimer recruits two UvrB molecules to its two sides. Each of the two UvrB molecules clamps a different DNA strand using its β-hairpin element. Both UvrB molecules then translocate to the lesion, and UvrA dissociates. The UvrB molecule that clamps the damaged strand gets stalled at the lesion to recruit UvrC. This mechanism allows UvrB to verify the DNA damage and identify its precise location triggering subsequent steps in the NER pathway. Show less
Wilson, M.D.; Renault, L.L.R.; Maskell, D.P.; Ghoneim, M.; Pye, V.E.; Nans, A.; ... ; Costa, A. 2019
Retroviral integrase can efficiently utilise nucleosomes for insertion of the reverse-transcribed viral DNA. In face of the structural constraints imposed by the nucleosomal structure, integrase... Show moreRetroviral integrase can efficiently utilise nucleosomes for insertion of the reverse-transcribed viral DNA. In face of the structural constraints imposed by the nucleosomal structure, integrase gains access to the scissile phosphodiester bonds by lifting DNA off the histone octamer at the site of integration. To clarify the mechanism of DNA looping by integrase, we determined a 3.9 Å resolution structure of the prototype foamy virus intasome engaged with a nucleosome core particle. The structural data along with complementary single-molecule Förster resonance energy transfer measurements reveal twisting and sliding of the nucleosomal DNA arm proximal to the integration site. Sliding the nucleosomal DNA by approximately two base pairs along the histone octamer accommodates the necessary DNA lifting from the histone H2A-H2B subunits to allow engagement with the intasome. Thus, retroviral integration into nucleosomes involves the looping-and-sliding mechanism for nucleosomal DNA repositioning, bearing unexpected similarities to chromatin remodelers. Show less
Balbi, C.; Lodder, K.; Costa, A.; Moimas, S.; Moccia, F.; Herwaarden, T. van; ... ; Bollini, S. 2019
The data and information presented here refer to the research article entitled: "Reactivating endogenous mechanisms of cardiac regeneration via paracrine boosting with the human amniotic fluid stem... Show moreThe data and information presented here refer to the research article entitled: "Reactivating endogenous mechanisms of cardiac regeneration via paracrine boosting with the human amniotic fluid stem cell secretome" (Balbi et al., 2019, Apr 04). This dataset illustrates the in vitro paracrine effect exerted by the human amniotic fluid stem cell secretome on rodent neonatal cardiomyocytes, human endothelial progenitors and different subsets of cardiac progenitor cells. Cytokine/chemokine profiling of the human amniotic fluid stem cell secretome is provided as well. This data can provide useful insights in regenerative medicine as demonstrating the in vitro cardioprotective and proliferative secretory paracrine potential of human fetal stem cells. (c) 2019 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Show less
Balbi, C.; Lodder, K.; Costa, A.; Moimas, S.; Moccia, F.; Herwaarden, T. van; ... ; Bollini, S. 2019
Background: The adult mammalian heart retains residual regenerative capability via endogenous cardiac progenitor cell (CPC) activation and cardiomyocyte proliferation. We previously reported the... Show moreBackground: The adult mammalian heart retains residual regenerative capability via endogenous cardiac progenitor cell (CPC) activation and cardiomyocyte proliferation. We previously reported the paracrine cardioprotective capacity of human amniotic fluid-derived stem cells (hAFS) following ischemia or cardiotoxicity. Here we analyse the potential of hAFS secretome fractions for cardiac regeneration and future clinical translation.Methods: hAFS were isolated from amniotic fluid leftover samples from prenatal screening. hAFS conditioned medium (hAFS-CM) was obtained following hypoxic preconditioning. Anti-apoptotic, angiogenic and proliferative effects were evaluated on rodent neonatal cardiomyocytes (r/mNVCM), human endothelial colony forming cells (hECFC) and human CPC. Mice undergoing myocardial infarction (MI) were treated with hAFS-CM, hAFS-extracellular vesicles (hAFS-EV), or EV-depleted hAFS-CM(hAFS-DM) by single intra-myocardial administration and evaluated in the short and long term.Results: hAFS-CM improved mNVCM survival under oxidative and hypoxic damage, induced Ca2+-dependent angiogenesis in hECFC and triggered hCPC and rNVCM proliferation. hAFS-CM treatment after MI counteracted scarring, supported cardiac function, angiogenesis and cardiomyocyte cell cycle progression in the long term. hAFS-DM had no effect. hAFS-CM and hAFS-EV equally induced epicardium WT1+CPC reactivation. Although no CPC cardiovascular differentiation was observed, our data suggests contribution to local angiogenesis by paracrine modulation. hAFS-EV alone were able to recapitulate all the beneficial effects exerted by hAFS-CM, except for stimulation of vessel formation.Conclusions: hAFS-CM and hAFS-EV can improve cardiac repair and trigger cardiac regeneration via paracrine modulation of endogenous mechanisms. While both formulations are effective in sustaining myocardial renewal, hAFS-CM retains higher pro-angiogenic potential, while hAFS-EV particularly enhances cardiac function. (c) 2019 Elsevier B.V. All rights reserved. Show less
