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Kristina Flägel,1 Marie-Therese Müller,1 Katja Goetz,1 Elisabeth Flum,2 Simon Schwill,3 Jost Steinhäuser1 1Institute of Family Medicine, University Hospital Schleswig-Holstein, Campus Lübeck, Lübeck, Schleswig-Holstein, Germany; 2Group Practice Dr. Katharina Mendler & Dr. Elisabeth Flum, Eppelheim, Baden-Württemberg, Germany; 3Department of General Practice and Health Services Research, University Hospital Heidelberg, Heidelberg, Baden-Württemberg, GermanyCorrespondence: Kristina Flägel, Institute of Family Medicine, University Hospital Schleswig-Holstein, Campus Lübeck, Ratzeburger Allee 160, Lübeck, Schleswig-Holstein, 23538, Germany, Tel +49 451 3101 8012, Fax +49 451 3101 8004, Email kristina.flaegel@uni-luebeck.dePurpose: One of the reasons for postgraduate trainees not to choose working in a rural area is uncertainty related to the lack of competencies. The aim of this study was to investigate the concept of uncertainty by measuring competencies and to examine the psychometric properties of an instrument that measures competencies related to uncertainty in the self-assessment of postgraduate trainees in family medicine.Patients and Methods: A questionnaire was created based on pre-existing instruments. It was distributed to participants of postgraduate training seminars in the federal states of Baden-Württemberg and Schleswig-Holstein, Germany in 2016. Descriptive statistics and a partial correlation analysis were calculated for measuring the degree of association between year of postgraduate training and items’ responses. Psychometric properties were assessed by calculating descriptive item analysis, factor analysis and internal consistency.Results: The response rate was 85% (105/124). More than one-fifth of the participants stated to show only seldom or sometimes the following skills: ability to balance work and life (N=25, 24%), letting a mild disorder run its own way (N=24, 23%) and ability to conduct interventions that decreased aggression from the patient (N=22, 21%). More than half of the participants felt (very) insecure in performing routine child check-ups, the application of a below elbow backslab and the partial removal of a toenail. Nine out of 21 items showed positive statistically significant correlation between level of competence and progress in training. Factor analysis led to a final instrument with 12 items (Cronbach’s α=0.736) and a three-factor-structure: “doctor well-being and resilience”, “communication” and “skills”.Conclusion: In order to reduce uncertainty with all its consequences, a focus on teaching of competencies regarding the physicians’ well-being and resilience, communication and skills has to be continued in postgraduate training.Keywords: general practice, family medicine, uncertainty, postgraduate training, competence-based training, entrustable professional activities

OBJECTIVE: To determine how often requests are made for euthanasia and physician-assisted suicide (EAS) in the absence of severe disease and how such requests are dealt with in medical practice in The Netherlands. METHOD: Retrospective interview study. Participants: 125 general practitioners (GPs), 77 nursing home physicians (NHPs), and 208 clinical specialists. RESULTS: In The Netherlands, each year approximately 400 people request EAS, because they are 'weary of life'. Thirty per cent of all physicians have at some time received an explicit request for EAS in the absence of severe disease; 3% of all physicians had granted a request for EAS in such a case. Most requests for EAS to GPs in the absence of severe disease (n = 29) were made by single people aged 80 years and over. While their problems were most frequently of a social nature, 79% had one or more non-severe illnesses. Most GPs refused the request; half of them proposed an alternative treatment, which the patient often refused. Nineteen people who did not receive any treatment persisted in their wish to die; the request for EAS from 5 out of 10 patients who received one or more types of treatment was withdrawn or became less explicit. CONCLUSIONS: Most physicians in The Netherlands refuse requests for EAS in the absence of severe disease. Most patients persist in their request. In an ageing population more research is needed to provide physicians with practical interventions to prevent suicide and to make life bearable and satisfactory for elderly people who wish to die. [ABSTRACT FROM AUTHOR]

The researchers wanted to obtain insight into the cooperation between physicians and nurses with regard to active euthanasia and physician-assisted suicide (EAS). In study I a stratified random sample of 203 clinical specialists, 152 general practitioners (GPs) and 50 nursing home physicians (NHPs) participated. In study II a random sample of 521 GPs was drawn from the province of North Holland and a random sample of 521 GPs was drawn from the rest of the Netherlands. For study III all NHPs were approached. Data were collected by means of an interview in study I. In studies II and III an anonymous, postal questionnaire was used. Approximately half of the GPs did not consult with nurses about a patient's request for EAS, the intention to administer EAS, and the actual administration. In 5% of cases, the NHPs and the specialists did not consult with nurses concerning these aspects. The GPs and NHPs indicated in 4% and 3% of the cases, respectively, that nurses administered the lethal drug(s) to the patients; the corresponding figure for the specialists was 21%. Almost all GPs and NHPs and about three-quarters of the specialists thought that nurses should never be allowed to administer EAS. [ABSTRACT FROM AUTHOR]

