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1.  Novel and Lost Forests in the Upper Midwestern United States, from New Estimates of Settlement-Era Composition, Stem Density, and Biomass 
PLoS ONE  2016;11(12):e0151935.
Background
EuroAmerican land-use and its legacies have transformed forest structure and composition across the United States (US). More accurate reconstructions of historical states are critical to understanding the processes governing past, current, and future forest dynamics. Here we present new gridded (8x8km) reconstructions of pre-settlement (1800s) forest composition and structure from the upper Midwestern US (Minnesota, Wisconsin, and most of Michigan), using 19th Century Public Land Survey System (PLSS), with estimates of relative composition, above-ground biomass, stem density, and basal area for 28 tree types. This mapping is more robust than past efforts, using spatially varying correction factors to accommodate sampling design, azimuthal censoring, and biases in tree selection.
Changes in Forest Structure
We compare pre-settlement to modern forests using US Forest Service Forest Inventory and Analysis (FIA) data to show the prevalence of lost forests (pre-settlement forests with no current analog), and novel forests (modern forests with no past analogs). Differences between pre-settlement and modern forests are spatially structured owing to differences in land-use impacts and accompanying ecological responses. Modern forests are more homogeneous, and ecotonal gradients are more diffuse today than in the past. Novel forest assemblages represent 28% of all FIA cells, and 28% of pre-settlement forests no longer exist in a modern context. Lost forests include tamarack forests in northeastern Minnesota, hemlock and cedar dominated forests in north-central Wisconsin and along the Upper Peninsula of Michigan, and elm, oak, basswood and ironwood forests along the forest-prairie boundary in south central Minnesota and eastern Wisconsin. Novel FIA forest assemblages are distributed evenly across the region, but novelty shows a strong relationship to spatial distance from remnant forests in the upper Midwest, with novelty predicted at between 20 to 60km from remnants, depending on historical forest type. The spatial relationships between remnant and novel forests, shifts in ecotone structure and the loss of historic forest types point to significant challenges for land managers if landscape restoration is a priority. The spatial signals of novelty and ecological change also point to potential challenges in using modern spatial distributions of species and communities and their relationship to underlying geophysical and climatic attributes in understanding potential responses to changing climate. The signal of human settlement on modern forests is broad, spatially varying and acts to homogenize modern forests relative to their historic counterparts, with significant implications for future management.
doi:10.1371/journal.pone.0151935
PMCID: PMC5147790  PMID: 27935944
2.  Prelamin A impairs 53BP1 nuclear entry by mislocalizing NUP153 and disrupting the Ran gradient 
Aging Cell  2016;15(6):1039-1050.
Summary
The nuclear lamina is essential for the proper structure and organization of the nucleus. Deregulation of A‐type lamins can compromise genomic stability, alter chromatin organization and cause premature vascular aging. Here, we show that accumulation of the lamin A precursor, prelamin A, inhibits 53BP1 recruitment to sites of DNA damage and increases basal levels of DNA damage in aged vascular smooth muscle cells. We identify that this genome instability arises through defective nuclear import of 53BP1 as a consequence of abnormal topological arrangement of nucleoporin NUP153. We show for the first time that this nucleoporin is important for the nuclear localization of Ran and that the deregulated Ran gradient is likely to be compromising the nuclear import of 53BP1. Importantly, many of the defects associated with prelamin A expression were significantly reduced upon treatment with Remodelin, a small molecule recently reported to reverse deficiencies associated with abnormal nuclear lamina.
doi:10.1111/acel.12506
PMCID: PMC5114580  PMID: 27464478
53BP1; cytoplasmic–nuclear trafficking; NUP153; prelamin A; Ran gradient; vascular disease
3.  Different DNA End Configurations Dictate Which NHEJ Components Are Most Important for Joining Efficiency* 
The Journal of Biological Chemistry  2016;291(47):24377-24389.
The nonhomologous DNA end-joining (NHEJ) pathway is a key mechanism for repairing dsDNA breaks that occur often in eukaryotic cells. In the simplest model, these breaks are first recognized by Ku, which then interacts with other NHEJ proteins to improve their affinity at DNA ends. These include DNA-PKcs and Artemis for trimming the DNA ends; DNA polymerase μ and λ to add nucleotides; and the DNA ligase IV complex to ligate the ends with the additional factors, XRCC4 (X-ray repair cross-complementing protein 4), XLF (XRCC4-like factor/Cernunos), and PAXX (paralog of XRCC4 and XLF). In vivo studies have demonstrated the degrees of importance of these NHEJ proteins in the mechanism of repair of dsDNA breaks, but interpretations can be confounded by other cellular processes. In vitro studies with NHEJ proteins have been performed to evaluate the nucleolytic resection, polymerization, and ligation steps, but a complete system has been elusive. Here we have developed a NHEJ reconstitution system that includes the nuclease, polymerase, and ligase components to evaluate relative NHEJ efficiency and analyze ligated junctional sequences for various types of DNA ends, including blunt, 5′ overhangs, and 3′ overhangs. We find that different dsDNA end structures have differential dependence on these enzymatic components. The dependence of some end joining on only Ku and XRCC4·DNA ligase IV allows us to formulate a physical model that incorporates nuclease and polymerase components as needed.
