A complex regional intervention to implement advance care planning in one town's nursing homes: Protocol of a controlled inter-regional study

<p>Abstract</p> <p>Background</p> <p>Advance Care Planning (ACP) is an emerging strategy to ensure that well-reflected, meaningful and clearly documented treatment preferences are available and respected when critical decisions about life-sustaining treatment need to be made for patients unable to consent. In Germany, recent legislation confirms that advance directives (AD) have to be followed if they apply to the medical situation, but implementation of ACP has not yet been described.</p> <p>Methods/Design</p> <p>In a longitudinal controlled study, we compare 1 intervention region (4 nursing homes [n/hs], altogether 421 residents) with 2 control regions (10 n/hs, altogether 985 residents). Inclusion went from 01.02.09 to 30.06.09, observation lasted until 30.06.10. Primary endpoint is the prevalence of ADs at follow-up, 17 (12) months after the first (last) possible inclusion. Secondary endpoints compare relevance and validity of ADs, process quality, the rate of life-sustaining interventions and, in deceased residents, location of death and intensity of treatment before death. The regional multifaceted intervention on the basis of the US program Respecting Choices^®comprises training of n/h staff as facilitators, training of General Practitioners, education of hospital and ambulance staff, and development of eligible tools, including Physician Orders for Life-Sustaining Treatment in case of Emergency (POLST-E).</p> <p><it>Participation data: </it>Of 1406 residents reported to live in the 14 n/hs plus an estimated turnover of 176 residents until the last possible inclusion date, 645 (41%) were willing to participate. Response rates were 38% in the intervention region and 42% in the control region. Non-responder analysis shows an equal distribution of sex and age but a bias towards dependency on nursing care in the responder group. <it>Outcome analysis </it>of this study will become available in the course of 2011.</p> <p>Discussion</p> <p>Implementing an ACP program for the n/hs and related health care providers of a region requires a complex community intervention with the effect of nothing less than a cultural shift in this health care sector. This study is to our knowledge the first to develop a strategy for regional implementation of ACP, and to evaluate its feasibility in a controlled design.</p> <p>Trial Registration</p> <p>ISRCTN: <a href="http://www.controlled-trials.com/ISRCTN99887420">ISRCTN99887420</a></p

DNA Structure Modulates the Oligomerization Properties of the AAV Initiator Protein Rep68

Rep68 is a multifunctional protein of the adeno-associated virus (AAV), a parvovirus that is mostly known for its promise as a gene therapy vector. In addition to its role as initiator in viral DNA replication, Rep68 is essential for site-specific integration of the AAV genome into human chromosome 19. Rep68 is a member of the superfamily 3 (SF3) helicases, along with the well-studied initiator proteins simian virus 40 large T antigen (SV40-LTag) and bovine papillomavirus (BPV) E1. Structurally, SF3 helicases share two domains, a DNA origin interaction domain (OID) and an AAA+ motor domain. The AAA+ motor domain is also a structural feature of cellular initiators and it functions as a platform for initiator oligomerization. Here, we studied Rep68 oligomerization in vitro in the presence of different DNA substrates using a variety of biophysical techniques and cryo-EM. We found that a dsDNA region of the AAV origin promotes the formation of a complex containing five Rep68 subunits. Interestingly, non-specific ssDNA promotes the formation of a double-ring Rep68, a known structure formed by the LTag and E1 initiator proteins. The Rep68 ring symmetry is 8-fold, thus differing from the hexameric rings formed by the other SF3 helicases. However, similiar to LTag and E1, Rep68 rings are oriented head-to-head, suggesting that DNA unwinding by the complex proceeds bidirectionally. This novel Rep68 quaternary structure requires both the DNA binding and AAA+ domains, indicating cooperativity between these regions during oligomerization in vitro. Our study clearly demonstrates that Rep68 can oligomerize through two distinct oligomerization pathways, which depend on both the DNA structure and cooperativity of Rep68 domains. These findings provide insight into the dynamics and oligomeric adaptability of Rep68 and serve as a step towards understanding the role of this multifunctional protein during AAV DNA replication and site-specific integration

Apoptosis is associated with triacylglycerol accumulation in Jurkat T-cells

Magnetic resonance spectroscopy is increasingly used as a non-invasive method to investigate apoptosis. Apoptosis was induced in Jurkat T-cells by Fas mAb. 1H magnetic resonance spectra of live cells showed an increase in methylene signal as well as methylene/methyl ratio of fatty acid side chains at 5 and 24 h following induction of apoptosis. To explain this observation, 1H magnetic resonance spectra of cell extracts were investigated. These demonstrated a 70.0±7.0%, 114.0±8.0% and 90.0±5.0% increase in the concentration of triacylglycerols following 3, 5 and 7 h of Fas mAb treatment (P<0.05). Confocal microscopy images of cells stained with the lipophilic dye Nile Red demonstrated the presence of lipid droplets in the cell cytoplasm. Quantification of the stained lipids by flow cytometry showed a good correlation with the magnetic resonance results (P⩾0.05 at 3, 5 and 7 h). 31P magnetic resonance spectra showed a drop in phosphatidylcholine content of apoptosing cells, indicating that alteration in phosphatidylcholine metabolism could be the source of triacylglycerol accumulation during apoptosis. In summary, apoptosis is associated with an early accumulation of mobile triacylglycerols mostly in the form of cytoplasmic lipid droplets. This is reflected in an increase in the methylene/methyl ratio which could be detected by magnetic resonance spectroscopy