INTRODUCTION AND METHOD: Recognition and treatment of depression is a quality indicator for nursing homes. Nurses and nursing assistants are in a particularly good position to recognize depression in long-term care. How well do nurses and nursing assistants recognize depression, compared with a DSM-IV diagnosis of depression? To answer this question a critical review of relevant literature in PubMed searches was performed. RESULTS: It was found that nurses and nursing assistants recognize true depression in about 55% (sensitivity 42% to 78%) and 'over-recognized' depression in about 40% of nondepressed patients (specificity 56% to 67%). DISCUSSION: The prominent role of nurses and nursing assistants in daily mental health care is not reflected in the number of methodologically sound studies. Sensitivity of the Geriatric Depression Scale (GDS) is about 50% higher than nurses' recognition of depression. A specific recommendation for specially trained nursing assistants in depression management and standard use of screening scales in an update of the American Medical Directors Association's Clinical Practice Guideline on Depression might improve depression recognition, as well as nursing assistants' work satisfaction and staff turnover. CONCLUSION: Recognition of depression by nurses and nursing assistants is low. Standard use of a screening scale like the GDS would improve recognition of depression in the elderly. More research is needed aimed at how nursing assistants can empower their role as mental health care provider in long-term care. [ABSTRACT FROM AUTHOR]

DNA topoisomerases are essential enzymes for all living organisms and important targets for anticancer drugs and antibiotics. Although DNA topoisomerases have been studied extensively, steady-state kinetics has not been systematically investigated because of the lack of an appropriate assay. Previously, we demonstrated that newly synthesized, fluorescently labeled plasmids pAB1_FL905 and pAB1_FL924 can be used to study DNA topoisomerase-catalyzed reactions by fluorescence resonance energy transfer (FRET) or supercoiling-dependent fluorescence quenching (SDFQ). With the FRET or SDFQ method, we performed steady-state kinetic studies for six different DNA topoisomerases including two type IA enzymes ( Escherichia coli and Mycobacterium smegmatis DNA topoisomerase I), two type IB enzymes (human and variola DNA topoisomerase I), and two type IIA enzymes ( E. coli DNA gyrase and human DNA topoisomerase IIα). Our results show that all DNA topoisomerases follow the classical Michaelis-Menten kinetics and have unique steady-state kinetic parameters, K M , V max , and k cat . We found that k cat for all topoisomerases are rather low and that such low values may stem from the tight binding of topoisomerases to DNA. Additionally, we confirmed that novobiocin is a competitive inhibitor for adenosine 5'-triphosphate binding to E. coli DNA gyrase, demonstrating the utility of our assay for studying topoisomerase inhibitors., Competing Interests: The authors declare no competing financial interest., (Copyright © 2019 American Chemical Society.)

In addition to genetic mutations, epigenetic revision plays a major role in the development and progression of cancer; specifically, inappropriate DNA methylation or demethylation of CpG residues may alter the expression of genes that promote tumorigenesis. We hypothesize that DNA repair, specifically the repair of DNA double strand breaks (DSB) by Non-Homologous End Joining (NHEJ) may play a role in this process. Using a GFP reporter system inserted into the genome of HeLa cells, we are able to induce targeted DNA damage that enables the cells, after successfully undergoing NHEJ repair, to express WT GFP. These GFP+ cells were segregated into two expression classes, one with robust expression (Bright) and the other with reduced expression (Dim). Using a DNA hypomethylating drug (AzadC) we demonstrated that the different GFP expression levels was due to differential methylation statuses of CpGs in regions on either side of the break site. Deep sequencing analysis of this area in sorted Bright and Dim populations revealed a collection of different epi-alleles that display patterns of DNA methylation following repair by NHEJ. These patterns differ between Bright and Dim cells which are hypo- and hypermethylated, respectively, and between the post-repair populations and the original, uncut cells. These data suggest that NHEJ repair facilitates a rewrite of the methylation landscape in repaired genes, elucidating a potential source for the altered methylation patterns seen in cancer cells, and understanding the mechanism by which this occurs could provide new therapeutic targets for preventing this process from contributing to tumorigenesis.

Genome-wide methylation analysis is limited by its low coverage and the inability to detect single variants below 10%. Quantitative analysis provides accurate information on the extent of methylation of single CpG dinucleotide, but it does not measure the actual polymorphism of the methylation profiles of single molecules. To understand the polymorphism of DNA methylation and to decode the methylation signatures before and after DNA damage and repair, we have deep sequenced in bisulfite-treated DNA a reporter gene undergoing site-specific DNA damage and homologous repair. In this paper, we provide information on the data generation, the rationale for the experiments and the type of assays used, such as cytofluorimetry and immunoblot data derived during a previous work published in Scientific Reports, describing the methylation and expression changes of a model gene (GFP) before and after formation of a double-strand break and repair by homologous-recombination or non-homologous-end-joining. These data provide: 1) a reference for the analysis of methylation polymorphism at selected loci in complex cell populations; 2) a platform and the tools to compare transcription and methylation profiles.

