[Health economic evaluation of rehabilitative short-term care : Analysis of the costs of geriatric patients with and without rehabilitative short-term care after inpatient hospital stay]. / Gesundheitsökonomische Evaluation einer rehabilitativen Kurzzeitpflege : Ermittlung der Kosten von geriatrischen Patienten mit und ohne rehabilitative Kurzzeitpflege nach stationärem Krankenhausaufenthalt.

BACKGROUND: Geriatric patients requiring rehabilitation and admitted to short-term care after an acute inpatient hospital stay seldom receive rehabilitative services later. Rehabilitative short-term care (REKUP) supplements short-term care with rehabilitative measures, aiming to prevent functional restrictions and long-term care. STUDY OBJECTIVE: To conduct a cost and cost-effectiveness analyses of REKUP and provide data for a nationwide rollout. MATERIAL AND METHODS: A non-randomized controlled prospective study was carried out. The intervention group (IG) was paired 1:2 with a control group (KG), resulting in the formation of three collectives with follow-up periods of either 30, 90 or 180 days (each with IG and KG). Using administrative claims data from the AOK Baden-Württemberg, the mean total costs from the perspective of the health insurance were calculated. A potential impact of the intervention on costs was analyzed using the difference in differences approach. RESULTS: The analysis comprised 129 patients (IG 43; KG 86). During the follow-up periods, the IG presented higher rates of rehabilitation and lower rates of long-term care and mortality. Regarding costs, no statistically significant differences were found between the IG and KG in any of the three collectives. For nursing care and medication costs, costs were significantly higher in the follow-up period for the KG, whereas costs for rehabilitation were significantly higher for the IG (p < 0.001). DISCUSSION: Patients receiving REKUP utilize rehabilitation services more often and have a lower likelihood of requiring nursing care or dying with no statistically significant differences in costs. There are potential advantages of REKUP in the target population, which warrant further investigation due to methodological limitations.

Upscaling e-mental health in Europe: a six-country qualitative analysis and policy recommendations from the eMEN project.

E-mental health (eMH) encompasses the use of digital technologies to deliver, support, or enhance mental health services. Despite the growing evidence for the effectiveness of eMH interventions, the process of implementation of eMH solutions in healthcare remains slow throughout Europe. To address this issue, the e-Mental Health Innovation and Transnational Implementation Platform North-West Europe (eMEN) project was initiated to increase the dissemination and quality of eMH services in Europe. In this project, status analyses regarding eMH in the six participating countries (i.e., Belgium, France, Germany, Ireland, The Netherlands, and the UK) were conducted and eight recommendations for eMH were developed. Expert teams from the six participating countries conducted status analyses regarding the uptake of eMH based on a narrative literature review and stakeholder interviews. Based on these status analyses, the eMEN consortium developed eight policy recommendations to further support the implementation of eMH in Europe. The status analyses showed that the participating countries are in different stages of implementing eMH into mental healthcare. Some barriers to implementing eMH were common among countries (e.g., a limited legal and regulatory framework), while others were country-specific (e.g., fragmented, federal policies). The policy recommendations included fostering awareness, creating strong political commitment, and setting reliable standards related to ethics and data security. The eMEN project has provided the initial recommendations to guide political and regulatory processes regarding eMH. Further research is needed to establish well-tailored implementation strategies and to assess the generalizability of the recommendations beyond the countries involved in the eMEN project.

Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

Linker histone H1 is present in centromeric chromatin of living human cells next to inner kinetochore proteins.

The vertebrate kinetochore complex assembles at the centromere on alpha-satellite DNA. In humans, alpha-satellite DNA has a repeat length of 171 bp slightly longer than the DNA in the chromatosome containing the linker histone H1. The centromere-binding protein CENP-B binds specifically to alpha-satellite DNA with properties of a centromeric-linker histone. Here, we analysed if linker histone H1 is present at or excluded from centromeric chromatin by CENP-B. By immunostaining we detected the presence, but no enrichment or depletion of five different H1 subtypes at centromeric chromatin. The binding dynamics of H1 at centromeric sites were similar to that at other locations in the genome. These dynamics did not change in CENP-B depleted cells, suggesting that CENP-B and H1 co-exist in centromeric chromatin with no or little functional overlap. By bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer (FRET), we revealed that the linker histone H1 subtypes H1 degrees and H1.2 bind to centromeric chromatin in interphase nuclei in direct neighbourhood to inner kinetochore proteins.

