RESUMO
BACKGROUND: Approximately 8.1 million outpatient surgical procedures were performed in Germany in 2021. Little is known about the quality of postoperative pain treatment in the outpatient sector. METHODS: The AQS1 project comprises a combined survey of patients and staff in the framework of quality control for ambulatory surgery. The primary endpoint of this study was the prevalence of relevant incisional pain (≥ 4/10 on the numerical rating scale) up to postoperative day 3. Secondary endpoints included prognostic variables for pain and pain-associated outcomes, based on the AQS1 patient questionnaire. Moreover, mixed regression models were used to analyze potential prognostic variables and associations of pain with other outcomes (study registration number DRKS00028052). RESULTS: Data from 330 008 patients were evaluated (from 1 July 2001 to 31 December 2021). The overall prevalence of relevant incisional pain up to postoperative day 3 was 22.5%, with major differences between different types of procedure (3.2%-51.2%). Pain was most common after hemorrhoid surgery (51.2%) and the laparoscopic lysis of large and small bowel adhesions (45.4%). The main associations of relevant pain were with younger age (odds ratio [OR] 1.87, 95% confidence interval [1.82; 1.91]), early postoperative pain (1.34, [1.30; 1.39]), inadequate provision of analgesics (2.90, [2.71; 3.09]), and surgical wound infections (2.60, [2.43; 2.78]). Patients with pain reported lower overall satisfaction with the procedure and a longer inability to work. CONCLUSION: These data have not been tested for representativeness. They can serve as a point of departure for the optimization of individualized perioperative pain therapy and for the planning of prospective studies.
Assuntos
Procedimentos Cirúrgicos Ambulatórios , Pacientes Ambulatoriais , Humanos , Estudos Prospectivos , Dor Pós-Operatória , AnalgésicosRESUMO
Ultrafast electron diffraction techniques that employ relativistic electrons as a probe have been in the spotlight as a key technology for visualizing structural dynamics which take place on a time scale of a few femtoseconds to hundreds femtoseconds. These applications highly demand not only extreme beam quality in 6-D phase space such as a few nanometer transverse emittances and femtosecond duration but also equivalent beam stability. Although these utmost requirements have been demonstrated by a compact setup with a high-gradient electron gun with state-of-the-art laser technologies, this approach is fundamentally restricted by its nature for compressing the electrons in a short distance by a ballistic bunching method. Here, we propose a new methodology that pushes the limit of timing jitter beyond the state-of-the-art by utilizing consecutive RF cavities. This layout already exists in reality for energy recovery linear accelerator demonstrators. Furthermore, the demonstrators are able to provide MHz repetition rates, which are out of reach for most conventional high-gradient electron guns.
RESUMO
Superconducting radio-frequency (SRF) cavities are one of the most important elements in modern particle accelerators as they are used for beam acceleration, bunch manipulation, bunch focusing and defocusing, etc. Nevertheless, the availability of these complex structures prior to their installation in the accelerator is limited, either due to a lack of a real cavity or due to the time needed for the experiment setup (vacuum, cryogenics, cabling, etc.), and thus it can block or delay the development of new algorithms such as low level RF control, quench detection, etc. In this paper, we present a hardware virtual cavity to be used in hardware-in-the-loop simulations. The system implements a cavity electrical model for the transmitted and reflected voltages and more advanced features such as mechanical vibration modes driven by Lorentz-force detuning or external microphonics, hard quenches, and the Q-slope. As viewed from the RF input and output, this virtual cavity acts like a real SRF cavity and can replace such a system in early-stage debugging and operation of ancillary control systems.
RESUMO
Alternative lengthening of telomeres (ALT) is a telomere lengthening pathway that predominates in aggressive tumors of mesenchymal origin; however, the underlying mechanism of telomere synthesis is not fully understood. Here, we show that the BLM-TOP3A-RMI (BTR) dissolvase complex is required for ALT-mediated telomere synthesis. We propose that recombination intermediates formed during strand invasion are processed by the BTR complex, initiating rapid and extensive POLD3-dependent telomere synthesis followed by dissolution, with no overall exchange of telomeric DNA. This process is counteracted by the SLX4-SLX1-ERCC4 complex, which promotes resolution of the recombination intermediate, resulting in telomere exchange in the absence of telomere extension. Our data are consistent with ALT being a conservative DNA replication process, analogous to break-induced replication, which is dependent on BTR and counteracted by SLX4 complex-mediated resolution events.