Astrocytes are important mediators of Aβ-induced neurotoxicity and tau phosphorylation in primary culture

Alzheimer's disease (AD) is pathologically characterised by the age-dependent deposition of β-amyloid (Aβ) in senile plaques, intraneuronal accumulation of tau as neurofibrillary tangles, synaptic dysfunction and neuronal death. Neuroinflammation, typified by the accumulation of activated microglia and reactive astrocytes, is believed to modulate the development and/or progression of AD. We have used primary rat neuronal, astrocytic and mixed cortical cultures to investigate the contribution of astrocyte-mediated inflammatory responses during Aβ-induced neuronal loss. We report that the presence of small numbers of astrocytes exacerbate Aβ-induced neuronal death, caspase-3 activation and the production of caspase-3-cleaved tau. Furthermore, we show that astrocytes are essential for the Aβ-induced tau phosphorylation observed in primary neurons. The release of soluble inflammatory factor(s) from astrocytes accompanies these events, and inhibition of astrocyte activation with the anti-inflammatory agent, minocycline, reduces astrocytic inflammatory responses and the associated neuronal loss. Aβ-induced increases in caspase-3 activation and the production of caspase-3-truncated tau species in neurons were reduced when the astrocytic response was attenuated with minocycline. Taken together, these results show that astrocytes are important mediators of the neurotoxic events downstream of elevated Aβ in models of AD, and suggest that mechanisms underlying pro-inflammatory cytokine release might be an important target for therapy

The frequency of genes encoding three putative group B streptococcal virulence factors among invasive and colonizing isolates

BACKGROUND: Group B Streptococcus (GBS) causes severe infections in very young infants and invasive disease in pregnant women and adults with underlying medical conditions. GBS pathogenicity varies between and within serotypes, with considerable variation in genetic content between strains. Three proteins, Rib encoded by rib, and alpha and beta C proteins encoded by bca and bac, respectively, have been suggested as potential vaccine candidates for GBS. It is not known, however, whether these genes occur more frequently in invasive versus colonizing GBS strains. METHODS: We screened 162 invasive and 338 colonizing GBS strains from different collections using dot blot hybridization to assess the frequency of bca, bac and rib. All strains were defined by serotyping for capsular type, and frequency differences were tested using the Chi square test. RESULTS: Genes encoding the beta C protein (bac) and Rib (rib) occurred at similar frequencies among invasive and colonizing isolates, bac (20% vs. 23%), and rib (28% vs. 20%), while the alpha (bca) C protein was more frequently found in colonizing strains (46%) vs, invasive (29%). Invasive strains were associated with specific serotype/gene combinations. CONCLUSION: Novel virulence factors must be identified to better understand GBS disease

C2-phytoceramide perturbs lipid rafts and cell integrity in Saccharomyces cerevisiae in a sterol-dependent manner

Specific ceramides are key regulators of cell fate, and extensive studies aimed to develop therapies based on ceramide-induced cell death. However, the mechanisms regulating ceramide cytotoxicity are not yet fully elucidated. Since ceramides also regulate growth and stress responses in yeast, we studied how different exogenous ceramides affect yeast cells. C2-phytoceramide, a soluble form of phytoceramides, the yeast counterparts of mammalian ceramides, greatly reduced clonogenic survival, particularly in the G2/M phase, but did not induce autophagy nor increase apoptotic markers. Rather, the loss of clonogenic survival was associated with PI positive staining, disorganization of lipid rafts and cell wall weakening. Sensitivity to C2-phytoceramide was exacerbated in mutants lacking Hog1p, the MAP kinase homolog of human p38 kinase. Decreasing sterol membrane content reduced sensitivity to C2-phytoceramide, suggesting sterols are the targets of this compound. This study identified a new function of C2-phytoceramide through disorganization of lipid rafts and induction of a necrotic cell death under hypo-osmotic conditions. Since lipid rafts are important in mammalian cell signaling and adhesion, our findings further support pursuing the exploitation of yeast to understand the basis of synthetic ceramides' cytotoxicity to provide novel strategies for therapeutic intervention in cancer and other diseases.This work was supported by Fundacao para a Ciencia e Tecnologia through projects PTDC/BIA-BCM/69448/2006 and PEst-C/BIA/UI4050/2011, and fellowships to A. P. (SFRH/BPD/65003) and F. A. (SFRH/BD/80934/2011), as well as by FEDER through POFC - COMPETE. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Survivin expression in ovarian cancer and its correlation with clinico-pathological, surgical and apoptosis-related parameters

