The BHLF1 Locus of Epstein-Barr Virus Contributes to Viral Latency and B-Cell Immortalization.

The Epstein-Barr virus (EBV) BHLF1 gene encodes an abundant linear and several circular RNAs believed to perform noncoding functions during virus replication, although an open reading frame (ORF) is retained among an unknown percentage of EBV isolates. Evidence suggests that BHLF1 is also transcribed during latent infection, which prompted us to investigate the contribution of this locus to latency. Analysis of transcripts transiting BHLF1 revealed that its transcription is widespread among B-cell lines supporting the latency I or III program of EBV protein expression and is more complex than originally presumed. EBV-negative Burkitt lymphoma cell lines infected with either wild-type or two different BHLF1 mutant EBVs were initially indistinguishable in supporting latency III. However, cells infected with BHLF1- virus ultimately transitioned to the more restrictive latency I program, whereas cells infected with wild-type virus either sustained latency III or transitioned more slowly to latency I. Upon infection of primary B cells, which require latency III for growth in vitro, both BHLF1- viruses exhibited variably reduced immortalization potential relative to the wild-type virus. Finally, in transfection experiments, efficient protein expression from an intact BHLF1 ORF required the EBV posttranscriptional regulator protein SM, whose expression is limited to the replicative cycle. Thus, one way in which BHLF1 may contribute to latency is through a mechanism, possibly mediated or regulated by a long noncoding RNA, that supports latency III critical for the establishment of EBV latency and lifelong persistence within its host, whereas any retained protein-dependent function of BHLF1 may be restricted to the replication cycle.IMPORTANCE Epstein-Barr virus (EBV) has significant oncogenic potential that is linked to its latent infection of B lymphocytes, during which virus replication is not supported. The establishment of latent infection, which is lifelong and can precede tumor development by years, requires the concerted actions of nearly a dozen EBV proteins and numerous small non-protein-coding RNAs. Elucidating how these EBV products contribute to latency is crucial for understanding EBV's role in specific malignancies and, ultimately, for clinical intervention. Historically, EBV genes that contribute to virus replication have been excluded from consideration of a role in latency, primarily because of the general incompatibility between virus production and cell survival. However, here, we provide evidence that the genetic locus containing one such gene, BHLF1, indeed contributes to key aspects of EBV latency, including its ability to promote the continuous growth of B lymphocytes, thus providing significant new insight into EBV biology and oncogenic potential.

PARP1 Stabilizes CTCF Binding and Chromatin Structure To Maintain Epstein-Barr Virus Latency Type.

Epstein Barr virus (EBV) is a potentially oncogenic gammaherpesvirus that establishes a chronic, latent infection in memory B cells. The EBV genome persists in infected host cells as a chromatinized episome and is subject to chromatin-mediated regulation. Binding of the host insulator protein CTCF to the EBV genome has an established role in maintaining viral latency type. CTCF is posttranslationally modified by the host enzyme PARP1. PARP1, or poly(ADP-ribose) polymerase 1, catalyzes the transfer of a poly(ADP-ribose) (PAR) moiety from NAD+ onto acceptor proteins, including itself, histone proteins, and CTCF. PARylation of CTCF by PARP1 can affect CTCF's insulator activity, DNA binding capacity, and ability to form chromatin loops. Both PARP1 and CTCF have been implicated in the regulation of EBV latency and lytic reactivation. Thus, we predicted that pharmacological inhibition with PARP1 inhibitors would affect EBV latency type through a chromatin-specific mechanism. Here, we show that PARP1 and CTCF colocalize at specific sites throughout the EBV genome and provide evidence to suggest that PARP1 acts to stabilize CTCF binding and maintain the open chromatin landscape at the active Cp promoter during type III latency. Further, PARP1 activity is important in maintaining latency type-specific viral gene expression. The data presented here provide a rationale for the use of PARP inhibitors in the treatment of EBV-associated cancers exhibiting type III latency and ultimately could contribute to an EBV-specific treatment strategy for AIDS-related or posttransplant lymphomas.IMPORTANCE EBV is a human gammaherpesvirus that infects more than 95% of individuals worldwide. Upon infection, EBV circularizes as an episome and establishes a chronic, latent infection in B cells. In doing so, the virus utilizes host cell machinery to regulate and maintain the viral genome. In otherwise healthy individuals, EBV infection is typically nonpathological; however, latent infection is potentially oncogenic and is responsible for 1% of human cancers. During latent infection, EBV expresses specific sets of proteins according to the given latency type, each of which is associated with specific types of cancers. For example, type III latency, in which the virus expresses its full repertoire of latent proteins, is characteristic of AIDS-associated and posttransplant lymphomas associated with EBV infection. Understanding how viral latency type is regulated at the chromatin level may reveal potential targets for EBV-specific pharmacological intervention in EBV-associated cancers.