doi:10.1074/jbc.M116.752329
PMCID: PMC5114395  PMID: 27703001
chromosomes; DNA damage; DNA recombination; DNA repair; enzyme; double-strand DNA break; ligase; nuclease
4.  G9a inhibition potentiates the anti-tumour activity of DNA double-strand break inducing agents by impairing DNA repair independent of p53 status 
Cancer Letters  2016;380(2):467-475.
Highlights
•Cancer cell growth inhibition screen with epigenetic chemical probes and phleomycin.•G9a inhibitor UNC0638 hypersensitises tumour cells to DNA-damaging agents.•Under low-level damage, G9a inhibitor induces p53-independent tumour cell death.•G9a depletion induces tumour cell death by impairing DNA double-strand repair.•G9a promotes DSB repair by non-homologous end joining.
Cancer cells often exhibit altered epigenetic signatures that can misregulate genes involved in processes such as transcription, proliferation, apoptosis and DNA repair. As regulation of chromatin structure is crucial for DNA repair processes, and both DNA repair and epigenetic controls are deregulated in many cancers, we speculated that simultaneously targeting both might provide new opportunities for cancer therapy. Here, we describe a focused screen that profiled small-molecule inhibitors targeting epigenetic regulators in combination with DNA double-strand break (DSB) inducing agents. We identify UNC0638, a catalytic inhibitor of histone lysine N-methyl-transferase G9a, as hypersensitising tumour cells to low doses of DSB-inducing agents without affecting the growth of the non-tumorigenic cells tested. Similar effects are also observed with another, structurally distinct, G9a inhibitor A-366. We also show that small-molecule inhibition of G9a or siRNA-mediated G9a depletion induces tumour cell death under low DNA damage conditions by impairing DSB repair in a p53 independent manner. Furthermore, we establish that G9a promotes DNA non-homologous end-joining in response to DSB-inducing genotoxic stress. This study thus highlights the potential for using G9a inhibitors as anti-cancer therapeutic agents in combination with DSB-inducing chemotherapeutic drugs such as etoposide.
doi:10.1016/j.canlet.2016.07.009
PMCID: PMC5011428  PMID: 27431310
Cancer epigenetics; Chemical probes; UNC0638; Chemotherapeutics; Non-homologous end joining; DSB, double strand break; NHEJ, non-homologous end joining; SGC, Structural Genomics Consortium; DDR, DNA damage response; IC50, inhibitory concentration 50%; WT, wild-type; KO, knockout; PI, propidium iodide; XRCC4, X-ray repair cross-complementing protein 4; ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3-related protein
5.  Coordinated nuclease activities counteract Ku at single-ended DNA double-strand breaks 
Nature Communications  2016;7:12889.
Repair of single-ended DNA double-strand breaks (seDSBs) by homologous recombination (HR) requires the generation of a 3′ single-strand DNA overhang by exonuclease activities in a process called DNA resection. However, it is anticipated that the highly abundant DNA end-binding protein Ku sequesters seDSBs and shields them from exonuclease activities. Despite pioneering works in yeast, it is unclear how mammalian cells counteract Ku at seDSBs to allow HR to proceed. Here we show that in human cells, ATM-dependent phosphorylation of CtIP and the epistatic and coordinated actions of MRE11 and CtIP nuclease activities are required to limit the stable loading of Ku on seDSBs. We also provide evidence for a hitherto unsuspected additional mechanism that contributes to prevent Ku accumulation at seDSBs, acting downstream of MRE11 endonuclease activity and in parallel with MRE11 exonuclease activity. Finally, we show that Ku persistence at seDSBs compromises Rad51 focus assembly but not DNA resection.
Homologous recombination requires end resection of the DNA at the site of the break, however the Ku dimer can sequester single-ended double-strand breaks. Here the authors show that ATM-dependent phosphorylation of CtIP, along with the actions of Mre11, impair the stable loading of Ku onto DNA.
doi:10.1038/ncomms12889
PMCID: PMC5031800  PMID: 27641979
6.  Specific Roles of XRCC4 Paralogs PAXX and XLF during V(D)J Recombination 
Cell Reports  2016;16(11):2967-2979.