Escherichia coli low-copy-number plasmid R1 centromere parC forms a U-shaped complex with its binding protein ParR.

The Escherichia coli low-copy-number plasmid R1 contains a segregation machinery composed of parC, ParR and parM. The R1 centromere-like site parC contains two separate sets of repeats. By atomic force microscopy (AFM) we show here that ParR molecules bind to each of the 5-fold repeated iterons separately with the intervening sequence unbound by ParR. The two ParR protein complexes on parC do not complex with each other. ParR binds with a stoichiometry of about one ParR dimer per each single iteron. The measured DNA fragment lengths agreed with B-form DNA and each of the two parC 5-fold interon DNA stretches adopts a linear path in its complex with ParR. However, the overall parC/ParR complex with both iteron repeats bound by ParR forms an overall U-shaped structure: the DNA folds back on itself nearly completely, including an angle of approximately 150 degrees . Analysing linear DNA fragments, we never observed dimerized ParR complexes on one parC DNA molecule (intramolecular) nor a dimerization between ParR complexes bound to two different parC DNA molecules (intermolecular). This bacterial segrosome is compared to other bacterial segregation complexes. We speculate that partition complexes might have a similar overall structural organization and, at least in part, common functional properties.

[Mammography screening in Germany]. / Mammographiescreening in Deutschland.

Available data suggest that early detection of breast cancer by mammography screening can reduce mortality by about 25%. Intensified monitoring of women with a family history of breast cancer and regular general screening have recently been introduced in Germany. The screening program is expected to be fully established by 2008. Following its successful introduction (participation rates between 65 and 80%), the German screening program will be conducted and evaluated in accordance with the European guidelines. At least in the screening trials that were conducted prior to the now established screening program the quality criteria were more than fulfilled (e.g. cancer detection rate in Bremen 8.7, Wiesbaden 9.4, Weser-Ems region 8.3/1000). Additional parameters that can be taken into account for quality assurance are the overdiagnosis bias, lead time bias, length bias and selection bias. Moreover, there are some factors that are specific to the German program compared with the breast cancer screening programs already established in other countries. One of these is the intensified screening program for high-risk women (ca. 5% of all carcinomas) and as a result fewer women with an increased genetic risk of breast cancer will be represented in the general screening program. The German screening program involves only a few university centers and hospital-based physicians, which may have adverse effects on research and development as well as mammography training in the future. Therefore, the screening program should also provide for the investigation of new techniques or emerging techniques (e.g. CAD systems in screening mammography) in the future.

Multifunctional centromere binding factor 1 is essential for chromosome segregation in the human pathogenic yeast Candida glabrata.

The CBF1 (centromere binding factor 1) gene of Candida glabrata was cloned by functional complementation of the methionine biosynthesis defect of a Saccharomyces cerevisiae cbf1 deletion mutant. The C. glabrata-coded protein, CgCbf1, contains a basic-helix-loop-helix leucine zipper domain and has features similar to those of other budding yeast Cbf1 proteins. CgCbf1p binds in vitro to the centromere DNA element I (CDEI) sequence GTCACATG with high affinity (0.9 x 10(9) M(-1)). Bandshift experiments revealed a pattern of protein-DNA complexes on CgCEN DNA different from that known for S. cerevisiae. We examined the effect of altering the CDEI binding site on CEN plasmid segregation, using a newly developed colony-sectoring assay. Internal deletion of the CDEI binding site led only to a fivefold increase in rates of plasmid loss, indicating that direct binding of Cbf1p to the centromere DNA is not required for full function. Additional deletion of sequences to the left of CDEI, however, led to a 70-fold increase in plasmid loss rates. Deletion of the CBF1 gene proved to be lethal in C. glabrata. C. glabrata cells containing the CBF1 gene under the influence of a shutdown promoter (tetO-ScHOP) arrested their growth after 5 h of cultivation in the presence of the reactive drug doxycycline. DAPI (4',6'-diamidino-2-phenylindole) staining of the arrested cells revealed a significant increase in the number of large-budded cells with single nuclei, 2C DNA content, and short spindles, indicating a defect in the G(2)/M transition of the cell cycle. Thus, we conclude that Cbf1p is required for chromosome segregation in C. glabrata.