Epstein-Barr Virus Oncoprotein LMP1 Mediates Epigenetic Changes in Host Gene Expression through PARP1.

UNLABELLED: The latent infection of Epstein-Barr virus (EBV) is associated with 1% of human cancer incidence. Poly(ADP-ribosyl)ation (PARylation) is a posttranslational modification catalyzed by poly(ADP-ribose) polymerases (PARPs) that mediate EBV replication during latency. In this study, we detail the mechanisms that drive cellular PARylation during latent EBV infection and the effects of PARylation on host gene expression and cellular function. EBV-infected B cells had higher PAR levels than EBV-negative B cells. Moreover, cellular PAR levels were up to 2-fold greater in type III than type I latently infected EBV B cells. We identified a positive association between expression of the EBV genome-encoded latency membrane protein 1 (LMP1) and PAR levels that was dependent upon PARP1. PARP1 regulates gene expression by numerous mechanisms, including modifying chromatin structure and altering the function of chromatin-modifying enzymes. Since LMP1 is essential in establishing EBV latency and promoting tumorigenesis, we explored the model that disruption in cellular PARylation, driven by LMP1 expression, subsequently promotes epigenetic alterations to elicit changes in host gene expression. PARP1 inhibition resulted in the accumulation of the repressive histone mark H3K27me3 at a subset of LMP1-regulated genes. Inhibition of PARP1, or abrogation of PARP1 expression, also suppressed the expression of LMP1-activated genes and LMP1-mediated cellular transformation, demonstrating an essential role for PARP1 activity in LMP1-induced gene expression and cellular transformation associated with LMP1. In summary, we identified a novel mechanism by which LMP1 drives expression of host tumor-promoting genes by blocking generation of the inhibitory histone modification H3K27me3 through PARP1 activation. IMPORTANCE: EBV is causally linked to several malignancies and is responsible for 1% of cancer incidence worldwide. The EBV-encoded protein LMP1 is essential for promoting viral tumorigenesis by aberrant activation of several well-known intracellular signaling pathways. We have identified and defined an additional novel molecular mechanism by which LMP1 regulates the expression of tumor-promoting host genes. We found that LMP1 activates the cellular protein PARP1, leading to a decrease in a repressive histone modification, accompanied by induction in expression of multiple cancer-related genes. PARP1 inhibition or depletion led to a decrease in LMP1-induced cellular transformation. Therefore, targeting PARP1 activity may be an effective treatment for EBV-associated malignancies.

Enhancers compete with a long non-coding RNA for regulation of the Kcnq1 domain.

The imprinted Kcnq1 domain contains a differentially methylated region (KvDMR) in intron 11 of Kcnq1. The Kcnq1ot1 non-coding RNA emerges from the unmethylated paternal KvDMR in antisense direction, resulting in cis-repression of neighboring genes. The KvDMR encompasses the Kcnq1ot1 promoter, CTCF sites and other DNA elements, whose individual contribution to regulation of the endogenous domain is unknown. We find that paternal inheritance of a deletion of the minimal Kcnq1ot1 promoter derepresses the upstream Cdkn1c gene. Surprisingly, Kcnq1ot1 transcripts continue to emerge from alternative sites, evidence that silencing depends, not on the ncRNA, but on the promoter sequence. Detailed analyses of Kcnq1ot during cardiogenesis show substantial chromatin reorganization coinciding with discontinuous RNA production in both wild-type and mutant mice, with loss of imprinting. We show that CTCF binds to both methylated and unmethylated alleles of the KvDMR. Furthermore, we report a multitude of enhancers within the Kcnq1ot1 region, and present conformational dynamics of a novel heart enhancer engaged in Kcnq1 expression. Our results have important implications on tissue-specific imprinting patterns and how transcriptional mechanisms compete to maximize the expression of vital genes, in addition to shifting our perception on the role of the long ncRNA in regulating this imprinted domain.