We investigated the association of survivin expression with prognosis and other apoptosis-related biological factors in 110 primary ovarian cancer patients admitted to the Division of Gynecologic Oncology, Catholic University of Rome. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections by using polyclonal antibody ab469 for survivin, and mouse monoclonal antibodies (clone 124 and DO-7), for bcl-2 and p53, respectively. Cytoplasmic survivin immunoreaction was observed in 84.5% cases, while nuclear survivin immunostaining was observed in 29.1% cases. We failed to find any relationship between cytoplasmic survivin positivity rate and any of the parameters examined. Serous tumours showed a lower percentage of nuclear survivin positivity with respect to other histotypes (20.5 vs 48.6%, respectively; P-value=0.004). The percentage of nuclear survivin positivity was higher in cases subjected to primary tumour cytoreduction (43.5%), with respect to patients subjected to exploratory laparotomy (20%) (P=0.024). Bcl-2 and p53 were, respectively, expressed in 27.3 and 60.0% of the cases and their expression was not correlated with survivin status. During the follow-up period, progression and death of disease were observed in 68 (61.8%) and 53 (48.2%) cases, respectively. There was no difference in time to progression and overall survival according to survivin status in ovarian cancer patients. In conclusion, in our experience, the immunohistochemical assessment of survivin status does not seem to be helpful in the prognostic characterisation of ovarian cancer. A more in depth investigation of the complex physiology of divergent survivin variants is needed in order to clarify the biological and the clinical role of differentially located survivin isoforms

Routine care provided by specialists to children and adolescents in the United States (2002-2006).

BACKGROUND: Specialist physicians provide a large share of outpatient health care for children and adolescents in the United States, but little is known about the nature and content of these services in the ambulatory setting. Our objective was to quantify and characterize routine and co-managed pediatric healthcare as provided by specialists in community settings. METHODS: Nationally representative data were obtained from the National Ambulatory Medical Care Survey for the years 2002-2006. We included office based physicians (excluding family physicians, general internists and general pediatricians), and a representative sample of their patients aged 18 or less. Visits were classified into mutually exclusive categories based on the major reason for the visit, previous knowledge of the health problem, and whether the visit was the result of a referral. Primary diagnoses were classified using Expanded Diagnostic Clusters. Physician report of sharing care for the patient with another physician and frequency of reappointments were also collected. RESULTS: Overall, 41.3% out of about 174 million visits were for routine follow up and preventive care of patients already known to the specialist. Psychiatry, immunology and allergy, and dermatology accounted for 54.5% of all routine and preventive care visits. Attention deficit disorder, allergic rhinitis and disorders of the sebaceous glands accounted for about a third of these visits. Overall, 73.2% of all visits resulted in a return appointment with the same physician, in half of all cases as a result of a routine or preventive care visit. CONCLUSION: Ambulatory office-based pediatric care provided by specialists includes a large share of non referred routine and preventive care for common problems for patients already known to the physician. It is likely that many of these services could be managed in primary care settings, lessening demand for specialists and improving coordination of care.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Assembly and Development of the Pseudomonas aeruginosa Biofilm Matrix

Virtually all cells living in multicellular structures such as tissues and organs are encased in an extracellular matrix. One of the most important features of a biofilm is the extracellular polymeric substance that functions as a matrix, holding bacterial cells together. Yet very little is known about how the matrix forms or how matrix components encase bacteria during biofilm development. Pseudomonas aeruginosa forms environmentally and clinically relevant biofilms and is a paradigm organism for the study of biofilms. The extracellular polymeric substance of P. aeruginosa biofilms is an ill-defined mix of polysaccharides, nucleic acids, and proteins. Here, we directly visualize the product of the polysaccharide synthesis locus (Psl exopolysaccharide) at different stages of biofilm development. During attachment, Psl is anchored on the cell surface in a helical pattern. This promotes cell–cell interactions and assembly of a matrix, which holds bacteria in the biofilm and on the surface. Chemical dissociation of Psl from the bacterial surface disrupted the Psl matrix as well as the biofilm structure. During biofilm maturation, Psl accumulates on the periphery of 3-D-structured microcolonies, resulting in a Psl matrix-free cavity in the microcolony center. At the dispersion stage, swimming cells appear in this matrix cavity. Dead cells and extracellular DNA (eDNA) are also concentrated in the Psl matrix-free area. Deletion of genes that control cell death and autolysis affects the formation of the matrix cavity and microcolony dispersion. These data provide a mechanism for how P. aeruginosa builds a matrix and subsequently a cavity to free a portion of cells for seeding dispersal. Direct visualization reveals that Psl is a key scaffolding matrix component and opens up avenues for therapeutics of biofilm-related complications