Promoter- and cell-specific epigenetic regulation of CD44, Cyclin D2, GLIPR1 and PTEN by Methyl-CpG binding proteins and histone modifications

<p>Abstract</p> <p><it>Background</it></p> <p>The aim of the current study was to analyze the involvement of methyl-CpG binding proteins (MBDs) and histone modifications on the regulation of CD44, Cyclin D2, GLIPR1 and PTEN in different cellular contexts such as the prostate cancer cells DU145 and LNCaP, and the breast cancer cells MCF-7. Since global chromatin changes have been shown to occur in tumours and regions of tumour-associated genes are affected by epigenetic modifications, these may constitute important regulatory mechanisms for the pathogenesis of malignant transformation.</p> <p><it>Methods</it></p> <p>In DU145, LNCaP and MCF-7 cells mRNA expression levels of CD44, Cyclin D2, GLIPR1 and PTEN were determined by quantitative RT-PCR at the basal status as well as after treatment with demethylating agent 5-aza-2'-deoxycytidine and/or histone deacetylase inhibitor Trichostatin A. Furthermore, genomic DNA was bisulfite-converted and sequenced. Chromatin immunoprecipitation was performed with the stimulated and unstimulated cells using antibodies for MBD1, MBD2 and MeCP2 as well as 17 different histone antibodies.</p> <p><it>Results</it></p> <p>Comparison of the different promoters showed that MeCP2 and MBD2a repressed promoter-specifically Cyclin D2 in all cell lines, whereas in MCF-7 cells MeCP2 repressed cell-specifically all methylated promoters. Chromatin immunoprecipitation showed that all methylated promoters associated with at least one MBD. Treatment of the cells by the demethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) caused dissociation of the MBDs from the promoters. Only MBD1v1 bound and repressed methylation-independently all promoters. Real-time amplification of DNA immunoprecipitated by 17 different antibodies showed a preferential enrichment for methylated lysine of histone H3 (H3K4me1, H3K4me2 and H3K4me3) at the particular promoters. Notably, the silent promoters were associated with unmodified histones which were acetylated following treatment by 5-aza-CdR.</p> <p><it>Conclusions</it></p> <p>This study is one of the first to reveal the histone code and MBD profile at the promoters of CD44, Cyclin D2, GLIPR1 and PTEN in different tumour cells and associated changes after stimulation with methylation inhibitor 5-aza-CdR.</p

Fatores de risco para quedas em pacientes adultos hospitalizados: um estudo caso-controle

Objective: to identify risk factors for falls in hospitalized adult patients. Methods: a matched case-control study (one control for each case). A quantitative study conducted in clinical and surgical units of a teaching hospital in Southern Brazil. The sample comprised 358 patients. Data were collected over 18 months between 2013-2014. Data analysis was performed with descriptive statistics and conditional logistic regression using Microsoft Excel and SPSS version 18.0. Results: risk factors identified were: disorientation/confusion [OR 4.25 (1.99 to 9.08), p&lt;0.001]; frequent urination [OR 4.50 (1.86 to 10.87), p=0.001]; walking limitation [OR 4.34 (2.05 to 9.14), p&lt;0.001]; absence of caregiver [OR 0.37 (0.22 to 0.63), p&lt;0.001]; postoperative period [OR 0.50 (0.26 to 0.94), p=0.03]; and number of medications administered within 72 hours prior the fall [OR 1.20 (1.04 to 1.39) p=0.01]. Conclusion: risk for falls is multifactorial. However, understanding these factors provides support to clinical decision-making and positively influences patient safety.Objetivo: identificar los factores de riesgo para la ocurrencia de caídas en pacientes adultos hospitalizados. Métodos: un estudio caso-control emparejado (un control para cada caso). Investigación cuantitativa llevada a cabo en unidades clínicas y quirúrgicas de un hospital universitario en el Sur de Brasil. La muestra constó de 358 pacientes. Se recopilaron datos durante 18 meses, entre 2013-2014. El análisis de los datos se realizó mediante estadística descriptiva y regresión logística condicional, utilizando el Microsoft Excel y el SPSS versión 18.0. Resultados: los factores de riesgo identificados fueron: desorientación/confusión [OR 4,25 (1,99 a 9,08), p&lt;0,001]; micción frecuente [OR 4,50 (1,86 a 10,87), p=0,001]; limitación para caminar [OR 4,34 (2,05 a 9,14), p&lt;0,001]; ausencia de cuidadores [OR 0,37 (0,22 a 0,63), p&lt;0,001]; período postoperatorio [OR 0,50 (0,26 a 0,94), p=0,03]; y número de medicamentos administrados dentro de las 72 horas previas a la caída [OR 1,20 (1,04 a 1,39) p=0,01]. Conclusión: los riesgos de caídas son multifactoriales. Sin embargo, la comprensión de estos factores respalda la toma de decisiones clínicas y tiene un impacto positivo en la seguridad del paciente.Objetivo: identificar os fatores de risco para a ocorrência de quedas em pacientes adultos hospitalizados. Métodos: estudo do tipo caso-controle pareado (um controle para cada caso). Pesquisa quantitativa realizada em unidades clínicas e cirúrgicas de um hospital universitário da região Sul do Brasil. A amostra incluiu 358 pacientes. Os dados foram coletados durante 18 meses, entre 2013-2014. A análise dos dados foi realizada por meio de estatística descritiva e regressão logística condicional, utilizando o Microsoft Excel e o SPSS versão 18.0. Resultados: os fatores de risco identificados foram: desorientação/confusão [OR 4,25 (1,99 a 9,08), p&lt;0,001]; micção frequente [OR 4,50 (1,86 a 10,87), p=0,001]; limitação para caminhar [OR 4,34 (2,05 a 9,14), p&lt;0,001]; ausência de cuidador [OR 0,37 (0,22 a 0,63), p&lt;0,001]; período pós-operatório [OR 0,50 (0,26 a 0,94), p=0,03]; e o número de medicamentos administrados nas 72 horas anteriores à queda [OR 1,20 (1,04 a 1,39) p=0,01]. Conclusão: os riscos para quedas são multifatoriais. Todavia, conhecê-los dá suporte à decisão clínica do enfermeiro, o que contribui para a busca das melhores intervenções preventivas e impacta positivamente na segurança dos pacientes