Summary
Paralog of XRCC4 and XLF (PAXX) is a member of the XRCC4 superfamily and plays a role in nonhomologous end-joining (NHEJ), a DNA repair pathway critical for lymphocyte antigen receptor gene assembly. Here, we find that the functions of PAXX and XLF in V(D)J recombination are masked by redundant joining activities. Thus, combined PAXX and XLF deficiency leads to an inability to join RAG-cleaved DNA ends. Additionally, we demonstrate that PAXX function in V(D)J recombination depends on its interaction with Ku. Importantly, we show that, unlike XLF, the role of PAXX during the repair of DNA breaks does not overlap with ATM and the RAG complex. Our findings illuminate the role of PAXX in V(D)J recombination and support a model in which PAXX and XLF function during NHEJ repair of DNA breaks, whereas XLF, the RAG complex, and the ATM-dependent DNA damage response promote end joining by stabilizing DNA ends.
Graphical Abstract
Image 1
Highlights
•PAXX-deficient pro-B cells support normal V(D)J recombination•PAXX and XLF are mutually redundant in repairing RAG-DNA breaks•PAXX function in V(D)J recombination depends on its interaction with Ku•Unlike XLF, PAXX is not redundant with ATM and the RAG complex in repairing DNA breaks
Developing lymphocytes rely on nonhomologous end joining (NHEJ) to repair programmed DNA double-strand breaks generated during antigen receptor gene assembly. Lescale et al. show that PAXX—a component of the NHEJ machinery—has a key role in V(D)J recombination that is masked by functional redundancy with its paralog XLF.
doi:10.1016/j.celrep.2016.08.069
PMCID: PMC5033762  PMID: 27601299
V(D)J recombination; DNA repair; NHEJ; PAXX; XLF; XRCC4
7.  When two is not enough: a CtIP tetramer is required for DNA repair by Homologous Recombination 
Nucleus  2015;6(5):344-348.
Homologous recombination (HR) is central to the repair of double-strand DNA breaks that occur in S/G2 phases of the cell cycle. HR relies on the CtIP protein (Ctp1 in fission yeast, Sae2 in budding yeast) for resection of DNA ends, a key step in generating the 3′-DNA overhangs that are required for the HR strand-exchange reaction. Although much has been learned about the biological importance of CtIP in DNA repair, our mechanistic insight into its molecular functions remains incomplete. It has been recently discovered that CtIP and Ctp1 share a conserved tetrameric architecture that is mediated by their N-terminal domains and is critical for their function in HR. The specific arrangement of protein chains in the CtIP/Ctp1 tetramer indicates that an ability to bridge DNA ends might be an important feature of CtIP/Ctp1 function, establishing an intriguing similarity with the known ability of the MRE11-RAD50-NBS1 complex to link DNA ends. Although the exact mechanism of action remains to be elucidated, the remarkable evolutionary conservation of CtIP/Ctp1 tetramerisation clearly points to its crucial role in HR.
doi:10.1080/19491034.2015.1086050
PMCID: PMC4915501  PMID: 26305173
CtIP; DNA repair; DNA-end resection; double strand break; Homologous Recombination; tetramerisation
8.  Premonitory urges are associated with decreased grey matter thickness within the insula and sensorimotor cortex in young people with Tourette syndrome 
Journal of Neuropsychology  2015;10(1):143-153.
Tourette syndrome (TS) is a neurological disorder characterized by vocal and motor tics and is associated with cortical–striatal–thalamic–cortical circuit (CSTC) dysfunction and hyperexcitability of cortical limbic and motor regions, which are thought to lead to the occurrence of tics. Importantly, individuals with TS often report that their tics are preceded by ‘premonitory sensory phenomena’ (PSP) that are described as uncomfortable cognitive or bodily sensations that precede the execution of a tic, and are experienced as a strong urge for motor discharge. While the precise role played by PSP in the occurrence of tics is controversial, PSP are nonetheless of considerable theoretical and clinical importance in TS, not least because they form the core component in many of the behavioural therapies that are currently used in the treatment of tic disorders. In this study, we investigated the brain structure correlates of PSP. Specifically, we conducted a whole‐brain analysis of cortical (grey matter) thickness in 29 children and young adults with TS and investigated the association between grey matter thickness and PSP. We demonstrate for the first time that PSP are inversely associated with grey matter thickness measurements within the insula and sensorimotor cortex. We also demonstrate that grey matter thickness is significantly reduced in these areas in individuals with TS relative to a closely age‐ and gender‐matched group of typically developing individuals and that PSP ratings are significantly correlated with tic severity.
doi:10.1111/jnp.12089
PMCID: PMC4982075  PMID: 26538289
Tourette syndrome; cortical thickness; premonitory urges; premonitory sensory phenomena; magnetic resonance imaging; insula; sensorimotor cortex
9.  Managing Climate Change Refugia for Climate Adaptation 
PLoS ONE  2016;11(8):e0159909.
