Recruitment of underage test persons: motivators and barriers in an anthropological EU-survey on a sensitive topic

Recruiting test persons is crucial in many scientific fields. The recognition of motivators or barriers to survey participation may support the design and recruitment strategy of future studies. The recruitment of under age test persons is very complex and sensitive. This paperpresents and analyses the experiences in recruitment of female juveniles (10-18 years old) in Germany, Italy and Lithuania within the context of an EU funded project concerning the extremely sensitive topic of "child pornography". The purpose of the project was to develop a method for age estimation of juveniles on photographs and videos. The faces of the test persons were photographed and anthropologically measured after an informed consent was signed by their parents and themselves. The analysis of the recruitment strategies and response rates revealed that culturally influenced factors played a significant role in the individual decision for or against a participation in the study. In all countries, the sensitive topic of child pornography had a great influence on the decision process. Many German parents agreed especially in light of this topic while in Italy and Lithuania the word "child pornography" triggered a very negative response. In Germany, the anthropological investigation of the faces provoked negative associations with the anthropometric surveys in the Third Reich. Social and political climate were further relevant factors for decision-making especially in Lithuania. The "top-down" principle of recruitment proved to be very effective for this kind of study. The authorities of headmasters, scientists and institutions positively influenced the decisions of the parents.     

The peroxisomal receptor Pex19p forms a helical mPTS recognition domain

The protein Pex19p functions as a receptor and chaperone of peroxisomal membrane proteins (PMPs). The crystal structure of the folded C‐terminal part of the receptor reveals a globular domain that displays a bundle of three long helices in an antiparallel arrangement. Complementary functional experiments, using a range of truncated Pex19p constructs, show that the structured α‐helical domain binds PMP‐targeting signal (mPTS) sequences with about 10 μM affinity. Removal of a conserved N‐terminal helical segment from the mPTS recognition domain impairs the ability for mPTS binding, indicating that it forms part of the mPTS‐binding site. Pex19p variants with mutations in the same sequence segment abolish correct cargo import. Our data indicate a divided N‐terminal and C‐terminal structural arrangement in Pex19p, which is reminiscent of a similar division in the Pex5p receptor, to allow separation of cargo‐targeting signal recognition and additional functions.     

Molecular characterization of isolated from murine adult tissues very small embryonic/epiblast like stem cells (VSELs)

Pluripotent very small embryonic/epiblast derived stem cells (VSELs) as we hypothesize are deposited at begin of gastrulation in developing tissues and play an important role as backup population of pluripotent stem cells (PSCs) for tissue committed stem cells (TCSCs). We envision that during steady state conditions these cells may be involved in tissue rejuvenation and in processes of regeneration/repair after organ injuries. Molecular analysis of adult bone marrow (BM)-derived purified VSELs revealed that they i) express pluripotent stem cells markers e.g., Oct4, Nanog, Klf-4, SSEA-1 ii) share several markers characteristic for epiblast as well as migratory primordial germ cells (PGCs), and iii) possess a unique pattern of genomic imprinting (e.g., erasure of differently methylated regions at Igf2-H19 and Rasgrf1 loci and hypermethylation at KCNQ1 and Igf2R loci). This supports that VSELs are related to epiblast-derived migrating PGC-like cells and, despite their pluripotent stem cell character, changes in the epigenetic signature of imprinted genes keep these cells quiescent in adult tissues and prevent them from teratoma formation. In contrast epigenetic changes/mutations that lead to activation of imprinted genes could potentially lead to tumor formation by these cells. Mounting evidence accumulates that perturbation of expression of imprinted genes is a common phenomenon observed in developing tumors.     

Association of Early Repolarization Pattern on ECG with Risk of Cardiac and All-Cause Mortality: A Population-Based Prospective Cohort Study (MONICA/KORA)

Background

Early repolarization pattern (ERP) on electrocardiogram was associated with idiopathic ventricular fibrillation and sudden cardiac arrest in a case-control study and with cardiovascular mortality in a Finnish community-based sample. We sought to determine ERP prevalence and its association with cardiac and all-cause mortality in a large, prospective, population-based case-cohort study (Monitoring of Cardiovascular Diseases and Conditions [MONICA]/KORA [Cooperative Health Research in the Region of Augsburg]) comprised of individuals of Central-European descent.

