Long-term care, care needs and wellbeing of individuals after cancer in childhood or adolescence (VersKiK): study protocol of a large scale multi-methods non-interventional study.

BACKGROUND: It has been shown previously that a relevant proportion of childhood cancer survivors suffers from late effects, which are often directly related to the cancer itself or its therapy, resulting in particular follow-up needs, additionally burdening healthcare systems. Being diagnosed with cancer at a vulnerable stage of development, this group of cancer survivors is at comparatively higher risk of relapse or subsequent cancer. Although national and international follow-up guidelines based on treatment modalities have been developed, their implementation seems to leave room for improvement. Additionally, they lack a sufficient consideration of the survivors' psychosocial needs, affecting their adherence to them. The aim of the VersKiK study is to provide representative information on late effects in childhood and adolescence cancer survivors in Germany. The main research objectives are: (1) to describe the state of follow-up care among survivors after a cancer diagnosis in childhood or adolescence; (2) to quantify the occurrence of late effects among this group of survivors; (3) to examine the adherence to selected audiological and cardiological follow-up guidelines and to identify factors affecting it; (4) to explore actual follow-up needs of paediatric cancer survivors; (5) to review selected follow-up guidelines with the aim to improve and expand them. METHODS: VersKiK is designed as a mixed-methods non-interventional study. We will use claims data from statutory health insurance companies in combination with individually linked population-based registry data from the German Childhood Cancer Registry (GCCR). This data base will permit us to quantify diagnoses and procedures in comparison to the general population as well as the adherence to existing follow-up guidelines. Additional information will be obtained through interviews with childhood and adolescence cancer survivors and their informal caregivers, as well as in focus groups with healthcare professionals. DISCUSSION: The present study aims to research the actual needs of individuals after cancer diagnosis and treatment in childhood or adolescence - physical, psychological and organisational - in order to improve existing follow-up guidelines. These improvements might further positively affect not only actual care provided to paediatric cancer survivors, but also benefit healthcare systems in general while decreasing consequent medical visits in this group of patients. TRIAL REGISTRATION: Registered at German Clinical Trial Register (ID: DRKS00025960 and DRKS00026092).

Sex related differences in therapy and outcome of patients with intermittent claudication in a real-world cohort.

BACKGROUND AND AIMS: The prevalence of lower extremity artery disease (LEAD) is increasing worldwide and sex-related differences are a current matter of debate. METHODS: We analysed claims data on unselected patients with in-patient treatment for LEAD with intermittent claudication (IC; Rutherford grade 1-3) from 01.01.2014 to 31.12.2015. Data files included diagnostic and procedural information from two years before index, and a five-year follow-up. RESULTS: Our analysis comprised 42,197 IC patients, thereof 28,520 (68%) male. Male patients were younger (median: 66.4 years vs. 72.6 years) but presented with higher frequency of cardiovascular risk factors such as diabetes (40% female vs. 46% male), atrial fibrillation (13% vs. 17%), chronic coronary syndrome (41% vs. 53%), chronic heart failure (23% vs. 27%), or chronic kidney disease (29% vs. 32%; all p < 0.001; age adjusted). Revascularisation applied in 80% of patients, thereof endovascular approach predominantly in female and surgery in male patients. Concomitant pharmacotherapy with statins (74% at 2 years) and platelet inhibitors (75% respectively) were long lasting below guideline recommendation, under-use being more pronounced in women. Two years after index, one-third of IC patients had subsequent revascularisation, one-quarter progressed to chronic limb threatening ischemia (CLTI), and 2% underwent amputation. Male sex was an independent risk factor for long-term mortality (female HR 0.75; 95%-CI 0.72-0.79; p < 0.001) and CLTI (female HR 0.89; 95%-CI 0.86-0.92; p < 0.001) during follow-up. CONCLUSIONS: The majority of in-patient treated patients for IC are male, presenting with worse cardiovascular risk profiles. In view of a general under-supply with statins and platelet inhibitors, women received somewhat less often preventive medication. Despite low LEAD stages at index, serious prognosis was observed in the long term. Particularly male patients were at high risk for all-cause mortality and the combined endpoint CLTI and death.

Use of site-specific recombination as a probe of nucleoprotein complex formation in chromatin.