Refugia have long been studied from paleontological and biogeographical perspectives to understand how populations persisted during past periods of unfavorable climate. Recently, researchers have applied the idea to contemporary landscapes to identify climate change refugia, here defined as areas relatively buffered from contemporary climate change over time that enable persistence of valued physical, ecological, and socio-cultural resources. We differentiate historical and contemporary views, and characterize physical and ecological processes that create and maintain climate change refugia. We then delineate how refugia can fit into existing decision support frameworks for climate adaptation and describe seven steps for managing them. Finally, we identify challenges and opportunities for operationalizing the concept of climate change refugia. Managing climate change refugia can be an important option for conservation in the face of ongoing climate change.
doi:10.1371/journal.pone.0159909
PMCID: PMC4980047  PMID: 27509088
10.  Molecular Insights into Division of Single Human Cancer Cells in On-Chip Transparent Microtubes 
ACS Nano  2016;10(6):5835-5846.
In vivo, mammalian cells proliferate within 3D environments consisting of numerous microcavities and channels, which contain a variety of chemical and physical cues. External environments often differ between normal and pathological states, such as the unique spatial constraints that metastasizing cancer cells experience as they circulate the vasculature through arterioles and narrow capillaries, where they can divide and acquire elongated cylindrical shapes. While metastatic tumors cause most cancer deaths, factors impacting early cancer cell proliferation inside the vasculature and those that can promote the formation of secondary tumors remain largely unknown. Prior studies investigating confined mitosis have mainly used 2D cell culture systems. Here, we mimic aspects of metastasizing tumor cells dividing inside blood capillaries by investigating single-cell divisions of living human cancer cells, trapped inside 3D rolled-up, transparent nanomembranes. We assess the molecular effects of tubular confinement on key mitotic features, using optical high- and super-resolution microscopy. Our experiments show that tubular confinement affects the morphology and dynamics of the mitotic spindle, chromosome arrangements, and the organization of the cell cortex. Moreover, we reveal that membrane blebbing and/or associated processes act as a potential genome-safety mechanism, limiting the extent of genomic instability caused by mitosis in confined circumstances, especially in tubular 3D microenvironments. Collectively, our study demonstrates the potential of rolled-up nanomembranes for gaining molecular insights into key cellular events occurring in tubular 3D microenvironments in vivo.
doi:10.1021/acsnano.6b00461
PMCID: PMC4961266  PMID: 27267364
rolled-up nanofilms/membranes; mitosis; chromosome segregation; membrane blebbing; actin cortex; 3D cell culture; metastasis
11.  Drug Utilization and Inappropriate Prescribing in Centenarians 
Objectives
To use primary care electronic health records (EHRs) to evaluate prescriptions and inappropriate prescribing in men and women at age 100.
Design
Population‐based cohort study.
Setting
Primary care database in the United Kingdom, 1990 to 2013.
Participants
Individuals reaching the age of 100 between 1990 and 2013 (N = 11,084; n = 8,982 women, n = 2,102 men).
Measurements
Main drug classes prescribed and potentially inappropriate prescribing according to the 2012 American Geriatrics Society Beers Criteria.
Results
At the age of 100, 73% of individuals (79% of women, 54% of men) had received one or more prescription drugs, with a median of 7 (interquartile range 0–12) prescription items. The most frequently prescribed drug classes were cardiovascular (53%), central nervous system (CNS) (53%), and gastrointestinal (47%). Overall, 32% of participants (28% of men, 32% of women) who received drug prescriptions may have received one or more potentially inappropriate prescriptions, with temazepam and amitriptyline being the most frequent. CNS prescriptions were potentially inappropriate in 23% of individuals, and anticholinergic prescriptions were potentially inappropriate in 18% of individuals.
Conclusion
The majority of centenarians are prescribed one or more drug therapies, and the prescription may be inappropriate for up to one‐third of these individuals. Research using EHRs offers opportunities to understand prescribing trends and improve pharmacological care of the oldest adults.
doi:10.1111/jgs.14106
PMCID: PMC4950321  PMID: 27130965
centenarians; epidemiology; inappropriate prescribing; aging; primary care
12.  Diversity of Natural Product Biosynthetic Genes in the Microbiome of the Deep Sea Sponges Inflatella pellicula, Poecillastra compressa, and Stelletta normani 
Three different deep sea sponge species, Inflatella pellicula, Poecillastra compressa, and Stelletta normani comprising seven individual samples, retrieved from depths of 760–2900 m below sea level, were investigated using 454 pyrosequencing for their secondary metabolomic potential targeting adenylation domain and ketosynthase domain sequences. The data obtained suggest a diverse microbial origin of nonribosomal peptide synthetases and polyketide synthase fragments that in part correlates with their respective microbial community structures that were previously described and reveals an untapped source of potential novelty. The sequences, especially the ketosynthase fragments, display extensive clade formations which are clearly distinct from sequences hosted in public databases, therefore highlighting the potential of the microbiome of these deep sea sponges to produce potentially novel small-molecule chemistry. Furthermore, sequence similarities to gene clusters known to be involved in the production of many classes of antibiotics and toxins including lipopeptides, glycopeptides, macrolides, and hepatotoxins were also identified.