Methods and Findings

Electrocardiograms of 1,945 participants aged 35–74 y, representing a source population of 6,213 individuals, were analyzed applying a case-cohort design. Mean follow-up was 18.9 y. Cause of death was ascertained by the 9th revision of the International Classification of Disease (ICD-9) codes as documented in death certificates. ERP-attributable effects on mortality were determined by a weighted Cox proportional hazard model adjusted for covariables. Prevalence of ERP was 13.1% in our study. ERP was associated with cardiac and all-cause mortality, most pronounced in those of younger age and male sex; a clear ERP-age interaction was detected (p = 0.005). Age-stratified analyses showed hazard ratios (HRs) for cardiac mortality of 1.96 (95% confidence interval [CI] 1.05–3.68, p = 0.035) for both sexes and 2.65 (95% CI 1.21–5.83, p = 0.015) for men between 35–54 y. An inferior localization of ERP further increased ERP-attributable cardiac mortality to HRs of 3.15 (95% CI 1.58–6.28, p = 0.001) for both sexes and to 4.27 (95% CI 1.90–9.61, p<0.001) for men between 35–54 y. HRs for all-cause mortality were weaker but reached significance.

Conclusions

We found a high prevalence of ERP in our population-based cohort of middle-aged individuals. ERP was associated with about a 2- to 4-fold increased risk of cardiac mortality in individuals between 35 and 54 y. An inferior localization of ERP was associated with a particularly increased risk. Please see later in the article for the Editors'' Summary     

Probing protein-chromophore interactions in Cph1 phytochrome by mutagenesis

We have investigated mutants of phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 in order to study chromophore-protein interactions. Cph1Delta2, the 514-residue N-terminal sensor module produced as a recombinant His6-tagged apoprotein in Escherichia coli, autoassembles in vitro to form a holoprotein photochemically indistinguishable from the full-length product. We generated 12 site-directed mutants of Cph1Delta2, focusing on conserved residues which might be involved in chromophore-protein autoassembly and photoconversion. Folding, phycocyanobilin-binding and Pr-->Pfr photoconversion were analysed using CD and UV-visible spectroscopy. MALDI-TOF-MS confirmed C259 as the chromophore attachment site. C259L is unable to attach the chromophore covalently but still autoassembles to form a red-shifted photochromic holoprotein. H260Q shows UV-visible properties similar to the wild-type at pH 7.0 but both Pr and Pfr (reversibly) bleach at pH 9.0, indicating that the imidazole side chain buffers chromophore protonation. Mutations at E189 disturbed folding but the residue is not essential for chromophore-protein autoassembly. In D207A, whereas red irradiation of the ground state leads to bleaching of the red Pr band as in the wild-type, a Pfr-like peak does not arise, implicating D207 as a proton donor for a deprotonated intermediate prior to Pfr. UV-Vis spectra of both H260Q under alkaline conditions and D207A point to a particular significance of protonation in the Pfr state, possibly implying proton migration (release and re-uptake) during Pr-->Pfr photoconversion. The findings are discussed in relation to the recently published 3D structure of a bacteriophytochrome fragment.     

Emerging concept of cancer as a stem cell disorder

Evidence has accumulated that malignancy arises from maturation arrest of stem cells — rather than the dedifferentiation of somatic cells. To support this notion, stem cells in contrast to somatic cells are long lived cells and thus may become the subject of accumulating mutations that are crucial for the initiation/progression of cancer. More importantly they may maintain these mutations and pass them to daughter stem cells. Cancer stem cells (CSC) that derive from transformed normal stem cells (NSC) are responsible not only for tumor initiation, but also for its re-growth and metastasis. Accumulating evidence also indicates that adult tissues may contain a population of very small embryonic like (VSEL) stem cells that may give rise to some very immature tumors e.g., pediatric sarcomas. Similar molecular mechanisms operating in NSC and CSC regulate resistance to radio-chemotherapy and promote migration/metastasis. Thus, by studying the biology of NSC we can learn more about cancer.     