We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5' and 3' ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells.

Novel combination therapies are desperately needed for combating lung infections caused by bacterial "superbugs". This study aimed to investigate the synergistic antibacterial activity of polymyxin B in combination with the cystic fibrosis (CF) drugs KALYDECO (ivacaftor) and ORKAMBI (ivacaftor + lumacaftor) against Gram-negative pathogens that commonly colonize the CF lung, in particular, the problematic Pseudomonas aeruginosa. The in vitro synergistic activity of polymyxin B combined with ivacaftor or lumacaftor was assessed using checkerboard and static time-kill assays against a panel of polymyxin-susceptible and polymyxin-resistant P. aeruginosa isolates from the lungs of CF patients. Polymyxin B, ivacaftor, and lumacaftor were ineffective when used individually against polymyxin-resistant (MIC ≥ 4 mg/L) isolates. However, when used together, the combination of clinically relevant concentrations of polymyxin B (2 mg/L) combined with ivacaftor (8 mg/L) or ivacaftor (8 mg/L) + lumacaftor (8 mg/L) displayed synergistic killing activity against polymyxin-resistant P. aeruginosa isolates as demonstrated by a 100-fold decrease in the bacterial count (CFU/mL) even after 24 h. The combinations also displayed excellent antibacterial activity against P. aeruginosa under CF relevant conditions in a sputum medium assay. The combination of lumacaftor (alone) with polymyxin B showed additivity against P. aeruginosa. The potential antimicrobial mode of action of the combinations against P. aeruginosa was investigated using different methods. Treatment with the combinations induced cytosolic GFP release from P. aeruginosa cells and showed permeabilizing activity in the nitrocefin assay, indicating damage to both the outer and inner Gram-negative cell membranes. Moreover, scanning and transmission electron micrographs revealed that the combinations produce outer membrane damage to P. aeruginosa cells that is distinct from the effect of each compound per se. Ivacaftor was also shown to be a weak inhibitor of the bacterial DNA gyrase and topoisomerase IV with no effect on either human type I or type IIα topoisomerases. Lumacaftor displayed the ability to increase the cellular production of damaging reactive oxygen species. In summary, the combination of polymyxin B with KALYDECO or ORKAMBI exhibited synergistic activity against highly polymyxin-resistant P. aeruginosa CF isolates and can be potentially useful for otherwise untreatable CF lung infections.

Non-small cell lung cancer (NSCLC) is the main cause of cancer-related death worldwide and new therapeutic strategies are urgently needed. In this study, we have characterized a panel of NSC lung cancer cell lines for the expression of coiled-coil-domain containing 6 (CCDC6), a tumor suppressor gene involved in apoptosis and DNA damage response. We show that low CCDC6 protein levels are associated with a weak response to DNA damage and a low number of Rad51 positive foci. Moreover, CCDC6 deficient lung cancer cells show defects in DNA repair via homologous recombination. In accordance with its role in the DNA damage response, CCDC6 attenuation confers resistance to cisplatinum, the current treatment of choice for NSCLC, but sensitizes the cells to olaparib, a small molecule inhibitor of the repair enzymes PARP1/2. Remarkably, the combination of the two drugs is more effective than each agent individually, as demonstrated by a combination index <1. Finally, CCDC6 is expressed at low levels in about 30% of the NSCL tumors we analyzed by TMA immunostaining. The weak CCDC6 protein staining is significatively correlated with the presence of lymph node metastasis (p ≤ 0.02) and negatively correlated to the disease free survival (p ≤ 0.01) and the overall survival (p ≤ 0.05). Collectively, the data indicate that CCDC6 levels provide valuable insight for OS. CCDC6 could represent a predictive biomarker of resistance to conventional single mode therapy and yield insight on tumor sensitivity to PARP inhibitors in NSCLC., (© 2014 UICC.)

Topoisomerase IB (Top1) is a key eukaryotic nuclear enzyme that regulates the topology of DNA during replication and gene transcription. Anticancer drugs that block Top1 are either well-characterized interfacial poisons or lesser-known catalytic inhibitor compounds. Here we describe a new class of cytotoxic redox-stable cationic Au(3+) macrocycles which, through hierarchical cluster analysis of cytotoxicity data for the lead compound, 3, were identified as either poisons or inhibitors of Top1. Two pivotal enzyme inhibition assays prove that the compounds are true catalytic inhibitors of Top1. Inhibition of human topoisomerase IIα (Top2α) by 3 was 2 orders of magnitude weaker than its inhibition of Top1, confirming that 3 is a type I-specific catalytic inhibitor. Importantly, Au(3+) is essential for both DNA intercalation and enzyme inhibition. Macromolecular simulations show that 3 intercalates directly at the 5'-TA-3' dinucleotide sequence targeted by Top1 via crucial electrostatic interactions, which include π-π stacking and an Au···O contact involving a thymine carbonyl group, resolving the ambiguity of conventional (drug binds protein) vs unconventional (drug binds substrate) catalytic inhibition of the enzyme. Surface plasmon resonance studies confirm the molecular mechanism of action elucidated by the simulations.