doi:10.3389/fmicb.2016.01027
PMCID: PMC4925706  PMID: 27446062
pyrosequencing; deep sea sponges; ketosynthase domain; adenylation domain; microbiome
13.  Longitudinal Trends in Hypertension Management and Mortality Among Octogenarians 
The role of hypertension management among octogenarians is controversial. In this long-term follow-up (>10 years) study, we estimated trends in hypertension prevalence, awareness, treatment, and control among octogenarians, and evaluated the relationship of systolic blood pressure (SBP) ranges with mortality. Data were based on the English Longitudinal Study of Ageing (ELSA). Outcome measures were hypertension prevalence, awareness, treatment and control, and cardiovascular disease, and all-cause mortality events. Participants were separated into 8 categories of SBP values (<110, 110–119, 120–129, 130–139, 140–149, 150–159, 160–169, and >169 mm Hg). Among 2692 octogenarians, mean SBP levels declined from 147 mm Hg in 1998/2000 to 134 mm Hg in 2012/2013. The decline was of lower magnitude in the 50 to 79 years old subgroup (n=22007). Hypertension prevalence and awareness were 40% and 13%, respectively, higher among octogenarians than the 50 to 79 years of age subgroup, but hypertension treatment rates were similar (≈90%). Around 47% of the treated octogenarians achieved conventional BP targets (<140/90 mm Hg), increasing to 59% when assessed against revised targets (<150/90 mm Hg). All-cause mortality rates were higher (hazard ratio, 1.55; 95% confidence interval, 0.89–2.72) at lower extremes of SBP values (<110 mm Hg). The lowest cardiovascular disease and all-cause mortality risk among treated octogenarians was observed for an SBP range of 140 to 149 mm Hg (1.04, 0.60–1.78) and 160 to 169 mm Hg (0.78, 0.51–1.21). An increasing trend in hypertension awareness and treatment was observed in a large sample of community-dwelling octogenarians. The results do not support the view that more stringent BP targets may be associated with lower mortality.
doi:10.1161/HYPERTENSIONAHA.116.07246
PMCID: PMC4900418  PMID: 27160194
aging; blood pressure; hypertension; mortality; prevalence
14.  Limonene ozonolysis in the presence of nitric oxide: Gas-phase reaction products and yields 
The reaction products from limonene ozonolysis were investigated using the new carbonyl derivatization agent, O-tert-butylhydroxylamine hydrochloride (TBOX). With ozone (O3) as the limiting reagent, five carbonyl compounds were detected. The yields of the carbonyl compounds are discussed with and without the presence of a hydroxyl radical (OH•) scavenger, giving insight into the influence secondary OH radicals have on limonene ozonolysis products. The observed reaction product yields for limonaketone (LimaKet), 7-hydroxyl-6-oxo-3-(prop-1-en-2-yl)heptanal (7H6O), and 2-acetyl-5-oxohexanal (2A5O) were unchanged suggesting OH• generated by the limonene + O3 reaction does not contribute to their formation. The molar yields of 3-isopropenyl-6-oxo-heptanal (IPOH) and 3-acetyl-6-oxoheptanal (3A6O) decreased by 68% and >95%; respectively, when OH• was removed. This suggests that OH• radicals significantly impact the formation of these products. Nitric oxide (NO) did not significantly affect the molar yields of limonaketone or IPOH. However, NO (20 ppb) considerably decreased the molar reaction product yields of 7H6O (62%), 2A5O (63%), and 3A6O (47%), suggesting NO reacted with peroxyl intermediates, generated during limonene ozonolysis, to form other carbonyls (not detected) or organic nitrates. These studies give insight into the transformation of limonene and its reaction products that can lead to indoor exposures.
doi:10.1016/j.atmosenv.2016.03.003
PMCID: PMC4920481  PMID: 27346977
Ozone; Reaction products; Oxygenated organic compounds; Derivatization
15.  Longitudinal Trends in Hypertension Management and Mortality Among Octogenarians 
Hypertension  2016;68(1):97-105.
Supplemental Digital Content is available in the text.