Biochemical, crystallographic, and mutagenic characterization of hint, the AMP-lysine hydrolase, with novel substrates and inhibitors

Hint, histidine triad nucleotide-binding protein, is a universally conserved enzyme that hydrolyzes AMP linked to lysine and, in yeast, functions as a positive regulator of the RNA polymerase II C-terminal domain kinase, Kin28. To explore the biochemical and structural bases for the adenosine phosphoramidate hydrolase activity of rabbit Hint, we synthesized novel substrates linking a p-nitroaniline group to adenylate (AMP-pNA) and inhibitors that consist of an adenosine group and 5'-sulfamoyl (AdoOSO(2)NH(2)) or N-ethylsulfamoyl (AdoOSO(2)NHCH(2)CH(3)) group. AMP-pNA is a suitable substrate for Hint that allowed characterization of the inhibitors; titration of each inhibitor into AMP-pNA assays revealed their K(i) values. The N-ethylsulfamoyl derivative has a 13-fold binding advantage over the sulfamoyl adenosine. The 1.8-A cocrystal structure of rabbit Hint with N-ethylsulfamoyl adenosine revealed a binding site for the ethyl group against Trp-123, a residue that reaches across the Hint dimer interface to interact with the alkyl portion of the inhibitor and, presumably, the alkyl portion of a lysyl substrate. Ser-107 is positioned to donate a hydrogen bond to the leaving group nitrogen. Consistent with a role in acid-base catalysis, the Hint S107A mutant protein displayed depressed catalytic activity.     

In situ identification of protein structural changes in prion-infected tissue

Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative disorders characterized by the conversion of the normal prion protein (PrP(C)) into aggregates of its pathological conformer (PrP(Sc)). The mechanism behind this structural conversion is unclear. We report the identification of disease-related protein structural differences directly within the tissue environment. Utilizing a synchrotron infrared (IR) light source, IR images of protein structure were obtained at a subcellular resolution, revealing regions of decreased alpha-helical content and elevated beta-sheet structure in and around infected neurons in the 263 K scrapie hamster model. PrP(Sc) immunostaining of the same tissue demonstrated that the elevated beta-sheet regions correspond to regions where the misfolded structure of PrP(Sc) is located. No evidence of these structural changes was observed in normal neurons.     

Local vascular pathway for progesterone transfer to the brain after nasal administration in gilts

In the present study we examined whether local transfer of intranasally administrated tritiated progesterone (3H-P4) would increase its concentration in blood supplying the brain and hypophysis in comparison with other organs. Additionally, the effect of estrous cycle on the P4 transfer was evaluated on isolated gilts' heads. In the first experiment 3H-P4 was instilled into the nasal cavities of anaesthetized, immature pigs (n=10). Simultaneous blood samples were collected for radioactivity measurement every minute from the same occluded carotid artery through two catheters; one catheter was pointed towards the head, the other one towards the heart. In eight animals the ratio calculated between the 'head' and 'heart' samples was significantly (p<0.05) higher than 1 and reached a mean (+/- SEM) level of 3.23 +/- 0.81. In two animals a much higher ratio was observed. A head/heart ratio>1 indicates an existence of local transfer of 3H-P4 from venous blood to the carotid blood. In the second experiment, heads of 26 mature, cycling gilts were perfused through the right carotid artery with autologous blood. The outflow from the left carotid artery was collected as 1 min samples. 3H-P4 was infused into the angularis oculi veins. Transfer of 3H-P4 from the venous blood into the arterial blood reached the mean (+/- SEM) level of 4.11 +/- 1.08 pg/ml on days 2-4, 3.2 +/- 0.70 on days 17-21 and 0.94 +/- 0.22 pg/ml on days 15-16 of the estrous cycle. No 3H-P4 transfer was observed on days 9-11. These findings demonstrate that nasally administered progesterone can reach the brain in locally higher concentration through the vascular pathway. Moreover, the between-vessel transfer of P4 is significantly affected by the stage of the estrous cycle.     

Density functional theory and surface enhanced Raman spectroscopy characterization of novel platinum drugs

There is considerable interest in the development of novel platinum-based anticancer drugs that overcome the disadvantages associated with the widely used drug cisplatin, which are its inactivity against some types of tumors and its toxic side effects. In this study we show the suitability of normal Raman spectroscopy (NRS) and surface enhanced Raman spectroscopy (SERS), assisted by density functional theoretical (DFT) calculations, for the characterization of Pt complexes. The Pt complexes studied include the established drugs cisplatin and carboplatin, as well as five novel Pt complexes with anticancer activity. DFT calculations at the B3LYP/LanL2DZ level are a good prediction of the experimental NRS spectra of small and medium sized Pt complexes. The use of SERS allows the investigation of Pt complexes at physiological concentrations, and the binding strengths of the different ligands can be determined. The formation of positively charged hydrolysis products may be necessary for SERS activity. The exiting group in the hydrolysis reaction can be identified.