Patients' knowledge of and participation in preventing pressure ulcers- an intervention study.

The aim of this study was to evaluate a patient information pamphlet on pressure ulcer (PU) prevention using a descriptive, comparative pre- and post-test study design. The patient information pamphlet 'How can you help to stop pressure ulcers?' developed by the European PU Advisory Panel in 2012 was implemented in two surgical wards in a university hospital. A total of 61 patients answered pre- and post-test questionnaires. Patients assessed their knowledge of the risks, causes and ways to prevent PUs significantly higher after the intervention than before. Twenty-eight patients (46%) reported that they had participated in PU prevention during the last 24 hours. The patients assessed the content of the PU pamphlet as useful, its language as quite easy to understand and its layout as good. Patients with a PU pamphlet during their hospital stay were more knowledgeable about and more active in their own care. It is important that nurses invite patients to be active partners in preventing PUs but also that they identify patients who need to have a more passive role. The PU pamphlet could be updated to increase its comprehensibility, meaningfulness and manageability for patients.

Amine Gradient Stationary Phases on In-House Built Monolithic Columns for Liquid Chromatography.

Stationary phase gradients on monolithic silica columns have been successfully and reproducibly prepared and characterized with comparisons made to uniformly modified stationary phases. Stationary phase gradients hold great potential for use in liquid chromatography (LC), both in terms of simplifying analysis as well as providing novel selectivity. In this work, we demonstrate the creation of a continuous stationary phase gradient on in-house synthesized monolithic columns by infusing an aminoalkoxysilane solution through the silica monoliths via controlled rate infusion. The presence of amine and its distribution along the length of gradient and uniformly modified columns were assessed via X-ray photoelectron spectroscopy (XPS). XPS showed a clear gradient in surface coverage along the length of the column for the gradient stationary phases while a near uniform distribution on the uniformly modified stationary phases. To demonstrate the application of these gradient stationary phases, the separations of both nucleobases and weak acids/weak bases on these gradient stationary phases have been compared to uniformly modified and unmodified silica columns. Of particular note, the retention characteristics of 11 gradient columns, 5 uniformly modified columns, and 5 unmodified columns have been tested to establish the reproducibility of the synthetic procedures. Standard deviations of the retention factors were in the range from 0.06 to 0.5, depending on the analyte species. We show that selectivity is achieved with the stationary phase gradients that are significantly different from either uniformly modified amine or unmodified columns. These results indicate the significant promise of this strategy for creating novel stationary phases for LC.

Epigenetic deregulation of the LMP1/LMP2 locus of Epstein-Barr virus by mutation of a single CTCF-cohesin binding site.

The chromatin regulatory factors CTCF and cohesin have been implicated in the coordinated control of multiple gene loci in Epstein-Barr virus (EBV) latency. We have found that CTCF and cohesin are highly enriched at the convergent and partially overlapping transcripts for the LMP1 and LMP2A genes, but it is not yet known how CTCF and cohesin may coordinately regulate these transcripts. We now show that genetic disruption of this CTCF binding site (EBVΔCTCF166) leads to a deregulation of LMP1, LMP2A, and LMP2B transcription in EBV-immortalized B lymphocytes. EBVΔCTCF166 virus-immortalized primary B lymphocytes showed a decrease in LMP1 and LMP2A mRNA and a corresponding increase in LMP2B mRNA. The reduction of LMP1 and LMP2A correlated with a loss of euchromatic histone modification H3K9ac and a corresponding increase in heterochromatic histone modification H3K9me3 at the LMP2A promoter region in EBVΔCTCF166. Chromosome conformation capture (3C) revealed that DNA loop formation with the origin of plasmid replication (OriP) enhancer was eliminated in EBVΔCTCF166. We also observed that the EBV episome copy number was elevated in EBVΔCTCF166 and that this was not due to increased lytic cycle activity. These findings suggest that a single CTCF binding site controls LMP2A and LMP1 promoter selection, chromatin boundary function, DNA loop formation, and episome copy number control during EBV latency.