Recent advances in FRET: distance determination in protein-DNA complexes.

Fluorescence resonance energy transfer (FRET) provides information on the distance between a donor and an acceptor dye in the range 10 to 100 A. Knowledge of the exact positions of some dyes with respect to nucleic acids now enables us to translate these data into precise structural information using molecular modeling. Advances in the preparation of dye-labeled nucleic acid molecules and in new techniques, such as the measurement of FRET in polyacrylamide gels or in vivo, will lead to an increasingly important role of FRET in structural and molecular biology.

The influence of different cultivation conditions on the metabolic functionality of encapsulated primary hepatocytes.

The clinical application of bioartificial liver support systems (BALS) is still limited because of technical problems associated with the storage, transport and scale-up of common systems. The encapsulation of primary hepatocytes could solve these problems since the scale-up depends only on the number of the beads and encapsulation leads to protection of the cells during the process of freezing and thawing. Many efforts have been made to find an appropriate material for the encapsulation of primary hepatocytes in terms of mechanical resistance as well as appropriate bio- and hemo-compatibility. This study focuses on the improvement of the metabolic functionality of encapsulated primary hepatocytes. A comparison between two different cultivation models showed that dynamic cultivation conditions lead to a 20.4-fold increase in the albumin production and a 5.21-fold increase in the urea synthesis of encapsulated hepatocytes. Furthermore, the influence of different ratios of the number of the cells to the volume of the media was analyzed. Encapsulated hepatocytes cultured with a high amount of medium were characterized by a significantly higher metabolic activity compared to encapsulated hepatocytes cultured with a low level of medium. Interestingly, the cell concentration per mL alginate has no significant influence on the metabolic activity of encapsulated hepatocytes. In conclusion, different optimization strategies are discussed and, finally, the functionality of encapsulated hepatocytes is compared to the standard model of hepatocyte culture, the collagen sandwich.

Determination of the binding constants of the centromere protein Cbf1 to all 16 centromere DNAs of Saccharomyces cerevisiae.

Cbf1p is a Saccharomyces cerevisiae chromatin protein belonging to the basic region helix-loop-helix leucine zipper (bHLHzip) family of DNA binding proteins. Cbf1p binds to a conserved element in the 5'-flanking region of methionine biosynthetic genes and to centromere DNA element I (CDEI) of S.cerevisiae centromeric DNA. We have determined the apparent equilibrium dissociation constants of Cbf1p binding to all 16 CDEI DNAs in gel retardation assays. Binding constants of full-length Cbf1p vary between 1.7 and 3.8 nM. However, the dissociation constants of a Cbf1p deletion variant that has been shown to be fully sufficient for Cbf1p function in vivo vary in a range between 3.2 and 12 nM. In addition, native polyacrylamide gel electrophoresis revealed distinct changes in the 3D structure of the Cbf1p/CEN complexes. We also show that the previously reported DNA binding stimulation activity of the centromere protein p64 functions on both the Cbf1 full-length protein and a deletion variant containing only the bHLHzip domain of Cbf1p. Our results suggest that centromeric DNA outside the consensus CDEI sequence and interaction of Cbf1p with adjacent centromere proteins contribute to the complex formation between Cbf1p and CEN DNA.

Conserved Senescence Associated Genes and Pathways in Primary Human Fibroblasts Detected by RNA-Seq.