Wouters, M.W.; Michielin, O.; Bastiaannet, E.; Beishon, M.; Catalano, O.; Marmol, V.; ... ; Naredi, P. 2018
ORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM... Show moreORC, Cdc6 and Cdt1 act together to load hexameric MCM, the motor of the eukaryotic replicative helicase, into double hexamers at replication origins. Here we show that Cdt1 interacts with MCM subunits Mcm2, 4 and 6, which both destabilizes the Mcm2-5 interface and inhibits MCM ATPase activity. Using X-ray crystallography, we show that Cdt1 contains two winged-helix domains in the C-terminal half of the protein and a catalytically inactive dioxygenase-related N-terminal domain, which is important for MCM loading, but not for subsequent replication. We used these structures together with single-particle electron microscopy to generate three-dimensional models of MCM complexes. These show that Cdt1 stabilizes MCM in a left-handed spiral open at the Mcm2-5 gate. We propose that Cdt1 acts as a brace, holding MCM open for DNA entry and bound to ATP until ORC-Cdc6 triggers ATP hydrolysis by MCM, promoting both Cdt1 ejection and MCM ring closure. Show less
The replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45–MCM–GINS (CMG) helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly. The... Show moreThe replisome unwinds and synthesizes DNA for genome duplication. In eukaryotes, the Cdc45–MCM–GINS (CMG) helicase and the leading-strand polymerase, Pol epsilon, form a stable assembly. The mechanism for coupling DNA unwinding with synthesis is starting to be elucidated, however the architecture and dynamics of the replication fork remain only partially understood, preventing a molecular understanding of chromosome replication. To address this issue, we conducted a systematic single-particle EM study on multiple permutations of the reconstituted CMG–Pol epsilon assembly. Pol epsilon contains two flexibly tethered lobes. The noncatalytic lobe is anchored to the motor of the helicase, whereas the polymerization domain extends toward the side of the helicase. We observe two alternate configurations of the DNA synthesis domain in the CMG-bound Pol epsilon. We propose that this conformational switch might control DNA template engagement and release, modulating replisome progression. Show less
Beets, G.; Sebag-Montefiore, D.; Andritsch, E.; Arnold, D.; Beishon, M.; Crul, M.; ... ; Naredi, P. 2017
Background: ECCO essential requirements for quality cancer care (ERQCC) are checklists and explanations of organisation and actions that are necessary to give high-quality care to patients who have... Show moreBackground: ECCO essential requirements for quality cancer care (ERQCC) are checklists and explanations of organisation and actions that are necessary to give high-quality care to patients who have a specific tumour type. They are written by European experts representing all disciplines involved in cancer care.ERQCC papers give oncology teams, patients, policymakers and managers an overview of the elements needed in any healthcare system to provide high quality of care throughout the patient journey. References are made to clinical guidelines and other resources where appropriate, and the focus is on care in Europe.Sarcoma: essential requirements for quality careSarcomas - which can be classified into soft tissue and bone sarcomas - are rare, but all rare cancers make up more than 20% of cancers in Europe, and there are substantial inequalities in access to high-quality care. Sarcomas, of which there are many subtypes, comprise a particularly complex and demanding challenge for healthcare systems and providers. This paper presents essential requirements for quality cancer care of soft tissue sarcomas in adults and bone sarcomas.High-quality care must only be carried out in specialised sarcoma centres (including paediatric cancer centres) which have both a core multidisciplinary team and an extended team of allied professionals, and which are subject to quality and audit procedures. Access to such units is far from universal in all European countries.It is essential that, to meet European aspirations for high-quality comprehensive cancer control, healthcare organisations implement the requirements in this paper, paying particular attention to multidisciplinarity and patient-centred pathways from diagnosis and follow-up, to treatment, to improve survival and quality of life for patients.Conclusion: Taken together, the information presented in this paper provides a comprehensive description of the essential requirements for establishing a high-quality service for soft tissue sarcomas in adults and bone sarcomas. The ECCO expert group is aware that it is not possible to propose a 'one size fits all' system for all countries, but urges that access to multidisciplinary teams is guaranteed to all patients with sarcoma. (C) 2016 The Authors. Published by Elsevier Ireland Ltd. Show less
Swuec, P.; Renault, L.L.R.; Borg, A.; Shah, F.; Murphy, V.J.; Twest, S. van; ... ; Costa, A. 2017