Morbidity and mortality in stranded Cook Inlet beluga whales Delphinapterus leucas.

The endangered Cook Inlet (Alaska, USA) stock of beluga whales Delphinapterus leucas declined 47% between 1994 and 1998, from an estimated 653 whales to 347 whales, with a continued decline to approximately 312 in 2012. Between 1998 and 2013, 164 known dead strandings were reported by the National Marine Fisheries Service. Only 38 of these animals, or 23% of the known stranded carcasses, were necropsied. Carcasses were found between April and October. The majority of animals necropsied were adults (n=25), followed by juveniles (n=6), calves (n=3), and aborted fetuses (n=4). Eight of the 11 mature females were pregnant, post-partum, or lactating. Many (82%) of these belugas were in moderate to advanced autolysis, which hampered determination of a cause of death (COD). Each animal had a single primary COD assigned within a broad set of categories. The CODs were unknown (29%), trauma (18%), perinatal mortality (13%), mass stranding (13%), single stranding (11%), malnutrition (8%), or disease (8%). Other disease processes were coded as contributory or incidental to COD. Multiple animals had mild to moderate verminous pneumonia due to Stenurus arctomarinus, renal granulomas due to Crassicauda giliakiana, and ulcerative gastritis due to Anisakis sp. Each stranding affords a unique opportunity to obtain natural history data and evidence of human interactions, and, by long-term monitoring, to characterize pathologies of importance to individual and population health.

Psychosocial reintegration post-traumatic spinal cord injury in Rwanda: An exploratory study.

Background: Traumatic spinal cord injury (TSCI) survivors are confronted by both physical and psychosocial barriers when returning to their communities. Therefore, reintegration is an important aspect of their journey back into social life. Objectives: To assess psychosocial reintegration after TSCI in Rwanda. Method: All community-dwelling adults who were registered in the previous epidemiological study were recruited and injury characteristics questionnaire and the Sydney Psychosocial Reintegration Scale version 2 (SPRS-2) were used to collect data through a telephone interview. Results: The study traced 58 participants, 77.6% (n = 45) were male and 56.9% (n = 33) were categorised with paraplegia. Overall, the results show poor community reintegration. The SPRS-2 and domain mean (SD) scores were: overall SPRS-2 of 20.95 (11.56), occupational activity (OA) of 3.68 (4.31), interpersonal relationship (IR) of 7.11(4.31) and living skills (LS) of 7.43 (5.32). Gender significantly influenced overall SPRS-2 (p = 0.011) and two domains: OA (p = 0.005) and LS (p = 0.012). Level of injury was significantly associated with an OA domain score of SPRS-2 (p = 0.002). Gender explained 29% of the variance in the LS domain of SPRS-2, with males reporting better psychosocial reintegration. Conclusion: Gender strongly predicted psychosocial reintegration following a TSCI, which is an indication of the role of social support. Clinical Implications: Traumatic SCI rehabilitation should be holistic to help prepare the person to return to the community. There should be an assessment of an individual's readiness to return to the community before discharge from the hospital.

Long-Range Atomic Order on Double-Stepped Al2O3(0001) Surfaces.

The deterministic preparation of highly ordered single-crystalline surfaces is a key step for studying and utilizing the physical properties of various advanced materials. This paper presents the fast and straightforward preparation of vicinal Al2O3(0001) surfaces with micrometer-scale atomic order. Crisp electron-diffraction spots up to at least 20th order evidence atomic coherence on terraces with widths exceeding 1 µm. The unique combination of three properties of Al2O3(0001) underlie this remarkable coherence: its high-temperature stability; the differences in the ionic bonding systems of the surface as compared to the bulk; and the fact that the terraces are non-polar whereas the step edges have a polar character. The step edges are furthermore found to have alternating configurations, which drive a step-doubling transition. On double-stepped surfaces, the Al-rich ( 31 × 31 ) R ± 9 $(\sqrt {31}\times \sqrt {31})\textrm {R}\pm 9$ ° surface reconstruction attains a singular in-plane orientation. These results set a benchmark for high-quality surface preparation and thus expand the scope for both fundamental studies on and the technological utilization of exciting material systems.