We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15-20 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression.

Aim: To gain insight in the way nursing home residents experience personal dignity and the factors that preserve or undermine it., Background: Nursing home residents are exposed to diverse factors which may be associated with the loss of personal dignity. To help them maintain their dignity, it is important to investigate this concept from the residents' perspective., Design: A qualitative descriptive study., Methods: In-depth interviews were conducted between May 2010-June 2011 with 30 recently admitted residents of the general medical wards of four nursing homes in The Netherlands., Results: Illness-related conditions were the starting point of a process which could affect personal dignity, by threatening aspects of one's individual self and social world. Living in a nursing home was not a reason in itself to feel less self-worth, but rather seen as a consequence of functional incapacity. Nevertheless, many residents felt discarded by society and not taken seriously, simply because of their age or illness. Waiting for help, being dictated to by nurses and not receiving enough attention could undermine personal dignity, whereas aspects of good professional care (e.g. being treated with respect), a supportive social network and adequate coping capacities could protect it., Conclusions: Contrary to the general view in society that living in a nursing home always undermines one's dignity, good professional care and a supportive social network can preserve dignity as well. To support residents in their challenge of maintaining dignity, nursing home staff, relatives and society should pay more attention to the way they treat them., (© 2013 John Wiley & Sons Ltd.)

Objectives: The aim of this study was to explore if and how spiritual needs are assessed and if spiritual care is provided to Dutch nursing home residents, including residents suffering from dementia, and if and how caregivers communicate and collaborate regarding the residents' spiritual needs., Design: Two researchers conducted an ethnographic participatory study in a Dutch nursing home between April 2010 and June 2011, on a psychogeriatric unit (mostly dementia) and a somatic unit for residents suffering from physical disabilities. Inductive thematic analysis was used to identify patterns and trends and to interpret the data., Results: The physicians did not actively address spiritual issues, nor was it part of the official job of care staff. There was no communication between the physicians and the spiritual counselor. When a resident was about to die, the nurses started an informal care process aimed at (spiritual) well-being, including cuddling, rituals, and music. This was not mentioned in the care plan or the medical chart. The nurses even supported the residents outside their professional role in their spare time. Furthermore, we identified different occupational subcultures (eg, nurses and physicians), in which behavior of residents was given different meaning, depending on the frame of reference within the subculture., Conclusion: Spiritual issues were addressed only informally and were not part of the formal care process, either for residents suffering from dementia or for those with physical disabilities. Our results raise questions about how the lack of communication about spiritual end-of-life care between disciplines, and the informal and formal care processes affect spiritual well-being., (Copyright © 2013 American Medical Directors Association, Inc. Published by Elsevier Inc. All rights reserved.)

The structures, spectroscopy, and cytotoxicity of four novel nominally square-planar gold(III) chelates 1-4 with the general formula cis-AuCl2(X), where the ligand X is an anionic bidentate pyridyl- or isoquinolylamido chelating agent, are described. The Au-N(amido), Au-N(pyridyl), and Au-N(isoquinolyl) distances are 2.002(9)-2.016(3), 2.01(1)-2.037(3), and 2.037(3) Å, respectively. Density functional theory simulations afforded accurate gold(III) coordination geometries for 1-4 (bond distances and angles to within 5% of the X-ray values), while accurate transition energies were limited to those calculated in the UV spectral region. The complexes had variable stability in dimethyl sulfoxide: compound 3 (relatively rigid) was indefinitely stable, compounds 1 and 2 (conformationally flexible) slowly demetalated over 30 days, and 4 (extensively aromatic) formed an insoluble precipitate after 10 days (72 h in an aqueous buffer). The isoquinolylamido derivative 4 was sufficiently cytotoxic in the NCI-60 screen to undergo full five-dose testing. Notably low GI50 (1.8, 2.3, and 3.2 μM) and IC50 (4.0, 9.8, and 15 μM) values were recorded for the OVCAR-3, IGROV1, and SW-620 cell lines, respectively. Hierarchical cluster analysis employing the National Cancer Institute (NCI) data for known anticancer drugs and 4 revealed that compound 4 is mechanistically identical with the topoisomerase IIα (Top2) poison zorubicin and statistically similar to the topoisomerase IB (Top1) poisons camptothecin and 9-methoxycamptothecin. The Top2-catalyzed decatenation reaction of kinetoplast DNA was studied as a function of the concentration of 4: the compound acts as an interfacial poison of Top2 at low concentrations (<1 μM) and a catalytic inhibitor of the enzyme above 5 μM. Gel mobility shift assays (plasmid DNA substrate) showed that the catalytic inhibition of Top2 likely correlates with DNA binding by 4 at concentrations >5 μM. Compound 4 is also a catalytic inhibitor of Top1 at higher concentrations, consistent with DNA binding by the complex.