Cellular senescence correlates with changes in the transcriptome. To obtain a complete view on senescence-associated transcription networks and pathways, we assessed by deep RNA sequencing the transcriptomes of five of the most commonly used laboratory strains of human fibroblasts during their transition into senescence. In a number of cases, we verified the RNA-seq data by real-time PCR. By determining cellular protein levels we observed that the age-related expression of most but not all genes is regulated at the transcriptional level. We found that 78% of the age-affected differentially expressed genes were commonly regulated in the same direction (either up- or down-regulated) in all five fibroblast strains, indicating a strong conservation of age-associated changes in the transcriptome. KEGG pathway analyses confirmed up-regulation of the senescence-associated secretory phenotype and down-regulation of DNA synthesis/repair and most cell cycle pathways common in all five cell strains. Newly identified senescence-induced pathways include up-regulation of endocytotic/phagocytic pathways and down-regulation of the mRNA metabolism and the mRNA splicing pathways. Our results provide an unprecedented comprehensive and deep view into the individual and common transcriptome and pathway changes during the transition into of senescence of five human fibroblast cell strains.

DNA curvature in native and modified EcoRI recognition sites and possible influence upon the endonuclease cleavage reaction.

The ligation of a decadeoxynucleotide containing the EcoRI recognition site forms a series of multimers which appear to be curved based on observed anomalous gel migration in polyacrylamide gels. The degree of DNA curvature present in the recognition sequence, based upon the observed migration anomaly, can be altered by modifications to the purine functional groups at the 2- and 6-positions. Deletion of the guanine 2-amino group, occurring in the minor groove of the B-DNA helix, is most effective in increasing the observed DNA curvature. Conversely, the displacement of an amino group from the major groove to the minor groove eliminates curvature. DNA curvature is also modulated by the exocyclic group at the purine 6-position with decreasing curvature observed when changing the amino group to a carbonyl or proton substituent. Differences in the kinetic parameters characterizing the cleavage reaction by the endonuclease for many of the modified sequences are the result of modifications of functional groups in the major groove, which are likely to contact the endonuclease during catalysis. However, with two examples, significant decreases in the observed specificity constant (kcat/Km), characterizing the protein-nucleic acid interaction, cannot be easily explained in terms of such functional group contacts. It is more likely in these cases that the functional group modifications affect the efficiency of the endonuclease-DNA interaction by modulation of the structure of the double-stranded DNA helix. With both examples, modifications have been made to minor groove substituents. The extent of DNA curvature is increased significantly for one and decreased for the other, compared with that observed for the native recognition site. The results suggest that curvature of the DNA helix axis is an intrinsic property of the d(GAATTC) sequence which helps to optimize the protein-nucleic acid interactions observed for the EcoRI restriction endonuclease.

On the sequence determinants and flexibility of the kinetoplast DNA fragment with abnormal gel electrophoretic mobilities.

A 410 base-pair (bp) Sau3A restriction fragment derived from a Leishmania tarentolae kinetoplast DNA minicircle, which is known to have slower than expected electrophoretic mobilities in polyacrylamide gels, has been cloned in a plasmid and deletions from one end of the cloned segment have been constructed. Analysis of the gel electrophoretic mobility data of a large number of restriction fragments derived from the kinetoplast DNA clone and its deletion subclones has led to the conclusion that two sequences, one in the region bp 100 to 170 and the other bp 190 to 250, both numbered from one end of the 410 bp kinetoplast DNA segment, are important for the abnormal gel electrophoretic behavior of the kinetoplast DNA fragment. One common feature of these sequences is the periodic presence of short runs of A residues (3 to 6 As in each); auto-correlation analysis of these runs of A residues shows a strong harmonic component with a period around 11 bp. These results support and extend the previous analysis of Wu & Crothers (1984). The abnormal electrophoretic behavior is accentuated at low temperature and by the addition of Mg2+ to the electrophoresis buffer; addition of Na+ has the opposite effect. Insertion of sequences derived from the kinetoplast DNA fragment into nicked circular DNA causes no unexpected change in its electrophoretic mobility in agarose gel, suggesting that the 410 bp sequence, or segments of it, has no significant spatial writhe. Abnormal shifts in agarose gel mobilities are observed, however, when certain segments of the kinetoplast DNA are inserted into positively or negatively supercoiled DNA topoisomers. These results are consistent with a bent structure of the kinetoplast DNA in which the bend has zero writhe in its undistorted form but is easily distorted.

The recognition of distorted DNA structures by HMG-D: a footprinting and molecular modelling study.