The association between perception of noise from a mechanical heart valve and symptoms of anxiety and depression.

AIM: Patients with symptomatic aortic valve stenosis are efficiently treated by aortic valve replacement (AVR), using a biological or mechanical valve. For some patients with mechanical valves, the metallic clicking sound may be problematic. The aim of this study was to investigate the perceived disturbance from the sound of a mechanical valve and the association between noise perception and symptoms of anxiety and depression. METHODS: and results: The study had a cross-sectional design. In April 2013, all patients who had undergone AVR at one university hospital during the period 2000-2012 were invited by post to participate. The primary variables were assessed using a valve-specific questionnaire and the Hospital Anxiety and Depression Scale (HADS).Of the 912 (77%) respondents, 245 had mechanical valves. Of these, fifty-nine (24%) were women, the mean (standard deviation: SD) age was 61 (11) years, and the mean time since surgery was 7 (3) years. The valve-specific questionnaire showed that 84% of the patients could sometimes or often hear the valve sound. A moderate positive correlation was found between valve prosthesis noise disturbance and anxiety, r = 0.35 (p = 0.001), and depression, r = 0.27 (p = 0.001). In a multiple linear regression analysis, valve noise perception was only significantly associated with anxiety among several other biopsychosocial factors. CONCLUSION: This study shows an association between valve noise disturbance and symptoms of anxiety, and highlights the importance of preparing all patients for the sound from the mechanical valves that arises after surgery.

Determination of chemical constituent yields in e-cigarette aerosol using partial and whole pod collections, a comparative analysis.

Literature reports the chemical constituent yields of electronic nicotine delivery systems (ENDS) aerosol collected using a range of aerosol collection strategies. The number of puffs to deplete an ENDS product varies widely, but collections often consist of data from the first 50-100 puffs. However, it is not clear whether these discrete puff blocks are representative of constituent yields over the life of a pod. We aimed to assess the effect of differing aerosol collection strategies on reported yields for select chemical constituents in the aerosol of closed pod-based ENDS products. Constituents analyzed were chosen to reflect important classes of compounds from the Final Premarket Tobacco Product Application Guidance. Yields were normalized to total device mass loss (DML). Collection strategies that consisted of partial pod collection were valid for determining yields of constituents whose DML normalized yields were consistent for the duration of pod life. These included primary aerosol constituents, such as propylene glycol, glycerol, and nicotine, and whole pod yields could be determined from initial puff blocks. However, changes were observed in the yields of some metals, some carbonyl compounds, and glycidol over pod life in a chemical constituent and product dependent manner. These results suggest that collection strategies consisting of initial puff block collections require validation per chemical constituent/product and are not appropriate for chemical constituents with variable yields over pod life. Whole pod collection increased sensitivity and accuracy in determining metal, carbonyl, and glycidol yields compared to puff block-based collection methodologies for all products tested.

ANTIBODIES AGAINST INFLUENZA VIRUS TYPES A AND B IN CANADIAN SEALS.

Influenza viruses have been reported from marine mammals worldwide, particularly in pinnipeds, and have caused mass mortalities of seals in North America and Europe. Because influenza viruses in marine mammals can be zoonotic, our objective was to examine Canadian phocids for exposure to influenza A and B viruses in order to understand health risks to wild populations as well as to humans who consume or handle these animals. Blood was collected from 394 seals in eastern Canada from 1994 to 2005. Sera were screened for exposure to influenza viruses in three resident species of seals: harbour, Phoca vitulina (n=66); grey, Halichoerus grypus (n=82); ringed, Phoca hispida (n=2); and two migrant species: harp, Pagophilus groenlandica (n=206) and hooded, Cystophora cristata (n=38). Included were samples from captive grey (n=1) and harbour seals (n=8) at two aquaria. Sera were prescreened using indirect enzyme-linked immunosorbent assay (ELISA), and antibodies against influenza A virus were confirmed using a commercial competitive ELISA (IDEXX Europe B.V.). A subset of influenza A virus positive sera was used to determine common virus subtypes recognized by sera using reference strains. All positive sera in the indirect ELISA reacted with influenza A virus subtypes H3, H4, and H10 using a hemagglutination inhibition assay. Sera from harbour, grey, harp, and hooded seals had antibodies against influenza A and influenza B viruses (some cross-reactivity occurred). Overall, 33% (128/385) of wild seals were seropositive to influenza viruses, with the highest seroprevalence in harp (42%) followed by harbour (33%), grey (23%), and hooded (11%) seals. Antibodies were detected in both sexes and most age classes of wild seals. Two of eight captive harbour seals were seropositive to influenza B virus and four had cross-reactions to influenza A and B viruses. This study reports antibodies against influenza A and B viruses in four seal species from the same geographic area in eastern Canada.