In this work, we examine regulation of DNA methyltransferase 1 (DNMT1) by the DNA damage inducible protein, GADD45α. We used a system to induce homologous recombination (HR) at a unique double-strand DNA break in a GFP reporter in mammalian cells. After HR, the repaired DNA is hypermethylated in recombinant clones showing low GFP expression (HR-L expressor class), while in high expressor recombinants (HR-H clones) previous methylation patterns are erased. GADD45α, which is transiently induced by double-strand breaks, binds to chromatin undergoing HR repair. Ectopic overexpression of GADD45α during repair increases the HR-H fraction of cells (hypomethylated repaired DNA), without altering the recombination frequency. Conversely, silencing of GADD45α increases methylation of the recombined segment and amplifies the HR-L expressor (hypermethylated) population. GADD45α specifically interacts with the catalytic site of DNMT1 and inhibits methylation activity in vitro. We propose that double-strand DNA damage and the resulting HR process involves precise, strand selected DNA methylation by DNMT1 that is regulated by GADD45α. Since GADD45α binds with high avidity to hemimethylated DNA intermediates, it may also provide a barrier to spreading of methylation during or after HR repair.

Serum PSA, Gleason score, pathological stage, and positive surgical margins are currently used as predictors for disease recurrence. However, these criteria are less than precise in predicting disease outcome, with only 10% specificity at the 90% sensitivity level. Keratins are intermediate filament proteins that are contained within normal epithelia. However, human prostate cancer tissue shows differential immunohistochemical staining of keratin 8 (K8) when compared to normal prostate tissue. Our immunofluorescence and flow cytometry data show that K8 is also present on the cell surface of transformed prostate cancer cell lines. K8 is expressed at high levels on the surfaces of DU-145 and PC-3 cells but is expressed at comparatively lower levels on the surfaces of LNCaP cells, BPH-1 cells, and RWPE-1 cells. We hypothesize that extracellular K8 (eK8) present on epithelial prostate cancer cells plays an integral role in migration and in vivo dissemination. We found that K8 increased the rate of activity of plasmin approximately fivefold over a 48-h period. Functionally, K8 also enhanced the plasmin-mediated proteolysis of vitronectin, an important component of the prostate extracellular matrix. Taken together, our data show that K8 enhances the proteolytic activity of the plasminogen activation system, indicating that eK8 may be an important distinguishing marker in prostate cancer and progression.

We report a mechanism-based screening technique to rapidly identify eukaryotic topoisomerase I targeting agents. The method is based on genetic tagging of topoisomerase I to immobilize the enzyme on a solid surface in a microtiter well format. DNA is added to the wells, and retained DNA is detected by Pico Green fluorescence. Compounds that result in an increase in Pico Green staining represent potential topoisomerase interfacial poisons, whereas those that reduce fluorescence report catalytic inhibitors; therefore, the solid phase assay represents a "bimodal" readout that reveals mechanisms of action. The method has been demonstrated to work with known interfacial poisons and catalytic inhibitors. This method is rapid, robust, economical, and scalable for large library screens., (Copyright © 2011 Elsevier Inc. All rights reserved.)

CCDC6 was originally identified in chimeric genes caused by chromosomal translocation involving the RET proto-oncogene in some thryoid tumors mostly upon ionizing radiation exposure. Recognised as a pro-apoptotic phosphoprotein that negatively regulates CREB1-dependent transcription, CCDC6 is an ATM substrate that is responsive to genotoxic stress. Here we report that following genotoxic stress, loss or inactivation of CCDC6 in cancers that carry the CCDC6 fusion, accelerates the dephosphorylation of pH2AX S139, resulting in defective G2 arrest and premature mitotic entry. Moreover, we show that CCDC6 depleted cells appear to repair DNA damaged in a shorter time compared to controls, based on reporter assays in cells. High-troughput proteomic screening predicted the interaction between the CCDC6 gene product and the catalytic subunit of Serin-Threonin Protein Phosphatase 4 (PP4c) recently identified as the evolutionarily conserved pH2AX S139 phosphatase that is activated upon DNA Damage. We describe the interaction between CCDC6 and PP4c and we report the modulation of PP4c enzymatic activity in CCDC6 depleted cells. We discuss the functional significance of CCDC6-PP4c interactions and hypothesize that CCDC6 may act in the DNA Damage Response by negatively modulating PP4c activity. Overall, our data suggest that in primary tumours the loss of CCDC6 function could influence genome stability and thereby contribute to carcinogenesis.