The high mobility group (HMG) domain is a DNA binding motif found in some eukaryotic chromosomal proteins and transcription factors. This domain binds in the minor groove of DNA inducing a sharp bend and also preferentially binds to certain distorted DNA structures. Although structures of sequence-specific HMG domains with their cognate double-helical DNA binding sites have been solved, the nature of the interaction of the domain with distorted DNA remains to be established. In this study we have investigated the interaction of HMG-D, a Drosophila counterpart of the vertebrate HMG1, with a DNA oligomer containing a bulge of two adenine residues. We show by footprinting that HMG-D binds preferentially on one side of the bulged DNA. Based on these data and on the published NMR structures of the HMG domain of HMG-D and the LEF-1-DNA complex, we modelled the HMG-D - bulged DNA complex. This model predicts that two residues, Val32 and Thr33, in the loop between alpha-helices I and II are inserted deep into the "hole" in the DNA formed by the two missing bases on one strand of the DNA bulge. Mutation of these residues confirmed that both are required for the efficient binding and bending of DNA by HMG-D. We discuss both the role of this loop in the recognition of distorted DNA structures by non-sequence specific HMG domain proteins and that of the basic tail in stabilising the induced DNA bend.

DNA curvature does not require bifurcated hydrogen bonds or pyrimidine methyl groups.

Short tracts of the homopolymer dA.dT confer intrinsic curvature on the axis of the DNA double helix. This phenomenon is assumed to be a consequence of such tracts adopting a stable B'-DNA conformation that is distinct from B-form structure normally assumed by other DNA sequences. The more stable B' structure of dA.dT tracts has been attributed to several possible stabilizing factors: (1) optimal base stacking interactions consequent upon the high propeller twist, (2) bifurcated hydrogen bonds between adjacent dA.dT base-pairs, (3) stacking interactions involving the dT methyl groups, and finally (4) a putative spine of ordered water molecules in the minor groove. DNA oligodeoxynucleotides have been synthesized that enable these hypotheses to be tested; of particular interest is the combination of effects due to bifurcation (2) and methylation of the pyrimidines nucleotides (3). The data indicate that neither bifurcated hydrogen bonds nor pyrimidine methyl groups nor both are essential for DNA curvature. The data further suggest that the influence of the minor groove spine of hydration on the B'-formation is small. The experiments favor the hypothesis that base stacking interactions are the dominant force in stabilizing the B'-form structure.

Model for the interaction of DNA junctions and resolving enzymes.

Four-way DNA junctions are thought to be important intermediates in a number of recombination processes. Resolution of these junctions occurs by cleavage of two strands of DNA to generate two duplex molecules. The interaction between DNA junctions and resolving enzymes appears to be largely structure-specific, reflecting a molecular recognition on a significant scale. We propose a working model for this interaction that takes account of the present state of knowledge of the structure of the DNA junction, and the substrate requirements of the enzymes. We note that three different enzymes introduce cleavages at phosphodiester bonds that are presented on one side of the molecule, suggesting that the enzymes selectively interact with this face of the junction. By forcing a junction of constant sequence to adopt one or other of the two possible antiparallel isomers, we show that the junction is cleaved in such a way as to suggest a constant mode of interaction with the protein that is dependent on structure rather than sequence. We propose that the feature that is recognized is a mutual inclination of two DNA helices at approximately 120 degrees. We show that a number of DNA substrates that contain similar inclined helices, such as a three-way junction, bulged duplexes and a duplex that is curved because of repeated runs of oligoadenine sequences, are each cleaved by phage T4 endonuclease VII. This mode of DNA-protein interaction could be significant in either recombination or DNA repair processes.

Long-lasting transneuronal changes in rat dentate granule cell dendrites after entorhinal cortex lesion. A combined intracellular injection and electron microscopy study.