Closed form approximations to predict retention times and peak widths in gradient elution under conditions of sample volume overload and sample solvent mismatch.

Closed form expressions for the prediction of retention times and peak widths for gradient liquid chromatography are particularly useful in understanding, rationalizing and optimizing separations. These expressions are obtained by integrating differential equations, in conjunction with a model of the variation of the retention factor as a function of mobile phase composition. Two of these models, the linear solvent strength (LSS) model and the Neue-Kuss (NK) model are explored in the present work. Here, we expand on these closed form expressions to account for effects of sample volume overload and a mismatch between the sample solvent and the initial mobile phase composition for the gradient. We show that there have been errors in expressions reported in the literature, and we have evaluated the accuracy of the predictions from the closed form expressions reported here using a recently developed liquid chromatography simulator. The expressions assume a constant plate height and consider elution across four zones of the gradient profile - elution in the sample solvent, elution in the initial (isocratic) mobile phase caused by the gradient delay volume, elution during a linear gradient, and elution post-gradient at the final (isocratic) mobile phase composition. The expressions generally give reasonably accurate predictions for retention times and peak widths, except for cases where the solute elutes during transitions between the different zones. The average magnitude of the prediction errors for retention time and peak width relative to simulation were 0.093% and 0.40% for the LSS expressions for ten amphetamine solutes at 36 different separation conditions, and 0.22% and 1.8% for the NK expressions for eight alkylbenzene solutes at 36 different separation conditions, respectively.

Dioctophyme renale (Goeze, 1782) in the abdominal cavity of a domestic cat from Brazil.

This study reports a case of parasitism by the giant kidney worm, Dioctophyme renale (Goeze, 1782), in the abdominal cavity of a domestic cat from Brazil. A female adult cat presenting prostration, dehydration, physical debility, pronounced jaundice and ascitis, was taken to the Department of Animal Parasitology of the Veterinary Institute of the Universidade Federal Rural do Rio de Janeiro, Brazil. Clinical signs suggested a case of peritonitis. The cat's clinical condition was grave and death occurred within a few days. During necropsy, a brownish-red nematode, 24.9cm long, was found in the abdominal cavity and was identified as a male adult D. renale. This study reports the first confirmed case of dioctophymatosis in the domestic cat. The parasite's aberrant location in the abdominal cavity suggests that the domestic cat is not a suitable host.

Atomic structure of PI3-kinase SH3 amyloid fibrils by cryo-electron microscopy.

High resolution structural information on amyloid fibrils is crucial for the understanding of their formation mechanisms and for the rational design of amyloid inhibitors in the context of protein misfolding diseases. The Src-homology 3 domain of phosphatidyl-inositol-3-kinase (PI3K-SH3) is a model amyloid system that plays a pivotal role in our basic understanding of protein misfolding and aggregation. Here, we present the atomic model of the PI3K-SH3 amyloid fibril with a resolution determined to 3.4 Å by cryo-electron microscopy (cryo-EM). The fibril is composed of two intertwined protofilaments that create an interface spanning 13 residues from each monomer. The model comprises residues 1-77 out of 86 amino acids in total, with the missing residues located in the highly flexible C-terminus. The fibril structure allows us to rationalise the effects of chemically conservative point mutations as well as of the previously reported sequence perturbations on PI3K-SH3 fibril formation and growth.

Simulation of elution profiles in liquid chromatography - III. Stationary phase gradients.