DNA transactions in eukaryotes require that proteins gain access to target sequences packaged in chromatin. Further, interactions between distinct nucleoprotein complexes are often required to generate higher-order structures. Here, we employed two prokaryotic site-specific recombination systems to investigate how chromatin packaging affects the assembly of nucleoprotein structures of different complexities at more than 30 genomic loci. The dynamic nature of chromatin permitted protein-DNA and DNA-DNA interactions for sites of at least 34 bp in length. However, the assembly of higher-order nucleoprotein structures on targets spanning 114 bp was impaired. This impediment was maintained over at least 72 h and was not affected by the transcriptional status of chromatin nor by inhibitors of histone deacetylases and topoisomerases. Our findings suggest that nucleosomal linker-sized DNA segments become accessible within hours for protein binding due to the dynamic nature of chromatin. Longer segments, however, appear refractory for complete occupancy by sequence-specific DNA-binding proteins. The results thus also provide an explanation why simple recombination systems such as Cre and Flp are proficient in eukaryotic chromatin.

Altered directionality in the Cre-LoxP site-specific recombination pathway.

The site-specific recombinase Cre must employ control mechanisms to impose directionality on recombination. When two recombination sites (locus of crossing over in phage P1, loxP) are placed as direct repeats on the same DNA molecule, collision between loxP-bound Cre dimers leads to excision of intervening DNA. If two sites are placed as inverted repeats, the intervening segment is flipped around. Cre catalyzes these reactions in the absence of protein co-factors. Current models suggest that directionality is controlled at two steps in the recombination pathway: the juxtaposition of loxP sites and the single-strand-transfer reactions within the synaptic complex. Here, we show that in Escherichia coli strain 294-Cre, directionality for recombination is altered when the expression of Cre is increased. This leads to deletion instead of inversion on substrates carrying two loxP sites as inverted repeats. The nucleotide sequence composition of loxP sites remaining in aberrant products indicates that site alignment and/or DNA strand transfer in the in vivo Cre-loxP recombination pathway are not always tightly controlled.

High local protein concentrations at promoters: strategies in prokaryotic and eukaryotic cells.

The speed of chemical reactions is proportional to the concentration of molecules involved. Since proteins catalyze most of the essential reactions inside a living cell, their concentration should be as high as possible. An economical way to achieve this is through the establishment of small cell compartments. We propose that within these compartments, two types of local concentration effects are at work. (1) With local concentration type I reactions, multimeric proteins bound to a specific DNA sequence have an increased local concentration for a second DNA site sufficiently close-by, or for proteins bound to such a site. (2) For type II effects, DNA can be used as a scaffold to build unique nucleoprotein complexes that would otherwise not exist free in solution. These complexes are proficient in establishing longer-range interactions with similarly unique complexes located far away on the genome. We discuss the consequences of these local concentration effects in the light of the markedly different sizes of prokaryotic and eukaryotic cells and of their genomes.

Site-specific recombination in mammalian cells catalyzed by gammadelta resolvase mutants: implications for the topology of episomal DNA.

We have transferred the prokaryotic gammadelta resolvase system to mammalian cells and present a comparative analysis of recombination by wild-type and two mutant resolvases (E124Q and E102Y/E124Q). Transient co-transfection assays using beta-galactosidase as reporter for recombination reveal that episomal DNA does not contain a significant level of unconstrained negative supercoiling, since only mutant resolvases are recombination-proficient. We also show that the efficiency of recombination by the resolvase double mutant is comparable to that observed with Cre, which indicates that resolvase can be used as a new tool for controlled manipulations of episomal DNAs.

Site-specific recombination in human cells catalyzed by phage lambda integrase mutants.

Phage lambda Integrase (Int) is the prototype of the so-called integrase family of conservative site-specific recombinases, which includes Cre and FLP. The natural function of Int is to execute integration and excision of the phage into and out of the Escherichia coli genome, respectively. In contrast to Cre and FLP, however, wild-type Int requires accessory proteins and DNA supercoiling of target sites to catalyze recombination. Here, we show that two mutant Int proteins, Int-h (E174 K) and its derivative Int-h/218 (E174 K/E218 K), which do not require accessory factors, are proficient to perform intramolecular integrative and excisive recombination in co-transfection assays inside human cells. Intramolecular integrative recombination is also detectable by Southern analysis in human reporter cell lines harboring target sites attB and attP as stable genomic sequences. Recombination by wild-type Int, however, is not detectable by this method. The latter result implies that eukaryotic co-factors, which could functionally replace the prokaryotic ones normally required for wild-type Int, are most likely not present in human cells.