Standardizing admission and discharge processes to improve patient flow: a cross sectional study

Background: The aim of this study was to evaluate how hospital capacity was managed focusing on standardizing the admission and discharge processes. Methods: This study was set in a 900-bed university affiliated hospital of the National Health Service, near Barcelona (Spain). This is a cross-sectional study of a set of interventions which were gradually implemented between April and December 2008. Mainly, they were focused on standardizing the admission and discharge processes to improve patient flow. Primary administrative data was obtained from the 2007 and 2009 Hospital Database. Main outcome measures were median length of stay, percentage of planned discharges, number of surgery cancellations and median number of delayed emergency admissions at 8:00 am. For statistical bivariate analysis, we used a Chi-squared for linear trend for qualitative variables and a Wilcoxon signed ranks test and a Mann-Whitney test for non-normal continuous variables. Results: The median patients' global length of stay was 8.56 days in 2007 and 7.93 days in 2009 (p < 0.051). The percentage of patients admitted the same day as surgery increased from 64.87% in 2007 to 86.01% in 2009 (p < 0.05). The number of cancelled interventions due to lack of beds was 216 patients in 2007 and 42 patients in 2009. The median number of planned discharges went from 43.05% in 2007 to 86.01% in 2009 (p < 0.01). The median number of emergency patients waiting for an in-hospital bed at 8:00 am was 5 patients in 2007 and 3 patients in 2009 (p < 0.01). Conclusions: In conclusion, standardization of admission and discharge processes are largely in our control. There is a significant opportunity to create important benefits for increasing bed capacity and hospital throughput

Methylation-Dependent Binding of the Epstein-Barr Virus BZLF1 Protein to Viral Promoters

The switch between latent and lytic Epstein-Barr virus (EBV) infection is mediated by the viral immediate-early (IE) protein, BZLF1 (Z). Z, a homologue of c-jun that binds to AP1-like motifs (ZREs), induces expression of the BRLF1 (R) and BRRF1 (Na) viral proteins, which cooperatively activate transcription of the Z promoter and thereby establish a positive autoregulatory loop. A unique feature of Z is its ability to preferentially bind to, and activate, the methylated form of the BRLF1 promoter (Rp). To date, however, Rp is the only EBV promoter known to be regulated in this unusual manner. We now demonstrate that the promoter driving transcription of the early BRRF1 gene (Nap) has two CpG-containing ZREs (ACGCTCA and TCGCCCG) that are only bound by Z in the methylated state. Both Nap ZREs are highly methylated in cells with latent EBV infection. Z efficiently activates the methylated, but not unmethylated, form of Nap in reporter gene assays, and both ZREs are required. Z serine residue 186, which was previously shown to be required for Z binding to methylated ZREs in Rp, but not for Z binding to the AP1 site, is required for Z binding to methylated Nap ZREs. The Z(S186A) mutant cannot activate methylated Nap in reporter gene assays and does not induce Na expression in cells with latent EBV infection. Molecular modeling studies of Z bound to the methylated Nap ZREs help to explain why methylation is required for Z binding, and the role of the Z Ser186 residue. Methylation-dependent Z binding to critical viral promoters may enhance lytic reactivation in latently infected cells, where the viral genome is heavily methylated. Conversely, since the incoming viral genome is initially unmethylated, methylation-dependent Z activation may also help the virus to establish latency following infection