A previously developed liquid chromatographic simulator (see parts I and II) [1-3] is extended to allow for simulations of stationary phase gradients with isocratic and gradient mobile phases. Gradient stationary phases have recently been proposed as means of engineering unique chromatographic selectivities. In the present work, the simulator provides retention times and peak widths that agree with closed form theory for a linear gradient in retention factor and provides accurate retention time predictions for experimentally implemented continuous and discontinuous gradients. Calculation of discontinuous gradients implemented using the commercially available POPLC system have shown good agreement with experiment, with the largest deviation of the simulated retention time from experiment of 4.5%. In addition, simulations of a novel continuous amine gradient column show good agreement with experiment, and give insights into synergistic interactions on column. With the exception of solutes that show evidence of synergistic interactions, the simulated retention times are in agreement with the 95% confidence limits of the experimental values.

Poly(ADP-ribose) Polymerase 1, PARP1, modifies EZH2 and inhibits EZH2 histone methyltransferase activity after DNA damage.

The enzyme Poly(ADP-ribose) polymerase 1 (PARP1) plays a very important role in the DNA damage response, but its role in numerous aspects is not fully understood. We recently showed that in the absence of DNA damage, PARP1 regulates the expression of the chromatin-modifying enzyme EZH2. Work from other groups has shown that EZH2 participates in the DNA damage response. These combined data suggest that EZH2 could be a target of PARP1 in both untreated and genotoxic agent-treated conditions. In this work we tested the hypothesis that, in response to DNA damage, PARP1 regulates EZH2 activity. Here we report that PARP1 regulates EZH2 activity after DNA damage. In particular, we find that EZH2 is a direct target of PARP1 upon induction of alkylating and UV-induced DNA damage in cells and in vitro. PARylation of EZH2 inhibits EZH2 histone methyltransferase (H3K27me) enzymatic activity. We observed in cells that the induction of PARP1 activity by DNA alkylating agents decreases the association of EZH2 with chromatin, and PARylation of histone H3 reduces EZH2 affinity for its target histone H3. Our findings establish that PARP1 and PARylation are important regulators of EZH2 function and link EZH2-mediated heterochromatin formation, DNA damage and PARylation. These findings may also have clinical implications, as they suggest that inhibitors of EZH2 can improve anti-tumor effects of PARP1 inhibitors in BRCA1/2-deficient cancers.

Simulation of elution profiles in liquid chromatography - II: Investigation of injection volume overload under gradient elution conditions applied to second dimension separations in two-dimensional liquid chromatography.

An important research direction in the continued development of two-dimensional liquid chromatography (2D-LC) is to improve the detection sensitivity of the method. This is especially important in applications where injection of large volumes of effluent from the first dimension (1D) column into the second dimension (2D) column leads to severe 2D peak broadening and peak shape distortion. For example, this is common when coupling two reversed-phase columns and the organic solvent content of the 1D mobile phase overwhelms the 2D column with each injection of 1D effluent, leading to low resolution in the second dimension. In a previous study we validated a simulation approach based on the Craig distribution model and adapted from the work of Czok and Guiochon [1] that enabled accurate simulation of simple isocratic and gradient separations with very small injection volumes, and isocratic separations with mismatched injection and mobile phase solvents [2]. In the present study we have extended this simulation approach to simulate separations relevant to 2D-LC. Specifically, we have focused on simulating 2D separations where gradient elution conditions are used, there is mismatch between the sample solvent and the starting point in the gradient elution program, injection volumes approach or even exceed the dead volume of the 2D column, and the extent of sample loop filling is varied. To validate this simulation we have compared results from simulations and experiments for 101 different conditions, including variation in injection volume (0.4-80µL), loop filling level (25-100%), and degree of mismatch between sample organic solvent and the starting point in the gradient elution program (-20 to +20% ACN). We find that that the simulation is accurate enough (median errors in retention time and peak width of -1.0 and -4.9%, without corrections for extra-column dispersion) to be useful in guiding optimization of 2D-LC separations. However, this requires that real injection profiles obtained from 2D-LC interface valves are used to simulate the introduction of samples into the 2D column. These profiles are highly asymmetric - simulation using simple rectangular pulses leads to peak widths that are far too narrow under many conditions. We believe the simulation approach developed here will be useful for addressing practical questions in the development of 2D-LC methods.