Intradermal DNA electroporation induces survivin-specific CTLs, suppresses angiogenesis and confers protection against mouse melanoma

Survivin is an intracellular tumor-associated antigen that is broadly expressed in a large variety of tumors and also in tumor associated endothelial cells but mostly absent in differentiated tissues. Naked DNA vaccines targeting survivin have been shown to induce T cell as well as humoral immune responses in mice. However, the lack of epitope-specific CD8+ T cell detection and modest tumor protection observed highlight the need for further improvements to develop effective survivin DNA vaccination approaches. Here, the efficacy of a human survivin DNA vaccine delivered by intradermal electroporation (EP) was tested. The CD8+ T cell epitope surv_20–28 restricted to H-2 Db was identified based on in-silico epitope prediction algorithms and binding to MHC class I molecules. Intradermal DNA EP of mice with a human survivin encoding plasmid generated CD8+ cytotoxic T lymphocyte (CTL) responses cross-reactive with the mouse epitope surv_20–28, as determined by intracellular IFN-γ staining, suggesting that self-tolerance has been broken. Survivin-specific CTLs displayed an activated effector phenotype as determined by CD44 and CD107 up-regulation. Vaccinated mice displayed specific cytotoxic activity against B16 and peptide-pulsed RMA-S cells in vitro as well as against surv_20–28 peptide-pulsed target cells in vivo. Importantly, intradermal EP with a survivin DNA vaccine suppressed angiogenesis in vivo and elicited protection against highly aggressive syngeneic B16 melanoma tumor challenge. We conclude that intradermal EP is an attractive method for delivering a survivin DNA vaccine that should be explored also in clinical studies.     

The miR-17-92 microRNA cluster regulates multiple components of the TGF-β pathway in neuroblastoma

The miR-17-92 microRNA cluster is often activated in cancer cells, but the identity of its targets remains elusive. Using SILAC and quantitative mass spectrometry, we examined the effects of activation of the miR-17-92 cluster on global protein expression in neuroblastoma (NB) cells. Our results reveal cooperation between individual miR-17-92 miRNAs and implicate miR-17-92 in multiple hallmarks of cancer, including proliferation and cell adhesion. Most importantly, we show that miR-17-92 is a potent inhibitor of TGF-β signaling. By functioning both upstream and downstream of pSMAD2, miR-17-92 activation triggers downregulation of multiple key effectors along the TGF-β signaling cascade as well as direct inhibition of TGF-β-responsive genes.     

Metal-Free ALS Variants of Dimeric Human Cu,Zn-Superoxide Dismutase Have Enhanced Populations of Monomeric Species

Amino acid replacements at dozens of positions in the dimeric protein human, Cu,Zn superoxide dismutase (SOD1) can cause amyotrophic lateral sclerosis (ALS). Although it has long been hypothesized that these mutations might enhance the populations of marginally-stable aggregation-prone species responsible for cellular toxicity, there has been little quantitative evidence to support this notion. Perturbations of the folding free energy landscapes of metal-free versions of five ALS-inducing variants, A4V, L38V, G93A, L106V and S134N SOD1, were determined with a global analysis of kinetic and thermodynamic folding data for dimeric and stable monomeric versions of these variants. Utilizing this global analysis approach, the perturbations on the global stability in response to mutation can be partitioned between the monomer folding and association steps, and the effects of mutation on the populations of the folded and unfolded monomeric states can be determined. The 2- to 10-fold increase in the population of the folded monomeric state for A4V, L38V and L106V and the 80- to 480-fold increase in the population of the unfolded monomeric states for all but S134N would dramatically increase their propensity for aggregation through high-order nucleation reactions. The wild-type-like populations of these states for the metal-binding region S134N variant suggest that even wild-type SOD1 may also be prone to aggregation in the absence of metals.     

The Escherichia coli BarA-UvrY two-component system is needed for efficient switching between glycolytic and gluconeogenic carbon sources

The Escherichia coli BarA and UvrY proteins were recently demonstrated to constitute a novel two-component system, although its function has remained largely elusive. Here we show that mutations in the sensor kinase gene, barA, or the response regulator gene, uvrY, in uropathogenic E. coli drastically affect survival in long-term competition cultures. Using media with gluconeogenic carbon sources, the mutants have a clear growth advantage when competing with the wild type, but using media with carbon sources feeding into the glycolysis leads to a clear growth advantage for the wild type. Results from competitions with mutants in the carbon storage regulation system, CsrA/B, known to be a master switch between glycolysis and gluconeogenesis, led us to propose that the BarA-UvrY two-component system controls the Csr system. Taking these results together, we propose the BarA-UvrY two-component system is crucial for efficient adaptation between different metabolic pathways, an essential function for adaptation to a new environment.     