Although spiritual caregiving is a key domain of palliative care, it lacks a clear definition, which impedes both caregiving and research in this domain. The aim of this study was to conceptualize spirituality by identifying dimensions, based on instruments measuring spirituality in end-of-life populations. A systematic literature review was conducted. Literature published between 1980 and 2009, focussing on instruments measuring spirituality at the end of life was collected from the PubMed, Cumulative Index to Nursing and Allied Health Literature (CINAHL), and PsycINFO databases. Inclusion criteria were: (1) the studies provide empirical data collected with an instrument measuring spirituality or aspects of spirituality at the end of life; (2) the data report on a (subgroup) of an end-of-life population, and (3) the instrument is available in the public domain. Content validity was assessed according to a consensus-based method. From the items of the instruments, three investigators independently derived dimensions of spirituality at the end of life. In 36 articles that met the inclusion criteria we identified 24 instruments. Nine instruments with adequate content validity were used to identify dimensions of spirituality. To adequately represent the items of the instruments and to describe the relationships between the dimensions, a model defining spirituality was constructed. The model distinguishes the dimensions of Spiritual Well-being (e.g., peace), Spiritual Cognitive Behavioral Context (Spiritual Beliefs, Spiritual Activities, and Spiritual Relationships), and Spiritual Coping, and also indicates relationships between the dimensions. This model may help researchers to plan studies and to choose appropriate outcomes, and assist caregivers in planning spiritual care.

Background: The clinical impact of risk score use in end-of-life settings is unknown, with reports limited to technical properties., Methods: We conducted a mixed-methods study to evaluate clinical impact of a validated mortality risk score aimed at informing prognosis and supporting clinicians in decision-making in dementia patients with pneumonia. We performed a trial (n = 69) with physician-reported outcomes referring to the score's aims. Subsequently, physician focus group discussions were planned to better understand barriers to clinical impact, and we surveyed families (n = 50) and nurses practicing in nursing homes (n = 29). We finally consulted with experts and key persons for implementation., Results: Most (71%) physicians who used the score considered it useful, but mainly for its learning effects. Families were never informed of numerical risk estimates. Two focus group discussions revealed a reluctance to use a numerical approach, and physicians found that outcomes conditional on antibiotic treatment were inadequate to support decision-making. Nurses varied in their perceived role in informing families. Most families (88%) wished to be informed, preferring a numerical (43%), verbalized (35%), or other approach (18%) or had no preference (5%). Revising the score, we added an ethical framework for decision-making to acknowledge its complexity, an explanatory note addressing barriers related to physicians' attitudes, and a nurses' form., Conclusion: The combined quantitative and qualitative studies elicited: substantial barriers to a numerical approach to physicians' end-of-life decision-making; crucial information for revisions and further score development; and a need for implementation strategies that focus on education.

Correction of double strand DNA breaks proceeds in an error-free pathway of homologous recombination (HR), which can result in gene silencing of half of the DNA molecules caused by action by DNA methyltransferase 1 (DNMT1) (Cuozzo, C., Porcellini, A., Angrisano, T., Morano, A., Lee, B., Di Pardo, A., Messina, S., Iuliano, R., Fusco, A., Santillo, M. R., Muller, M. T., Chiariotti, L., Gottesman, M. E., and Avvedimento, E. V. (2007) PLoS Genet. 3, e110). To explore the mechanism that leads to HR-induced silencing, a genetic screen was carried out based on the silencing of a GFP reporter to identify potential partners. DMAP1, a DNMT1 interacting protein, was identified as a mediator of this process. DMAP1 is a potent activator of DNMT1 methylation in vitro, suggesting that DMAP1 is a co-repressor that supports the maintenance and de novo action of DNMT1. To examine critical roles for DMAP1 in vivo, lentiviral shRNA was used to conditionally reduce cellular DMAP1 levels. The shRNA transduced cells grew poorly and eventually ceased their growth. Analysis of the tumor suppressor gene p16 methylation status revealed a clear reduction in methylated CpGs in the shRNA cells, suggesting that reactivation of a tumor suppressor gene pathway caused the slow growth phenotype. Analysis of HR, using a fluorescence-based reporter, revealed that knocking down DMAP1 also caused hypomethylation of the DNA repair products following gene conversion. DMAP1 was selectively enriched in recombinant GFP chromatin based on chromatin immunoprecipitation analysis. The picture that emerges is that DMAP1 activates DNMT1 preferentially at sites of HR repair. Because DMAP1 depleted cells display enhanced HR, we conclude that it has additional roles in genomic stability.

To understand telomere homeostasis, a significant aspect of cancer and growth control, it is important to examine telomerase induction as well as mechanisms of regulated elimination. Makorin-1 (MKRN1) was previously shown to be an E3 ubiquitin ligase that targets the telomerase catalytic subunit (hTERT) for proteasome processing (Kim et al., Genes Dev 19:776-781, 2005). In this study we examined expression and regulation of endogenous MKRN1 during the cell cycle and terminal differentiation. When WI-38 cells transition from active growth into a resting G1 state, basal levels of MKRN1 were found to increase by sixfold. In contrast, cancer cells typically contained low or in some cases undetectable levels of MKRN1 protein. HL-60 cells growing exponentially in culture contain no detectable MKRN1; however, following terminal differentiation, MKRN1 mRNA and protein levels are strongly up-regulated while hTERT mRNA, hTERC, and telomerase are shut down. The initial decrease in telomerase activity is due to a gradual reduction in transcription of the hTERT gene that occurs during the first 12 h of terminal differentiation. MKRN1 protein appears between 12 and 24 h and is attended by a more rapid loss of telomerase activity. As more MKRN1 protein accumulates, significantly less telomerase activity is seen. Addition of the proteasome inhibitor, MG132, reverses the loss of telomerase activity; therefore, reductions in telomerase activity are dynamic, ongoing, and correlated with robust up-regulation of MKRN1 as the cells terminally differentiate. The data are consistent with the idea that MKRN1 represents a telomerase elimination pathway to rapidly draw down the activity during differentiation or cell cycle arrest when telomerase action at chromosome ends is no longer necessary.