A DNA-binding domain swap converts the invertase gin into a resolvase.

DNA resolvases and invertases are closely related, yet catalyze recombination within two distinct nucleoprotein structures termed synaptosomes and invertasomes, respectively. Different protein-protein and protein-DNA interactions guide the assembly of each type of recombinogenic complex, as well as the subsequent activation of DNA strand exchange. Here we show that invertase Gin catalyzes factor for inversion stimulation dependent inversion on isolated copies of sites I from ISXc5 res, which is typically utilized by the corresponding resolvase. The concomitant binding of Gin to sites I and III in res, however, inhibits recombination. A chimeric recombinase, composed of the catalytic domain of Gin and the DNA-binding domain of ISXc5 resolvase, recombines two res with high efficiency. Gin must therefore contain residues proficient for both synaptosome formation and activation of strand exchange. Surprisingly, this chimera is unable to assemble a productive invertasome; a result which implies a role for the C-terminal domain in invertasome formation that goes beyond DNA binding.

Alterations in the directionality of lambda site-specific recombination catalyzed by mutant integrases in vivo.

Phage lambda integrative and excisive recombination normally proceeds by a pair of sequential strand exchanges. During the first exchange reaction, the "top" strand in each recombination site is cleaved, exchanged, and religated generating a Holliday junction intermediate. This intermediate DNA structure is resolved through a pair of reciprocal "bottom" strand exchanges, leading to recombinant products. The strict co-ordination of exchange reactions ensures religation between correct partner strands only. Here we show that the directionality of recombination is altered in vivo by two mutant integrases, Int-h (E174 K) and a double mutant Int-h/218 (E174 K/E218 K). This change in directionality leads to deletion instead of inversion on substrates that carry inverted attachment sites and, depending on the pair of target sites employed, requires the presence or absence of integration host factor. Neither Fis nor Xis is involved in deletion. Sequence analyses of deletion products reveal that the newly generated hybrid attachment site exhibits a reversed genetic polarity. We demonstrate that only one of two possible hybrid site configurations is generated and discuss two pathways leading to deletion. In the first, deletion results from a wrong alignment of the two recombination sites within the synaptic complex. In the second pathway, the unco-ordinated cleavage by the mutant integrases of all four DNA strands present in a conventional Holliday junction intermediate leads to two double-stranded breaks, whereby the subsequent rejoining between "wrong" partner strands appears restricted to only two strands.

RNA splicing of bacterial genes in eukaryotes.

The presence of intervening sequences or introns in eukaryotic genes has been known for more than 20 years, and the mechanisms underlying RNA splicing have been studied in depth both genetically and biochemically. In recent years, however, an increasing number of bacterial genes have been introduced into higher eukaryotes as important tools for genetic studies. Their gene products are frequently used as an indirect measure for cell type-specific promoter activity, as, for example, in the case of chloramphenicol acetyl transferase (CAT assay) or beta-galactosidase. Here we show that RNA splicing of two prokaryotic genes encoding site-specific DNA recombinases occurs in eukaryotic cells. In one case, splicing is only observed after treatment of cells with the cytokine alpha interferon. We further demonstrate that mutating an intragenic donor splice site in a bacterial gene apparently activates a second, alternative splicing pathway. In conjunction with previous reports, our findings should also be regarded as a warning that splicing of bacterial genes in higher eukaryotes is a more common phenomenon than presently recognized, which may be difficult to overcome and may cause problems in the interpretation of experimental results.

The resolvase encoded by Xanthomonas campestris transposable element ISXc5 constitutes a new subfamily closely related to DNA invertases.