Germfree status of mice obtained by embryo transfer in an isolator environment

The technique of embryo transfer has been evaluated for the purpose of changing the mouse stocks to a germfree (GF) status. Our results show reproducible and quality-assured conversion of animals to those which are negative for the presence of microorganisms. Rapid and easy access to GF mice is advantageous for studies of selected microflora and their cross-talks with the host, when applying, e.g. genomic, proteomic and metabolic methodology.The study involved embryo transfer in an isolator environment, thereby allowing implantation of cleansed embryos into GF recipients under well-controlled conditions. The recipient females gave birth normally and took care of the offspring as if they were their own pups, thus enhancing the survival rate. Access to full technical resources required to maintain GF isolators are, however, a prerequisite. In this study, we used stainless steel isolators designed by Gustafsson (1959), on which a stereomicroscope was mounted to facilitate embryo transfer inside the isolator.The use of embryo transfer and isolator techniques will facilitate the availability of various mouse mutant models under different gnotobiotic conditions, GF, monoxenic or polyxenic animals, to enable comparison with conventional animals for physiological and pathophysiological studies.     

Adolescents' Utilisation of Psychiatric Care,Neighbourhoods and Neighbourhood Socioeconomic Deprivation: A Multilevel Analysis

Mental health problems among adolescents have become a major public health issue, and it is therefore important to increase knowledge on the contextual determinants of adolescent mental health. One such determinant is the socioeconomic structure of the neighbourhood. The present study has two central objectives, (i) to examine if neighbourhood socioeconomic deprivation is associated to individual variations in utilisation of psychiatric care in a Swedish context, and (ii) to investigate if neighbourhood boundaries are a valid construct for identifying contexts that influence individual variations in psychiatric care utilization. Data were obtained from the Longitudinal Multilevel Analysis in Scania (LOMAS) database. The study population consists of all boys and girls aged 13–18 years (N=18,417), who were living in the city of Malmö, Sweden, in 2005. Multilevel logistic regression analysis was applied to estimate the probability of psychiatric care utilisation. The results from the study indicate that the neighbourhood of residence had little influence on psychiatric care utilisation. Although we initially found a variation between neighbourhoods, this general contextual effect was very small (i.e. 1.6 %). The initial conclusive association between the neighbourhood level of disadvantage and psychiatric care utilisation (specific contextual effect) disappeared following adjustment for individual and family level variables. Our results suggest the neighbourhoods in Malmö (at least measured in terms of SAMS-areas), do not provide accurate information for discriminating adolescents utilisation of psychiatric care. The SAMS-areas appears to be an inappropriate construct of the social environment that influences adolescent utilisation of psychiatric care. Therefore, public health interventions should be directed to the whole city rather than to specific neighbourhoods. However, since geographical, social or cultural contexts may be important for our understanding of adolescent mental health further research is needed to identify such contexts.     

Mechanisms that Trigger a Good Health-Care Response to Intimate Partner Violence in Spain. Combining Realist Evaluation and Qualitative Comparative Analysis Approaches

BackgroundHealth care professionals, especially those working in primary health-care services, can play a key role in preventing and responding to intimate partner violence. However, there are huge variations in the way health care professionals and primary health care teams respond to intimate partner violence. In this study we tested a previously developed programme theory on 15 primary health care center teams located in four different Spanish regions: Murcia, C Valenciana, Castilla-León and Cantabria. The aim was to identify the key combinations of contextual factors and mechanisms that trigger a good primary health care center team response to intimate partner violence.MethodsA multiple case-study design was used. Qualitative and quantitative information was collected from each of the 15 centers (cases). In order to handle the large amount of information without losing familiarity with each case, qualitative comparative analysis was undertaken. Conditions (context and mechanisms) and outcomes, were identified and assessed for each of the 15 cases, and solution formulae were calculated using qualitative comparative analysis software.ResultsThe emerging programme theory highlighted the importance of the combination of each team’s self-efficacy, perceived preparation and women-centredness in generating a good team response to intimate partner violence. The use of the protocol and accumulated experience in primary health care were the most relevant contextual/intervention conditions to trigger a good response. However in order to achieve this, they must be combined with other conditions, such as an enabling team climate, having a champion social worker and having staff with training in intimate partner violence.ConclusionsInterventions to improve primary health care teams’ response to intimate partner violence should focus on strengthening team’s self-efficacy, perceived preparation and the implementation of a woman-centred approach. The use of the protocol combined with a large working experience in primary health care, and other factors such as training, a good team climate, and having a champion social worker on the team, also played a key role. Measures to sustain such interventions and promote these contextual factors should be encouraged.     