The Quality of Life in Late Stage Dementia (QUALID) Scale is an instrument to measure quality of life in patients with severe dementia over the last week by means of an interview with a nurse or nurse aid. We interviewed nurse aids on 48 patients with severe dementia in two nursing homes in The Netherlands to determine the psychometric properties of the Dutch translation of the QUALID. Test- retest reliability was good with an Concordance Correlation Coefficient (CCC) of 0.82, (95% Confidence Interval, CI 0.70 - 0.90); interrater reliability was moderate, with an CCC of 0.49 (CI: 0.17-0.72). There was a significant, but low correlation between QUALID ratings and discomfort ratings as observed with the Discomfort Scale-Dementia of Alzheimer Type (DS-DAT), amounting 0.32 (CI: 0.04 - 0.56). The QUALID seems appropriate for measuring quality of life in patients with advanced dementia, when one and the same rater monitors a patient over time in a longitudinal study.

DNA methylation regulates gene expression through a complex network of protein-protein and protein-DNA interactions in chromatin. The maintenance methylase, DNMT1 (DNA methyltransferase 1), is a prominent enzyme in the process that is linked to DNA replication and drives the heritable nature of epigenetic modifications. The mechanistic details that explain how DNMT1 catalytic action is directed and regulated in chromatin are important in our overall understanding of gene control. In this work, we show that DNMT1 is modified by SUMOylation and we have mapped these SUMOylation sites by defined mutations. SUMOylated DNMT1 is catalytically active on genomic DNA in vivo and we find that SUMOylation significantly enhances the methylase activity of DNMT1 both in vitro and in chromatin. These data suggest that SUMOylation modulates the endogenous activity of a prominent epigenetic maintenance pathway in somatic cells.

Background: The End-of-Life in Dementia (EOLD) scales comprise the most specific set of instruments developed for evaluations of patients' end of life by their families. It is not known whether the EOLD scales are useful for cross-national comparisons., Methods: We used a mortality follow-back design in multi-center studies in the Netherlands (pilot study 2005-2007) and the U.S.A. (1999), and we compared EOLD Satisfaction With Care (SWC; last three months of life), Symptom Management (SM; last three months) and Comfort Assessment in Dying (CAD) scores for 54 Dutch and 76 U.S. nursing home residents., Results: SWC total scores did not differ significantly between the Dutch and U.S. studies (31.9, SD 4.7 versus 30.4, SD 6.1), but three of ten items were rated more favorable for Dutch residents, as were SM total scores (29.1, SD 9.2 versus 20.4, SD 10.6). CAD total scores did not differ (32.0, SD 5.4 versus 30.5, SD 5.9, respectively), but the "well-being" subscale was rated more favorably for Dutch residents. Results were similar after adjustment for demographics and dementia severity., Conclusion: The Dutch families rated end of life with dementia in nursing homes as somewhat better than did U.S. families. Although differences were small, the observed patterns were consistent. This suggests validity of the SM and CAD to assess differences in quality of dying and possible sensitivity to differences between countries or time frames. Larger, simultaneous, cross-national studies are needed to confirm usefulness of the scales and to detect areas which need improvement in the respective countries.

Every year more than 20,000 people with dementia die in Dutch nursing homes and this number steadily increases. Therefore, the importance of good end-of-life care for these patients including physical, psychosocial and spiritual care is evident. Although the training standards for Dutch nursing home physicians and nurses share a common standard, the philosophy of a nursing home may affect end-of-life care strategies for the residents. We compared end of life of nursing home residents with dementia in two anthroposophic and two traditional nursing homes in a retrospective study using the most specific instrument available: the End-of-Life in Dementia scales (EOLD). Family caregivers completed the EOLD questionnaire. There was no difference in mean Satisfaction With Care scale scores between both types of nursing homes: 32.9 (SD 4.3) and 31.6 (SD 4.9), respectively. The anthroposophic nursing homes had significant higher scores on the 'Symptom Management' ((32.9 (SD 7.5) versus 26.9 (SD 9.5)), and 'Comfort Assessment in Dying' scales (34.0 (SD 3.9) versus 30.8 (SD 5.8)) and on its subscale Well Being (7.7 (SD 1.2) versus 6.7 (SD 2.1)). Our results suggest that death with dementia was more favourable in anthroposophic nursing homes than in regular homes. The results inform further prospective studies on nursing homes how this and other philosophies are translated into daily nursing home practice, including decision making in multi-disciplinary teams, family consultation, and complementary non-pharmacological therapies.