Assuntos
Replicação do DNA/genética , RecQ Helicases/fisiologia , Recombinases/fisiologia , Recombinação Genética/genética , Homeostase do Telômero/genética , Células Cultivadas , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo I/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/fisiologia , Humanos , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/fisiologia , RecQ Helicases/metabolismo , Recombinases/metabolismo , Telômero/metabolismoRESUMO
Acquisition of replicative immortality is currently regarded as essential for malignant transformation. This is achieved by activating a telomere lengthening mechanism (TLM), either telomerase or alternative lengthening of telomeres, to counter normal telomere attrition. However, a substantial proportion of some cancer types, including glioblastomas, liposarcomas, retinoblastomas, and osteosarcomas, are reportedly TLM-negative. As serial samples of human tumors cannot usually be obtained to monitor telomere length changes, it has previously been impossible to determine whether tumors are truly TLM-deficient, there is a previously unrecognized TLM, or the assay results are false-negative. Here, we show that a subset of high-risk neuroblastomas (with â¼50% 5-year mortality) lacked significant TLM activity. Cancer cells derived from these highly aggressive tumors initially had long telomeres and proliferated for >200 population doublings with ever-shorter telomeres. This indicates that prevention of telomere shortening is not always required for oncogenesis, which has implications for inhibiting TLMs for cancer therapy.
Assuntos
Proliferação de Células , Encurtamento do Telômero , Linhagem Celular Tumoral , Ativação Enzimática , Amplificação de Genes , Humanos , Proteína Proto-Oncogênica N-Myc/genética , Neuroblastoma/genética , Neuroblastoma/patologia , Telomerase/metabolismoRESUMO
Some cancers use alternative lengthening of telomeres (ALT), a mechanism whereby new telomeric DNA is synthesized from a DNA template. To determine whether normal mammalian tissues have ALT activity, we generated a mouse strain containing a DNA tag in a single telomere. We found that the tagged telomere was copied by other telomeres in somatic tissues but not the germline. The tagged telomere was also copied by other telomeres when introgressed into CAST/EiJ mice, which have telomeres more similar in length to those of humans. We conclude that ALT activity occurs in normal mouse somatic tissues.
Assuntos
Queratinócitos/fisiologia , Homeostase do Telômero/genética , Animais , Linfócitos B/citologia , Cruzamento , Linhagem Celular , Quimera/genética , Cromossomos/genética , Cromossomos/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Técnicas de Genotipagem , Queratinócitos/citologia , Queratinócitos/metabolismo , Masculino , Mamíferos , Camundongos , Camundongos Endogâmicos C57BL , Espermatócitos/citologia , Espermatócitos/fisiologia , Coloração e Rotulagem , Linfócitos T/citologiaRESUMO
Telomeres in cells that use the recombination-mediated alternative lengthening of telomeres (ALT) pathway elicit a DNA damage response that is partly independent of telomere length. We therefore investigated whether ALT telomeres contain structural abnormalities that contribute to ALT activity. Here we used next generation sequencing to analyze the DNA content of ALT telomeres. We discovered that variant repeats were interspersed throughout the telomeres of ALT cells. We found that the C-type (TCAGGG) variant repeat predominated and created a high-affinity binding site for the nuclear receptors COUP-TF2 and TR4. Nuclear receptors were directly recruited to telomeres and ALT-associated characteristics were induced after incorporation of the C-type variant repeat by a mutant telomerase. We propose that the presence of variant repeats throughout ALT telomeres results from recombination-mediated telomere replication and spreading of variant repeats from the proximal regions of the telomeres and that the consequent binding of nuclear receptors alters the architecture of telomeres to facilitate further recombination.