Anti- Japanese-Encephalitis-Viral Effects of Kaempferol and Daidzin and Their RNA-Binding Characteristics

Background: New therapeutic tools and molecular targets are needed for treatment of Japanese encephalitis virus (JEV) infections. JEV requires an a-1 translational frameshift to synthesize the NS1 ’ protein required for viral neuroinvasiveness. Several flavonoids have been shown to possess antiviral activity in vitro against a wide spectrum of viruses. To date, the antiviral activities of flavonol kaempferol (Kae) and isoflavonoid daidzin (Dai) against JEV have not been described. Methodology/Principal Findings: The 50 % cytotoxic concentration (CC50) and 50 % effective concentration (EC50) against JEV were investigated in BHK21 cells by MTS reduction. Activity against viral genomic RNA and proteins was measured by real-time RT-PCR and western blotting. The frameshift site RNA-binding characterization was also determined by electrospray ionization mass spectrometry, isothermal titration calorimetry and autodocking analysis. EC 50 values of Kae and Dai were 12.6 and 25.9 mM against JEV in cells pretreated before infection, whereas in cells infected before treatment, EC50 was 21.5 and 40.4 mM, respectively. Kae exhibited more potent activity against JEV and RNA binding in cells following internalization through direct inhibition of viral replication and protein expression, indicating that its antiviral activity was principally due to direct virucidal effects. The JEV frameshift site RNA (fsRNA) was selected as a target for assaying Kae and Dai. ITC of fsRNA revealed an apparent Kb value for Kae that was nine fold stronger than that for Dai. This binding was confirmed and localized to the RNA using ESI-MS and autodock analysis. Kae could form non-covalent complexes wit

DNA Barcoding for Community Ecology - How to Tackle a Hyperdiverse, Mostly Undescribed Melanesian Fauna

Trigonopterus weevils are widely distributed throughout Melanesia and hyperdiverse in New Guinea. They are a dominant feature in natural forests, with narrow altitudinal zonation. Their use in community ecology has been precluded by the "taxonomic impediment". We sampled >6,500 specimens from seven areas across New Guinea; 1,002 specimens assigned to 270 morphospecies were DNA sequenced. Objective clustering of a refined dataset (excluding nine cryptic species) at 3% threshold revealed 324 genetic clusters (DNA group count relative to number of morphospecies = 20.0% overestimation of species diversity, or 120.0% agreement) and 85.6% taxonomic accuracy (the proportion of DNA groups that "perfectly" agree with morphology-based species hypotheses). Agreement and accuracy were best at an 8% threshold. GMYC analysis revealed 328 entities (21.5% overestimation) with 227 perfect GMYC entities (84.1% taxonomic accuracy). Both methods outperform the parataxonomist (19% underestimation; 31.6% taxonomic accuracy). The number of species found in more than one sampling area was highest in the Eastern Highlands and Huon (Sørensen similarity index 0.07, 4 shared species); ⅓ of all areas had no species overlap. Success rates of DNA barcoding methods were lowest when species showed a pronounced geographical structure. In general, Trigonopterus show high α and β-diversity across New Guinea. DNA barcoding is an excellent tool for biodiversity surveys but success rates might drop when closer localities are included. Hyperdiverse Trigonopterus are a useful taxon for evaluating forest remnants in Melanesia, allowing finer-grained analyses than would be possible with vertebrate taxa commonly used to date. Our protocol should help establish other groups of hyperdiverse fauna as target taxa for community ecology. Sequencing delivers objective data on taxa of incredible diversity but mostly without a solid taxonomic foundation and should help pave the road for the eventual formal naming of new species

Implementation fidelity of a nurse-led falls prevention program in acute hospitals during the 6-PACK trial