To explore the link between DNA damage and gene silencing, we induced a DNA double-strand break in the genome of Hela or mouse embryonic stem (ES) cells using I-SceI restriction endonuclease. The I-SceI site lies within one copy of two inactivated tandem repeated green fluorescent protein (GFP) genes (DR-GFP). A total of 2%-4% of the cells generated a functional GFP by homology-directed repair (HR) and gene conversion. However, approximately 50% of these recombinants expressed GFP poorly. Silencing was rapid and associated with HR and DNA methylation of the recombinant gene, since it was prevented in Hela cells by 5-aza-2'-deoxycytidine. ES cells deficient in DNA methyl transferase 1 yielded as many recombinants as wild-type cells, but most of these recombinants expressed GFP robustly. Half of the HR DNA molecules were de novo methylated, principally downstream to the double-strand break, and half were undermethylated relative to the uncut DNA. Methylation of the repaired gene was independent of the methylation status of the converting template. The methylation pattern of recombinant molecules derived from pools of cells carrying DR-GFP at different loci, or from an individual clone carrying DR-GFP at a single locus, was comparable. ClustalW analysis of the sequenced GFP molecules in Hela and ES cells distinguished recombinant and nonrecombinant DNA solely on the basis of their methylation profile and indicated that HR superimposed novel methylation profiles on top of the old patterns. Chromatin immunoprecipitation and RNA analysis revealed that DNA methyl transferase 1 was bound specifically to HR GFP DNA and that methylation of the repaired segment contributed to the silencing of GFP expression. Taken together, our data support a mechanistic link between HR and DNA methylation and suggest that DNA methylation in eukaryotes marks homologous recombined segments., Competing Interests: Competing interests. The authors have declared that no competing interests exist.

This study used retrospective interviews with 87 relatives to describe the experiences of patients who died by euthanasia or physician-assisted suicide (EAS) in the Netherlands. Most of the patients suffered from cancer (85%). The relatives were most often a partner (63%) or a child (28%) of the patient. Before explicitly requesting EAS most patients (79%) had spoken about their wishes concerning medical end-of-life decisions to be made at a later date. Hopeless suffering, loss of dignity, and no prospect of recovery were the most prevalent reasons for explicitly requesting EAS. According to the relative, in 92% of patients EAS had contributed favourably to the quality of the end of life, mainly by preventing or ending suffering.

Carbohydrate moiety is found in many anticancer nature products. To explore the carbohydrate moiety of daunorubicin in enhancing anticancer efficacy, several daunorubicin derivatives bearing disaccharide (1-8) have been synthesized. Their cytotoxicities were tested in leukemia K562 and colon cancer SW620 cells. Topoisomerase II (topo II) poisoning was performed with the in vivo complex of topoisomerase bioassay. In both cell lines, compounds with various terminal 2,6-dideoxy sugars (compounds 1, 3, 5, and 8) showed 30- to 60-fold higher anticancer activity than compounds with 2-deoxy- or 6-deoxy sugar (compounds 6 and 7). Compounds with an alpha-linkage between two sugar units (compound 3) showed 35-fold higher anticancer activity than compounds with a beta-linkage (compound 4). In addition, the anticancer activities of these compounds correlated with their ability to target topo II mediated genomic DNA damage in vivo. Compounds 1 and 3 with 2,6-dideoxy sugars produced more covalent topo-DNA complex than compounds with 2-deoxy sugar (6) and 6-deoxy sugar (7). Compounds with an alpha-configuration of terminal 2,6-dideoxy sugar (compounds 1 and 3) showed higher topo II poisoning than their counterparts with the beta-configuration (compounds 2 and 4). These results indicate that sugar moieties in daunorubicin play a significant role in its anticancer activity and topo II inhibition. The second sugar of disaccharide daunorubicin should possess 2,6-dideoxy with alpha-linkage to the first sugar to exhibit better anticancer activity.

Rebeccamycin analogues containing uncommon sugars and substitutions on the imide nitrogen have been synthesized. Their cytotoxicities were tested in colon cancer and leukemia cells. Their ability to target topoisomerase I was examined using the in vivo complex of the topoisomerase bioassay in Hela cells. Compared with aglycon 1, the modified compounds with various sugar moieties showed more potent cytotoxicities and topo I targeting ability. In addition, the rebeccamycin analogues with various uncommon sugars showed distinct cytotoxicities and topo I targeting activities. The activity of compounds with 2-deoxyglucose (8 and 9) > compounds with 2,6-deoxyglucose (5 and 6) > compounds with 2,3,6-deoxyglucose (10). Furthermore, the anticancer activity of compounds correlated with their ability to target endogenous topo I. These results suggest that the sugar moiety, especially the 2-OH and 6-OH group of the sugar, rather than the modifications in imide structure on the indolocarbazole ring, is a key element for its activity.