Assuntos
Fator II de Transcrição COUP/metabolismo , Membro 2 do Grupo C da Subfamília 2 de Receptores Nucleares/metabolismo , Homeostase do Telômero/genética , Telômero/genética , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Humanos , Mutação , Ligação Proteica/genética , Sequências Repetitivas de Ácido Nucleico/genética , Análise de Sequência de DNA , Telomerase/genéticaRESUMO
Telomeres, specialised structures that protect chromosome ends, play a critical role in preserving chromosome integrity. Telomere dynamics in the Tasmanian devil (Sarcophilus harrisii) are of particular interest in light of the emergence of devil facial tumour disease (DFTD), a transmissible malignancy that causes rapid mortality and threatens the species with extinction. We used fluorescent in situ hybridisation to investigate telomere length in DFTD cells, in healthy Tasmanian devils and in four closely related marsupial species. Here we report that animals in the Order Dasyuromorphia have chromosomes characterised by striking telomere length dimorphism between homologues. Findings in sex chromosomes suggest that telomere length dimorphism may be regulated by events in the parental germlines. Long telomeres on the Y chromosome imply that telomere lengthening occurs during spermatogenesis, whereas telomere diminution occurs during oogenesis. Although found in several somatic cell tissue types, telomere length dimorphism was not found in DFTD cancer cells, which are characterised by uniformly short telomeres. This is, to our knowledge, the first report of naturally occurring telomere length dimorphism in any species and suggests a novel strategy of telomere length control. Comparative studies in five distantly related marsupials and a monotreme indicate that telomere dimorphism evolved at least 50 million years ago.
Assuntos
Marsupiais/genética , Telômero/genética , Animais , Hibridização In Situ , Cromossomos Sexuais/genética , Homeostase do Telômero/genéticaRESUMO
Replicative senescence is accompanied by a telomere-specific DNA damage response (DDR). We found that DDR+ telomeres occur spontaneously in early-passage normal human cells and increase in number with increasing cumulative cell divisions. DDR+ telomeres at replicative senescence retain TRF2 and RAP1 proteins, are not associated with end-to-end fusions and mostly result from strand-independent, postreplicative dysfunction. On the basis of the calculated number of DDR+ telomeres in G1-phase cells just before senescence and after bypassing senescence by inactivation of wild-type p53 function, we conclude that the accrual of five telomeres in G1 that are DDR+ but nonfusogenic is associated with p53-dependent senescence.
Assuntos
Senescência Celular/genética , Telômero/metabolismo , Células Cultivadas , Dano ao DNA , Humanos , Homeostase do TelômeroRESUMO
In human cancer cells with telomeres that have been over-lengthened by exogenous telomerase activity, telomere shortening can occur by a process that generates circles of double-stranded telomeric DNA (t-circles). Here, we demonstrate that this telomeretrimming process occurs in cells of the male germline and in normal lymphocytes following mitogen-stimulated upregulation of telomerase activity. Mouse tissues also contain abundant t-circles, suggesting that telomere trimming also contributes to telomere length regulation in mice. In cancer cells and stimulated lymphocytes, the mechanism involves the XRCC3 homologous recombination (HR) protein and generates single-stranded C-rich telomeric DNA. This suggests that, in addition to the well-documented gradual telomere attrition that accompanies cellular replication, there is also a more rapid form of negative telomere length control in normal mammalian cells, which most likely involves HR-mediated removal of telomere loops in the form of t-circles. We therefore propose that this telomere trimming mechanism is an additional factor in the balance between telomere lengthening and telomere shortening in normal human germline and somatic cells that may prevent excessive lengthening by processes such as telomerase activity.