The role of hypertension management among octogenarians is controversial. In this long-term follow-up (>10 years) study, we estimated trends in hypertension prevalence, awareness, treatment, and control among octogenarians, and evaluated the relationship of systolic blood pressure (SBP) ranges with mortality. Data were based on the English Longitudinal Study of Ageing (ELSA). Outcome measures were hypertension prevalence, awareness, treatment and control, and cardiovascular disease, and all-cause mortality events. Participants were separated into 8 categories of SBP values (<110, 110–119, 120–129, 130–139, 140–149, 150–159, 160–169, and >169 mm Hg). Among 2692 octogenarians, mean SBP levels declined from 147 mm Hg in 1998/2000 to 134 mm Hg in 2012/2013. The decline was of lower magnitude in the 50 to 79 years old subgroup (n=22007). Hypertension prevalence and awareness were 40% and 13%, respectively, higher among octogenarians than the 50 to 79 years of age subgroup, but hypertension treatment rates were similar (≈90%). Around 47% of the treated octogenarians achieved conventional BP targets (<140/90 mm Hg), increasing to 59% when assessed against revised targets (<150/90 mm Hg). All-cause mortality rates were higher (hazard ratio, 1.55; 95% confidence interval, 0.89–2.72) at lower extremes of SBP values (<110 mm Hg). The lowest cardiovascular disease and all-cause mortality risk among treated octogenarians was observed for an SBP range of 140 to 149 mm Hg (1.04, 0.60–1.78) and 160 to 169 mm Hg (0.78, 0.51–1.21). An increasing trend in hypertension awareness and treatment was observed in a large sample of community-dwelling octogenarians. The results do not support the view that more stringent BP targets may be associated with lower mortality.
doi:10.1161/HYPERTENSIONAHA.116.07246
PMCID: PMC4900418  PMID: 27160194
aging; blood pressure; hypertension; mortality; prevalence
16.  Systematic E2 screening reveals a UBE2D-RNF138-CtIP axis promoting DNA repair 
Nature cell biology  2015;17(11):1458-1470.
Ubiquitylation is crucial for proper cellular responses to DNA double-strand breaks (DSBs). If unrepaired, these highly cytotoxic lesions cause genome instability, tumourigenesis, neurodegeneration or premature ageing. Here, we conduct a comprehensive, multilayered screen to systematically profile all human ubiquitin E2-enzymes for impacts on cellular DSB responses. Applying a widely applicable approach, we use an exemplary E2 family, UBE2Ds, to identify ubiquitylation-cascade components downstream of E2s. Thus, we uncover the nuclear E3-ligase RNF138 as a key homologous recombination (HR)-promoting factor that functions with UBE2Ds in cells. Mechanistically, UBE2Ds and RNF138 accumulate at DNA-damage sites and act at early resection stages by promoting CtIP ubiquitylation and accrual. This work supplies insights into regulation of DSB repair by HR. Moreover, it provides a rich information resource on E2s that can be exploited by follow-on studies.
doi:10.1038/ncb3260
PMCID: PMC4894550  PMID: 26502057
17.  Synthetic viability genomic screening defines Sae2 function in DNA repair 
The EMBO Journal  2015;34(11):1509-1522.
DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3′ single-stranded DNA (ssDNA) generation by 5′ DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2Δ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR.
doi:10.15252/embj.201590973
PMCID: PMC4474527  PMID: 25899817
Mre11; Sae2; suppressor screening; synthetic viability; whole-genome sequencing
18.  TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism 
Nature genetics  2015;48(1):36-43.
DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism/Seckel syndrome. We establish that TRAIP relocalizes to sites of DNA damage where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to UV irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a novel component of the DNA damage response to replication-blocking DNA lesions.
doi:10.1038/ng.3451
PMCID: PMC4697364  PMID: 26595769
19.  CRISPR-Cas9D10A nickase-based genotypic and phenotypic screening to enhance genome editing 
Scientific Reports  2016;6:24356.
The RNA-guided Cas9 nuclease is being widely employed to engineer the genomes of various cells and organisms. Despite the efficient mutagenesis induced by Cas9, off-target effects have raised concerns over the system’s specificity. Recently a “double-nicking” strategy using catalytic mutant Cas9D10A nickase has been developed to minimise off-target effects. Here, we describe a Cas9D10A-based screening approach that combines an All-in-One Cas9D10A nickase vector with fluorescence-activated cell sorting enrichment followed by high-throughput genotypic and phenotypic clonal screening strategies to generate isogenic knockouts and knock-ins highly efficiently, with minimal off-target effects. We validated this approach by targeting genes for the DNA-damage response (DDR) proteins MDC1, 53BP1, RIF1 and P53, plus the nuclear architecture proteins Lamin A/C, in three different human cell lines. We also efficiently obtained biallelic knock-in clones, using single-stranded oligodeoxynucleotides as homologous templates, for insertion of an EcoRI recognition site at the RIF1 locus and introduction of a point mutation at the histone H2AFX locus to abolish assembly of DDR factors at sites of DNA double-strand breaks. This versatile screening approach should facilitate research aimed at defining gene functions, modelling of cancers and other diseases underpinned by genetic factors, and exploring new therapeutic opportunities.
doi:10.1038/srep24356
PMCID: PMC4832145  PMID: 27079678
20.  A high-throughput in vivo micronucleus assay for genome instability screening in mice 
Nature protocols  2014;10(1):205-215.