Following entorhinal cortex lesion, inhibitory hippocampal neurons show a persistent rarefication of those dendrites formally receiving entorhinal input. Physiological data indicate a long lasting disequilibrium of inhibition and excitation in the de-entorhinated hippocampus. We analyzed the intracellularly-stained dendritic tree of de-entorhinated excitatory rat granule cells. Granule cells of controls and animals surviving 2, 8, 60 and 270 days after unilateral entorhinal cortex lesion were impaled. Dendrites of control cells were of typical shape, traced to the hippocampal fissure and a complete dye filling of dendrites was ascertained by EM-analysis. Conversely, 60 and 270 days following lesioning, dendrites were only rarely seen to extend into the outer portions of the molecular layer and the dendritic architecture became significantly rarefied. Sixty days post-lesion, intracellularly filled dendrites extending to the middle molecular layer were surrounded by cell clusters resembling glia. Some of these contained the neuronally applied dye, suggesting a close association of the cytosolic compartments with the altered dendrites. These observed alterations exceed the process of sprouting and de novo synaptogenesis of remaining afference for long periods of time. The dendritic morphology of both inhibitory and excitatory neurons seems to require specific input from the entorhinal cortex. Moreover, sprouting of remaining afferents is apparently not sufficient to compensate for this loss of input.

Sequence specificity of curved DNA.

Anomalously slow migration of DNA fragments on polyacrylamide gels is interpreted as resulting from curvature of the DNA fragment. Different models have been suggested to explain DNA curvature. In this work a number of DNA fragments were synthesized, cloned, and electrophoretically characterized to distinguish between these models. Strong anomaly of migration is found for sequence stretches (dA)n repeated in phase with the helix turn with n at least 4. For n smaller than 4 only negligible anomaly is observed. The results contradict the purine-clash hypothesis. The data can be explained by assuming longer stretches of As to be in a B'-form, and that tilt of this structure might be the reason for its curvature.

Low and infrequent expression of nitric oxide synthase/NADPH-diaphorase in neurons of the human supraoptic nucleus: a histochemical study.

The gas nitric oxide is a messenger in brain signaling. In the hypothalamo-hypophyseal system nitric oxide is involved in the control of the expression and/or release of peptide hormones (corticotropin-releasing hormone, gonadotropin-releasing hormone, vasopressin and oxytocin). Nitric oxide synthase (NOS), the enzyme generating nitric oxide, is abundantly present in the magnocellular nuclei of the rat hypothalamus. Its localization in the human hypothalamus is less well studied. Hence, we investigated the anatomical distribution of neuronal nitric oxide synthase in the human supraoptic nucleus by use of immunohistochemical and enzyme histochemical techniques. The immunohistochemical localization of NOS was studied in 31 matched human hypothalami (13 control cases, eight depressed patients and ten schizophrenics). NADPH-diaphorase studies were carried out on seven additional hypothalami (three normal brains, four schizophrenics). Apparent inter-individual differences exist with regard to the occurrence of the enzyme in supraoptic neurons. In a majority of cases no immunostaining or histochemical reaction for the enzyme was observed. In seven cases (three controls, two schizophrenics, two depressives) a population of nitrergic nerve cells was seen in the dorsomedial part of the nucleus. This group of cells also stained for NADPH-diaphorase. Also, there were a few NOS-immunopositive neurons scattered throughout the nucleus. Additionally, thin NADPH-diaphorase positive fibers were observed to cross the nucleus. Our data show that, unlike the rat, the human supraoptic nucleus contains only a small number of nitrergic neurons. No correlation was found between the expression of the enzyme in supraoptic neurons and the psychiatric status of the patients.

Morphometric studies of the entorhinal cortex in neuropsychiatric patients and controls: clusters of heterotopically displaced lamina II neurons are not indicative of schizophrenia.

Subtle cytoarchitectural abnormalities of the rostral entorhinal cortex have been reported to be characteristic for schizophrenics. A main finding was the heterotopic displacement of clusters of lamina II neurons (which appeared morphologically immature) into layer III. Recent findings suggesting a considerable cytoarchitectural heterogeneity of the normal entorhinal cortex renewed interest in this problem and prompted the question of the extent to which 'aberrantly' located cell clusters are really disease-related. Being blind to the clinical diagnosis, we analyzed quantitatively the amount of entorhinal heterotopias in schizophrenics (n = 31), controls (n = 45) and depressive patients (n = 7). It was revealed that heterotopic cell clusters occurred in schizophrenics and controls with roughly the same frequency (amount of clusters per section). In depressive, there were significantly more heterotopias than in controls (right hemisphere). Our data show that heterotopias are common to the rostral part of the human entorhinal cortex and are not a suitable neuroanatomical marker of schizophrenia.