Assuntos
Cromossomos de Mamíferos/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Animais , Cromossomos de Mamíferos/genética , DNA Circular/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Recombinação Homóloga , Humanos , Ativação Linfocitária/imunologia , Masculino , Mamíferos , Camundongos , Modelos Biológicos , Especificidade de Órgãos , Espermatozoides/metabolismo , Telomerase/metabolismoRESUMO
Telomere dysfunction is typically studied under conditions in which a component of the six-subunit shelterin complex that protects chromosome ends is disrupted. The nature of spontaneous telomere dysfunction is less well understood. Here we report that immortalized human cell lines lacking wild-type p53 function spontaneously show many telomeres with a DNA damage response (DDR), commonly affecting only one sister chromatid and not associated with increased chromosome end-joining. DDR(+) telomeres represent an intermediate configuration between the fully capped and uncapped (fusogenic) states. In telomerase activity-positive (TA(+)) cells, DDR is associated with low TA and short telomeres. In cells using the alternative lengthening of telomeres mechanism (ALT(+)), DDR is partly independent of telomere length, mostly affects leading strand-replicated telomeres, and can be partly suppressed by TRF2 overexpression. In ALT(+) (but not TA(+)) cells, DDR(+) telomeres preferentially associate with large foci of extrachromosomal telomeric DNA and recombination proteins. DDR(+) telomeres therefore arise through different mechanisms in TA(+) and ALT(+) cells and have different consequences.
Assuntos
Cromossomos Humanos/metabolismo , Dano ao DNA , Telômero/metabolismo , Linhagem Celular , Humanos , Modelos Biológicos , Recombinases/metabolismo , Telomerase/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas/metabolismoRESUMO
Telomere lengths are maintained in many cancer cells by the ribonucleoprotein enzyme telomerase but can be further elongated by increasing telomerase activity through the overexpression of telomerase components. We report here that increased telomerase activity results in increased telomere length that eventually reaches a plateau, accompanied by the generation of telomere length heterogeneity and the accumulation of extrachromosomal telomeric repeat DNA, principally in the form of telomeric circles (t-circles). Telomeric DNA was observed in promyelocytic leukemia bodies, but no intertelomeric copying or telomere exchange events were identified, and there was no increase in telomere dysfunction-induced foci. These data indicate that human cells possess a mechanism to negatively regulate telomere length by trimming telomeric DNA from the chromosome ends, most likely by t-loop resolution to form t-circles. Additionally, these results indicate that some phenotypic characteristics attributed to alternative lengthening of telomeres (ALT) result from increased mean telomere length, rather than from the ALT mechanism itself.
Assuntos
Telômero/química , Telômero/metabolismo , Linhagem Celular Tumoral , DNA Circular/metabolismo , Células HeLa , Humanos , Leucemia Promielocítica Aguda/metabolismo , Telomerase/metabolismoRESUMO
Alternative lengthening of telomeres (ALT) is a telomere length maintenance mechanism based on recombination, where telomeres use other telomeric DNA as a template for DNA synthesis. About 10% of all human tumors depend on ALT for their continued growth, and understanding its molecular details is critically important for the development of cancer treatments that target this mechanism. We have previously shown that telomeres of ALT-positive human cells can become lengthened via inter-telomeric copying, i.e. by copying the telomere of another chromosome. The possibility that such telomeres could elongate by using other sources of telomeric DNA as copy templates has not been investigated previously. In this study, we have determined whether a telomere can become lengthened by copying its own sequences, without the need for using another telomere as a copy template. To test this, we transduced an ALT cell line with a telomere-targeting construct and obtained clones with a single tagged telomere. We showed that the telomere tag can be amplified without the involvement of other telomeres, indicating that telomere elongation can also occur by intra-telomeric DNA copying. This is the first direct evidence that the ALT mechanism involves more than one method of telomere elongation.
Assuntos
Replicação do DNA , Telômero/metabolismo , Sequência de Bases , Células Clonais , Duplicação Gênica , Humanos , Hibridização in Situ Fluorescente , Plasmídeos/genética , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Telômero/genética , TransfecçãoRESUMO
Activation of telomerase is a crucial step during cellular immortalization, and in some tumors this results from amplification of the human telomerase reverse transcriptase (hTERT) gene. Immortalization of normal human cells has been achieved by transduction with hTERT cDNA under the control of a strong heterologous enhancer/promoter, but this is sometimes an inefficient process, with periods of poor growth or even crisis occurring before immortalization. Here, we showed that normal human mammary epithelial cells expressing exogenous hTERT amplified the transgene extensively and expressed high levels of hTERT mRNA and protein. Paradoxically, the cells had low levels of telomerase activity and very short telomeres, indicating that telomerase activity did not correlate with hTERT expression. These cells contained only approximately 20 human telomerase RNA (hTR) molecules/cell (compared with approximately 120 hTR molecules per 293 cell). Expression of exogenous hTR caused increased telomerase activity and telomere lengthening. These data indicate that some hTERT-transduced normal cells may express high levels of the transgene but fail to up-regulate endogenous hTR expression sufficiently to enable expression of robust levels of telomerase activity.