We describe a sensitive, robust, high-throughput method for quantifying the formation of micronuclei, markers of genome instability, in mouse erythrocytes. Micronuclei are whole chromosomes or chromosome segments that have been separated from the nucleus. Other methods of detection rely on labour-intensive, microscopy-based techniques. Here, we describe a 2-d, 96-well plate-based flow cytometric method of micronucleus scoring that is simple enough for a research technician experienced in flow cytometry to perform. The assay detects low levels of genome instability that cannot be readily identified by classic phenotyping, using 25 μl of blood. By using this assay, we have screened >10,000 blood samples and discovered novel genes that contribute to vertebrate genome maintenance, as well as novel disease models and mechanisms of genome instability disorders. We discuss experimental design considerations, including statistical power calculation, we provide troubleshooting tips, and we discuss factors that contribute to a false-positive increase in the number of micronucleated red blood cells and to experimental variability.
doi:10.1038/nprot.2015.010
PMCID: PMC4806852  PMID: 25551665
Genome instability; DNA damage; micronucleus; mouse; in vivo; assay
21.  Selection of reference genes for diurnal and developmental time-course real-time PCR expression analyses in lettuce 
Plant Methods  2016;12:21.
Background
Real-time quantitative polymerase chain reaction (RT-qPCR) analysis is a low cost and sensitive technique that is widely used to measure levels of gene expression. Selecting and validating appropriate reference genes for normalising target gene expression should be the first step in any expression study to avoid inaccurate results.
Results
In this study, ten candidate genes were tested for their suitability for use as reference genes in diurnal and developmental timecourse experiments in lettuce. The candidate reference genes were then used to normalise the expression pattern of the FLOWERING LOCUS T (FT) gene, one of key genes involved in the flowering time pathway whose expression is known to vary throughout the day and at different stages of development. Three reference genes, LsPP2A-1 (PROTEIN PHOSPHATASE 2A-1), LsPP2AA3 (PROTEIN PHOSPHATASE 2A REGULATORY SUBUNIT A3) and LsTIP41 (TAP42-INTERACTING PROTEIN OF 41 kDa), were the most stably expressed candidate reference genes throughout both the diurnal and developmental timecourse experiments. In the developmental experiment using just LsPP2A-1 and LsTIP41 as reference genes would be sufficient for accurate normalisation, whilst in the diurnal experiment all three reference genes, LsPP2A-1, LsPP2AA3 and LsTIP41, would be necessary. The FT expression pattern obtained demonstrates that the use of multiple and robust reference genes for RT-qPCR expression analyses results in a more accurate and reliable expression profile.
Conclusions
Reference genes suitable for use in diurnal and developmental timecourse experiments in lettuce were identified and used to produce a more accurate and reliable analysis of lsFT expression levels than previously obtained in such timecourse experiments.
Electronic supplementary material
The online version of this article (doi:10.1186/s13007-016-0121-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s13007-016-0121-y
PMCID: PMC4804537  PMID: 27011764
Reference gene; Normalisation; qRT-PCR; Lettuce; Flowering time; FT
22.  A flow-cytometry-based method to simplify the analysis and quantification of protein association to chromatin in mammalian cells 
Nature protocols  2015;10(9):1297-1307.
Protein accumulation on chromatin has traditionally been studied using immunofluorescence microscopy or biochemical cellular fractionation followed by western immunoblot analysis. As a way to improve the reproducibility of this kind of analysis, make it easier to quantify and allow a stream-lined application in high-throughput screens, we recently combined a classical immunofluorescence microscopy detection technique with flow cytometry1. In addition to the features described above, and by combining it with detection of both DNA content and DNA replication, this method allows unequivocal and direct assignment of cell-cycle distribution of protein association to chromatin without the need for cell culture synchronization. Furthermore, it is relatively quick (no more than a working day from sample collection to quantification), requires less starting material compared to standard biochemical fractionation methods and overcomes the need for flat, adherent cell types that are required for immunofluorescence microscopy.
doi:10.1038/nprot.2015.066
PMCID: PMC4743064  PMID: 26226461
flow cytometry; chromatin; chromatin association; chromatin-bound; immunofluorescence microscopy
23.  Differences in Health at Age 100 According to Sex: Population‐Based Cohort Study of Centenarians Using Electronic Health Records 
Objectives
To use primary care electronic health records (EHRs) to evaluate the health of men and women at age 100.
Design
Population‐based cohort study.
Setting
Primary care database in the United Kingdom, 1990–2013.
Participants
Individuals reaching the age of 100 between 1990 and 2013 (N = 11,084, n = 8,982 women, n = 2,102 men).
Measurements
Main categories of morbidity and an index of multiple morbidities, geriatric syndromes and an index of multiple impairments, cardiovascular risk factors.