Defining the Determinants for Dual Targeting of Amino Acyl-tRNA Synthetases to Mitochondria and Chloroplasts

Most of the organellar amino acyl-tRNA synthetases (aaRSs) are dually targeted to both mitochondria and chloroplasts using dual targeting peptides (dTPs). We have investigated the targeting properties and domain structure of dTPs of seven aaRSs by studying the in vitro and in vivo import of N-terminal deleted constructs of dTPs fused to green fluorescent protein. The deletion constructs were designed based on prediction programs, TargetP and Predotar, as well as LogoPlots derived from organellar proteomes in Arabidopsis thaliana. In vitro import was performed either into a single isolated organelle or as dual import (i.e., into a mixture of isolated mitochondria and chloroplasts followed by reisolation of the organelles). In vivo import was investigated as transient expression of the green fluorescent protein constructs in Nicotiana benthamiana protoplasts. Characterization of recognition determinants showed that the N-terminal portions of TyrRS-, ValRS- and ThrRS-dTPs (27, 22 and 23 amino acids, respectively) are required for targeting into both mitochondria and chloroplasts. Surprisingly, these N-terminal portions contain no or very few arginines (or lysines) but very high number of hydroxylated residues (26-51%). For two aaRSs, a domain structure of the dTP became evident. Removal of 20 residues from the dTP of ProRS abolished chloroplastic import, indicating that the N-terminal region was required for chloroplast targeting, whereas deletion of 16 N-terminal amino acids from AspRS-dTP inhibited the mitochondrial import, showing that in this case, the N-terminal portion was required for the mitochondrial import. Finally, deletion of N-terminal regions of dTPs for IleRS and LysRS did not affect dual targeting. In summary, it can be concluded that there is no general rule for how the determinants for dual targeting are distributed within dTPs; in most cases, the N-terminal portion is essential for import into both organelles, but in a few cases, a domain structure was observed.     

Prevalence,Pharmacological Treatment,and Control of Cardiometabolic Risk Factors among Older People in Central Stockholm: A Population-Based Study

Background

Cardiometabolic risk factors and related cardiovascular diseases represent major threats to healthy aging.

Objective

We aimed to estimate distribution, pharmacological treatment, and control of main cardiometabolic risk factors among older people.

Methods

This population-based study included 3363 participants (age≥60 years, 64.9% women) in the Swedish National study on Aging and Care in Kungsholmen, in central Stockholm, Sweden (2001-2004). Data on demographics, cardiometabolic risk factors (hypertension, obesity, diabetes, and high cholesterol), and medication use were collected through face-to-face interviews, clinical examinations, laboratory tests, and the inpatient register. Cardiometabolic risk factors were defined following the most commonly used criteria. Prevalence was standardized using local census data.

Results

The age- and sex-standardized prevalence of diabetes, obesity, high cholesterol, and hypertension was 9.5%, 12.8%, 49.7%, and 74.9%, respectively. The prevalence of hypertension and diabetes increased with age, whereas the prevalence of obesity and high cholesterol decreased with age. Forty-nine percent of older adults had two or more cardiometabolic risk factors; 9.8% had three or more. Overall, 55.5% of people with hypertension, 50.3% with diabetes, and 25.0% with high cholesterol received pharmacological treatment. Of those treated pharmacologically, 49.4%, 38.1%, and 85.5% reached therapeutic goals for hypertension (blood pressure<150/90 mmHg), diabetes (glycated haemoglobin<7%), and high cholesterol (total cholesterol<6.22 mmol/l), respectively.

Conclusions

Hypertension, high cholesterol, and clustering of cardiometabolic risk factors were common among older people in Stockholm, but pharmacological treatment and control of these major factors can be improved. Appropriate management of cardiometabolic profiles among older people may help improve cardiovascular health and achieve healthy aging.