BACKGROUND: Conservative site-specific recombination is responsible for the resolution of cointegrates which result during the transposition of class II transposable elements. Resolution is catalysed by a transposon-encoded recombinase, resolvase, that belongs to a large family of recombinases, including DNA invertases. Resolvases and the related invertases are likely to employ similar reaction mechanisms during recombination. There are important differences, however. Resolvases require two accessory DNA binding sites within each of the two directly repeated recombination sites. Invertases instead need a host factor, Fis, and an enhancer type DNA sequence, in addition to two inversely orientated recombination sites. RESULTS: The resolvase encoded by transposable element ISXc5 from the gram-negative phytopathogen Xanthomonas campestris shows two features which distinguish it from other known resolvases. First, it is more closely phylogenetically related to invertases than other resolvases. In particular, two functionally important regions seem highly conserved between this resolvase and members of the invertase subfamily. Second, the enzyme exhibits a large extension of its carboxy-terminal domain with unknown function. We purified ISXc5 resolvase and analysed its resolution reaction in vitro. Our biochemical and DNA topological analysis reveals that critical features of resolution are similar, if not identical, to that carried out by gammadelta resolvase. However, despite its apparent similarity to invertases, we were unable to detect recombination on standard substrates for DNA inversion, in either the presence or absence of Fis. CONCLUSIONS: ISXc5 resolvase employs a reaction mechanism which is common to members of the resolvase family. Its position near the evolutionary borderline to invertases and its high degree of identity within two functionally important regions with members of the DNA invertase subfamily suggest that only a few replacements of critical residues may suffice to convert this resolvase into a functional, possibly Fis-dependent invertase.

Long-range effects in a supercoiled DNA domain generated by transcription in vitro.

The translocation of a transcription complex can transiently introduce positive and negative superhelical windings into the template DNA. To gain further insight into this dynamic DNA supercoiling mechanism and its possible involvement in biological processes, we employed an in vitro system in which site-specific recombination by gammadelta resolvase is topologically coupled to transcription-induced negative supercoiling. Our kinetic experiments suggest that recombination is closely linked to the process of supercoiling by transcription. We utilized the known high speed at which two resolvase-bound recombination sites can pair to form a synaptic complex in kinetic experiments with DNA substrates containing three recombination sites. Our data provide evidence for the existence of a transient gradient of negative supercoiling. Such a gradient seems to be predominantly a consequence of DNA double helix rotation behind a translocating RNA polymerase and originates within a broad region up to two kilobase-pairs upstream of the transcriptional start site. We further demonstrate that the topological coupling between transcription and recombination is not affected when the DNA-bending protein integration host factor from E. coli is bound to multiple sites within the phage lambda attachment region. We discuss implications of our in vitro findings with respect to possible in vivo functions of the dynamic nature of transcription-induced supercoiling.

Differential control of transcription-induced and overall DNA supercoiling by eukaryotic topoisomerases in vitro.

The global superhelical state of intracellular DNA is stringently controlled by topoisomerase action. Little is know, however, about topoisomerase-directed relaxation of localized DNA supercoiling generated by protein tracking processes such as transcription. Here we use transcription by a yeast Gal4 and phage T7 RNA polymerase fusion protein to induce localized supercoiling which, in turn, triggers site-specific DNA recombination by gamma delta resolvase. We demonstrate that only large amounts of eukaryotic topoisomerase I interfere, through supercoiling relaxation, with the topological coupling between transcription and recombination. The additional presence of a strong cleavage site for topoisomerase I has little influence on the relaxation of localized supercoiling. We also show that high levels of human topoisomerase II fail to compete with transcription-driven recombination. However, drastically reduced amounts of either enzyme completely suppress recombination of overall supercoiled DNA. Together, our results reveal a marked difference in topoisomerase requirement to relax transcription-induced and global DNA supercoiling. We discuss possible reasons for this difference and conclude that localized supercoiling frequently may escape relaxation by eukaryotic topoisomerases to mediate topological couplings between DNA transactions.

Protein tracking-induced supercoiling of DNA: a tool to regulate DNA transactions in vivo?

An interplay between DNA-dependent biological processes appears to be crucial for cell viability. At the molecular level, this interplay relies heavily on the communication between DNA-bound proteins, which can be facilitated and controlled by the dynamic structure of double-stranded DNA. Hence, DNA structural alterations are recognized as potential tools to transfer biological information over some distance within a genome. Until recently, however, direct evidence for DNA structural information as a mediator between cellular processes was lacking. This changed when the concept of transient waves of DNA supercoiling, induced by proteins tracking along the right-handed DNA double helix, came into the limelight. Indeed, a number of observations now suggest that helix tracking-induced DNA structural information might be exploited to participate in the regulation of a variety of DNA transactions in vivo.