Results
The number of new female centenarians per year increased from 16 per 100,000 in 1990–94 to 25 per 100,000 in 2010–13 (P < .001) and of male centenarians from four per 100,000 to six per 100,000 (P = .06). The most prevalent morbidities at the age of 100 were musculoskeletal diseases, disorders of the senses, and digestive diseases. Women had greater multiple morbidity than men (odds ratio (OR) = 1.64, 95% confidence interval (CI) = 1.42–1.89, P < .001). Geriatric syndromes, including falls, fractures, hearing and vision impairment, and dementia, were frequent; 30% of women and 49% of men had no recorded geriatric syndromes. Women had greater likelihood of having multiple geriatric syndromes (OR = 2.14, 95% CI = 1.90–2.41, P < .001).
Conclusion
Fewer men than women reach the age of 100, but male centenarians have lower morbidity and fewer geriatric syndromes than women. Research using EHRs offers opportunities to understand the epidemiology of aging and improve care of the oldest old.
doi:10.1111/jgs.13484
PMCID: PMC4745036  PMID: 26096699
centenarians; epidemiology; aging; general practice; primary care
24.  Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds 
Molecular Cell  2016;61(3):449-460.
Summary
G-quadruplex (G4)-forming genomic sequences, including telomeres, represent natural replication fork barriers. Stalled replication forks can be stabilized and restarted by homologous recombination (HR), which also repairs DNA double-strand breaks (DSBs) arising at collapsed forks. We have previously shown that HR facilitates telomere replication. Here, we demonstrate that the replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased significantly in cells lacking HR. Treatment with the G4-stabilizing compound pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells, suggesting that G4 formation drives telomere instability. Remarkably, PDS reduces proliferation of HR-defective cells by inducing DSB accumulation, checkpoint activation, and deregulated G2/M progression and by enhancing the replication defect intrinsic to HR deficiency. PDS toxicity extends to HR-defective cells that have acquired olaparib resistance through loss of 53BP1 or REV7. Altogether, these results highlight the therapeutic potential of G4-stabilizing drugs to selectively eliminate HR-compromised cells and tumors, including those resistant to PARP inhibition.
Graphical Abstract
Highlights
•G4 formation on the G-rich strand drives telomere fragility in HR-deficient cells•G4-stabilizing compounds reduce viability of cells lacking BRCA1, BRCA2, or RAD51•G4 toxicity stems from excessive replication stress and DNA damage accumulation•Olaparib-resistant, BRCA-defective cells are sensitive to G4-stabilizing compounds
Zimmer et al. discovered that homologous recombination activities of BRCA1 and BRCA2 facilitate replication of genomic regions with G-quadruplex-forming potential, including telomeres, and suppress genomic instability stemming from inefficient replication of these sites. G4-stabilizing compounds are toxic to BRCA1- and BRCA2-deficient cells, highlighting their therapeutic potential in targeting BRCA deficiency.
doi:10.1016/j.molcel.2015.12.004
PMCID: PMC4747901  PMID: 26748828
25.  Vertex Stimulation as a Control Site for Transcranial Magnetic Stimulation: A Concurrent TMS/fMRI Study 
Brain Stimulation  2016;9(1):58-64.
Highlights
•Combines simultaneous whole-brain fMRI recording with TMS stimulation.•Investigates the immediate and remote neural correlates of TMS stimulation to the vertex.•Vertex stimulation leads to widespread decreases in fMRI BOLD, particularly within the brain ‘Default Mode Network’.
Background
A common control condition for transcranial magnetic stimulation (TMS) studies is to apply stimulation at the vertex. An assumption of vertex stimulation is that it has relatively little influence over on-going brain processes involved in most experimental tasks, however there has been little attempt to measure neural changes linked to vertex TMS. Here we directly test this assumption by using a concurrent TMS/fMRI paradigm in which we investigate fMRI blood-oxygenation-level-dependent (BOLD) signal changes across the whole brain linked to vertex stimulation.
Methods
Thirty-two healthy participants to part in this study. Twenty-one were stimulated at the vertex, at 120% of resting motor threshold (RMT), with short bursts of 1 Hz TMS, while functional magnetic resonance imaging (fMRI) BOLD images were acquired. As a control condition, we delivered TMS pulses over the left primary motor cortex using identical parameters to 11 other participants.
Results
Vertex stimulation did not evoke increased BOLD activation at the stimulated site. By contrast we observed widespread BOLD deactivations across the brain, including regions within the default mode network (DMN). To examine the effects of vertex stimulation a functional connectivity analysis was conducted.
Conclusion
The results demonstrated that stimulating the vertex with suprathreshold TMS reduced neural activity in brain regions related to the DMN but did not influence the functional connectivity of this network. Our findings provide brain imaging evidence in support of the use of vertex simulation as a control condition in TMS but confirm that vertex TMS induces regional widespread decreases in BOLD activation.
doi:10.1016/j.brs.2015.09.008
PMCID: PMC4720218  PMID: 26508284
Transcranial magnetic stimulation; Vertex stimulation; Concurrent TMS/fMRI

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