Activation of the main DNA interstrand crosslink repair pathway in higher eukaryotes requires mono-ubiquitination of FANCI and FANCD2 by FANCL, the E3 ligase subunit of the Fanconi anemia core... Show moreActivation of the main DNA interstrand crosslink repair pathway in higher eukaryotes requires mono-ubiquitination of FANCI and FANCD2 by FANCL, the E3 ligase subunit of the Fanconi anemia core complex. FANCI and FANCD2 form a stable complex; however, the molecular basis of their ubiquitination is ill defined. FANCD2 mono-ubiquitination by FANCL is stimulated by the presence of the FANCB and FAAP100 core complex components, through an unknown mechanism. How FANCI mono-ubiquitination is achieved remains unclear. Here, we use structural electron microscopy, combined with crosslink-coupled mass spectrometry, to find that FANCB, FANCL, and FAAP100 form a dimer of trimers, containing two FANCL molecules that are ideally poised to target both FANCI and FANCD2 for mono-ubiquitination. The FANCC-FANCE-FANCF subunits bridge between FANCB-FANCL-FAAP100 and the FANCI-FANCD2 substrate. A transient interaction with FANCC-FANCE-FANCF alters the FANCI-FANCD2 configuration, stabilizing the dimerization interface. Our data provide a model to explain how equivalent mono-ubiquitination of FANCI and FANCD2 occurs. Show less
The Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function. Whether CMG translocation occurs on... Show moreThe Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function. Whether CMG translocation occurs on single- or double-stranded DNA and how ATP hydrolysis drives DNA unwinding remain open questions. Here we use cryo-electron microscopy to describe two subnanometre resolution structures of the CMG helicase trapped on a DNA fork. In the predominant state, the ring-shaped C-terminal ATPase of MCM is compact and contacts single-stranded DNA, via a set of pre-sensor 1 hairpins that spiral around the translocation substrate. In the second state, the ATPase module is relaxed and apparently substrate free, while DNA intimately contacts the downstream amino-terminal tier of the MCM motor ring. These results, supported by single-molecule FRET measurements, lead us to suggest a replication fork unwinding mechanism whereby the N-terminal and AAA+ tiers of the MCM work in concert to translocate on single-stranded DNA. Show less
Retroviral integration is catalysed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome. How the intasome interfaces with chromosomal DNA, which... Show moreRetroviral integration is catalysed by a tetramer of integrase (IN) assembled on viral DNA ends in a stable complex, known as the intasome. How the intasome interfaces with chromosomal DNA, which exists in the form of nucleosomal arrays, is currently unknown. Here we show that the prototype foamy virus (PFV) intasome is proficient at stable capture of nucleosomes as targets for integration. Single-particle cryo-electron microscopy reveals a multivalent intasome-nucleosome interface involving both gyres of nucleosomal DNA and one H2A-H2B heterodimer. While the histone octamer remains intact, the DNA is lifted from the surface of the H2A-H2B heterodimer to allow integration at strongly preferred superhelix location ±3.5 positions. Amino acid substitutions disrupting these contacts impinge on the ability of the intasome to engage nucleosomes in vitro and redistribute viral integration sites on the genomic scale. Our findings elucidate the molecular basis for nucleosome capture by the viral DNA recombination machinery and the underlying nucleosome plasticity that allows integration. Show less
Pike, A.C.; Gomathinayagam, S.; Swuec, P.; Berti, M.; Zhang, Y.; Schnecke, C.; ... ; Vindigni, A. 2015
RecQ helicases are a widely conserved family of ATP-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. However, the physical mechanisms by... Show moreRecQ helicases are a widely conserved family of ATP-dependent motors with diverse roles in nearly every aspect of bacterial and eukaryotic genome maintenance. However, the physical mechanisms by which RecQ helicases recognize and process specific DNA replication and repair intermediates are largely unknown. Here, we solved crystal structures of the human RECQ1 helicase in complexes with tailed-duplex DNA and ssDNA. The structures map the interactions of the ssDNA tail and the branch point along the helicase and Zn-binding domains, which, together with reported structures of other helicases, define the catalytic stages of helicase action. We also identify a strand-separating pin, which (uniquely in RECQ1) is buttressed by the protein dimer interface. A duplex DNA-binding surface on the C-terminal domain is shown to play a role in DNA unwinding, strand annealing, and Holliday junction (HJ) branch migration. We have combined EM and analytical ultracentrifugation approaches to show that RECQ1 can form what appears to be a flat, homotetrameric complex and propose that RECQ1 tetramers are involved in HJ recognition. This tetrameric arrangement suggests a platform for coordinated activity at the advancing and receding duplexes of an HJ during branch migration. Show less
The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have... Show moreThe Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3′ single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase. Show less