Background: When tested in a randomized controlled trial (RCT) of 31,411 patients, the nurse-led 6-PACK falls prevention program did not reduce falls. Poor implementation fidelity (i.e., program not implemented as intended) may explain this result. Despite repeated calls for the examination of implementation fidelity as an essential component of evaluating interventions designed to improve the delivery of care, it has been neglected in prior falls prevention studies. This study examined implementation fidelity of the 6-PACK program during a large multi-site RCT. Methods: Based on the 6-PACK implementation framework and intervention description, implementation fidelity was examined by quantifying adherence to program components and organizational support. Adherence indicators were: 1) falls-risk tool completion; and for patients classified as high-risk, provision of 2) a ‘Falls alert’ sign; and 3) at least one additional 6-PACK intervention. Organizational support indicators were: 1) provision of resources (executive sponsorship, site clinical leaders and equipment); 2) implementation activities (modification of patient care plans; training; implementation tailoring; audits, reminders and feedback; and provision of data); and 3) program acceptability. Data were collected from daily bedside observation, medical records, resource utilization diaries and nurse surveys. Results: All seven intervention components were delivered on the 12 intervention wards. Program adherence data were collected from 103,398 observations and medical record audits. The falls-risk tool was completed each day for 75% of patients. Of the 38% of patients classified as high-risk, 79% had a ‘Falls alert’ sign and 63% were provided with at least one additional 6-PACK intervention, as recommended. All hospitals provided the recommended resources and undertook the nine outlined program implementation activities. Most of the nurses surveyed considered program components important for falls prevention. Conclusions: While implementation fidelity was variable across wards, overall it was found to be acceptable during the RCT. Implementation failure is unlikely to be a key factor for the observed lack of program effectiveness in the 6-PACK trial. Trial registration: The 6-PACK cluster RCT is registered with the Australian New Zealand Clinical Trials Registry, number ACTRN12611000332921 (29 March 2011)

A systematic review of physiological methods in rodent pharmacological MRI studies

Rationale: Pharmacological magnetic resonance imaging (phMRI) provides an approach to study effects of drug challenges on brain processes. Elucidating mechanisms of drug action helps us to better understand the workings of neurotransmitter systems, map brain function or facilitate drug development. phMRI is increasingly used in preclinical research employing rodent models; however, data interpretation and integration are complicated by the use of different experimental approaches between laboratories. In particular, the effects of different anaesthetic regimes upon neuronal and haemodynamic processes and baseline physiology could be problematic. Objectives: This paper investigates how differences in phMRI research methodologies are manifested and considers associated implications, placing particular emphasis on choice of anaesthetic regimes. Methods: A systematic review of rodent phMRI studies was conducted. Factors such as those describing anaesthetic regimes (e.g. agent, dosage) and parameters relating to physiological maintenance (e.g. ventilatory gases) and MRI method were recorded. Results: We identified 126 eligible studies and found that the volatile agents isoflurane (43.7 %) and halothane (33.3 %) were most commonly used for anaesthesia, but dosage and mixture of ventilatory gases varied substantially between laboratories. Relevant physiological parameters were usually recorded, although 32 % of studies did not provide cardiovascular measures. Conclusions: Anaesthesia and animal preparation can influence phMRI data profoundly. The variation of anaesthetic type, dosage regime and ventilatory gases makes consolidation of research findings (e.g. within a specific neurotransmitter system) difficult. Standardisation of a small(er) number of preclinical phMRI research methodologies and/or increased consideration of approaches that do not require anaesthesia is necessary to address these challenges

DNA methylation and methyl-CpG binding proteins: developmental requirements and function

DNA methylation is a major epigenetic modification in the genomes of higher eukaryotes. In vertebrates, DNA methylation occurs predominantly on the CpG dinucleotide, and approximately 60% to 90% of these dinucleotides are modified. Distinct DNA methylation patterns, which can vary between different tissues and developmental stages, exist on specific loci. Sites of DNA methylation are occupied by various proteins, including methyl-CpG binding domain (MBD) proteins which recruit the enzymatic machinery to establish silent chromatin. Mutations in the MBD family member MeCP2 are the cause of Rett syndrome, a severe neurodevelopmental disorder, whereas other MBDs are known to bind sites of hypermethylation in human cancer cell lines. Here, we review the advances in our understanding of the function of DNA methylation, DNA methyltransferases, and methyl-CpG binding proteins in vertebrate embryonic development. MBDs function in transcriptional repression and long-range interactions in chromatin and also appear to play a role in genomic stability, neural signaling, and transcriptional activation. DNA methylation makes an essential and versatile epigenetic contribution to genome integrity and function