Transcription-driven site-specific DNA recombination in vitro.

Transcription of a topologically relaxed, circular DNA triggers recombination between two directly repeated res sites by gamma delta resolvase in vitro. This activation of recombination depends on the res site-to-site distance and the orientation of sites with respect to the direction of RNA polymerase tracking. In addition to functioning as a site-specific recombinase, gamma delta resolvase acts as a site-specific topoisomerase and increases the topological linking number of templates during transcription. The data suggest that the link between transcription and recombination could be negative DNA supercoiling that transiently builds up on a relatively short DNA segment in the wake of an advancing RNA polymerase. Surprisingly, transcription-driven recombination is not inhibited by the presence of large amounts of eukaryotic topoisomerase type I, indicating that site-specific recombination can override relaxation by diffusible topoisomerases. This in vitro system might therefore serve as a model for some transcription-directed recombination events observed in vivo.

Recombination of nicked DNA knots by gamma delta resolvase suggests a variant model for the mechanism of strand exchange.

Fast and efficient recombination catalyzed by gamma delta resolvase in vitro requires negative DNA supercoiling of plasmid substrates. The current model for recombination suggests that supercoiling is required to drive DNA strand exchange within a synaptic complex by 'simple rotation' of DNA-linked resolvase promoters. Surprisingly, DNA knots are recombined efficiently in the absence of supercoiling, whereby the rate of recombination increases with the number of irreducible DNA segment crossings, or nodes, within each substrate knot. Recombination products contain three knot nodes less than substrates, suggesting that a reduction in writhe drives the reaction. However, the proposed protomer rotation model predicts that writhe is not altered during the process of strand transfer but, instead, is reduced only when a synaptic complex disassembles after strand exchange. I present evidence that recombination of knotted and of linear substrates coincides with a disassembly of synaptic complexes. The results lead to a variant model for strand exchange on non-supercoiled substrates in which a specific disassembly of the synaptic complex, triggered by a reduction in writhe, guides the cleaved DNA into the recombinant configuration.

The influence of an alternate template conformation on elongating phage T7 RNA polymerase.

We investigated the effect of left-handed Z-DNA on transcription by bacteriophage T7 RNA polymerase in vitro and, surprisingly, found that the enzyme can efficiently utilize a template containing a stretch of left-handed DNA close to the promoter. Analysis of transcription products revealed that only a small fraction of elongating polymerases abort transcription either at the promoter proximal or at the distal B-to-Z junction and, even less frequently, within the stretch of left-handed DNA. Our results indicate that, unlike E. coli RNA polymerase, T7 RNA polymerase can utilize a template with a CG stretch in an alternate conformation. In contrast, polymerases are completely blocked at the promoter proximal junction by a monoclonal antibody directed against Z-DNA. This blockage remains stable over a remarkable time, even when negative supercoiling is released by linearization of the template. Together with our recent finding of transcription-induced formation of Z-DNA (3), our data provide an example for a possible auto-regulatory mechanism that employs a change in DNA conformation.

Transcription-induced conformational change in a topologically closed DNA domain.

We have tested in vitro the occurrence of a B-to-Z transition in a region of alternating purines and pyrimidines as a consequence of transcription-induced negative supercoiling. By using a monoclonal antibody as a specific Z-DNA stabilizing agent, we demonstrate that the formation of left-handed DNA can transiently occur when a topologically unconstrained template is transcribed. The B-to-Z transition, observed in a subpopulation of templates, appears to be induced by negative supercoiling generated in the wake of an elongating T7 RNA polymerase. Consistent with this, the presence of topoisomerases during the transcription period prevents the change in DNA conformation. These data agree with the 'twin-supercoiled-domain' model for transcription of Liu and Wang (1). Interestingly, our results suggest that the diffusion rate of transcription-induced superhelical twists must be relatively slow compared to their generation, and that under in vitro conditions localized transient supercoiling can reach unexpectedly high levels.