Assuntos
Amplificação de Genes , Glândulas Mamárias Humanas/metabolismo , RNA Mensageiro/metabolismo , Telomerase/genética , Telomerase/metabolismo , Linhagem Celular Transformada , Dosagem de Genes/fisiologia , Regulação da Expressão Gênica , Humanos , Telômero/metabolismo , TransfecçãoRESUMO
Immortalized human cells are able to maintain their telomeres by telomerase or by a recombination-mediated DNA replication mechanism known as alternative lengthening of telomeres (ALT). We showed previously that overexpression of Sp100 protein can suppress ALT and that this was associated with sequestration of the MRE11/RAD50/NBS1 (MRN) recombination protein complex by Sp100. In the present study, we determined whether MRN proteins are required for ALT activity. ALT cells were depleted of MRN proteins by small hairpin RNA-mediated knockdown, which was maintained for up to 100 population doublings. Knockdown of NBS1 had no effect on the level of RAD50 or MRE11, but knockdown of RAD50 also depleted cells of NBS1, and knockdown of MRE11 depleted cells of all three MRN proteins. Depletion of NBS1, with or without depletion of other members of the complex, resulted in inhibition of ALT-mediated telomere maintenance, as evidenced by decreased numbers of ALT-associated promyelocytic leukemia bodies and decreased telomere length. In some clones there was an initial period of rapid shortening followed by stabilization of telomere length, whereas in others there was continuous shortening at a rate within the reported range for normal human somatic cells lacking a telomere maintenance mechanism. In contrast, depletion of NBS1 in telomerase-positive cells did not result in telomere shortening. A recent study showed that NBS1 was required for the formation of extrachromosomal telomeric circles (Compton, S. A., Choi, J. H., Cesare, A. J., Ozgur, S., and Griffith, J. D. (2007) Cancer Res. 67, 1513-1519), also a marker for ALT. We conclude that the MRN complex, and especially NBS1, is required for the ALT mechanism.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Telômero/ultraestrutura , Hidrolases Anidrido Ácido , Linhagem Celular Tumoral , Reparo do DNA , Humanos , Hibridização in Situ Fluorescente , Proteína Homóloga a MRE11 , RNA/metabolismo , RNA Interferente Pequeno/metabolismo , Telômero/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , TransfecçãoRESUMO
The linear chromosomes of vertebrates terminate in telomeres that consist of a tandemly repeated hexameric sequence, 5'TTAGGG3'. Telomeres form a protective loop structure (t-loop), which is thought to prevent them from being recognized as a double-strand break. Approximately 10% of human tumors prevent shortening of their telomeres by using a recombination-mediated alternative lengthening of telomeres (ALT) mechanism. ALT-positive human cells contain extrachromosomal telomere repeat (ECTR) DNA that may either be circular or linear. It has been proposed that ECTR may be generated by recombination events involving the t-loop. A proportion of the cells within ALT-positive cell populations contain promyelocytic leukemia (PML) nuclear bodies that contain telomeric DNA and telomere-binding proteins that are called ALT-associated PML bodies (APB). Although the presence of APBs is very useful for determining whether tumors and cell lines use the ALT mechanism, the function of APBs is unknown. It has previously been shown that telomeric DNA is particularly susceptible to damage by hydrogen peroxide and N-methyl-N'-nitro-N-nitrosoguanidine. We report here that these DNA-damaging agents induce both linear and circular ECTR DNA in ALT cells and increase the proportion of cells that contain APBs. We partially purified APBs and showed that the telomeric repeat DNA they contain is predominantly linear. We propose that a function of APBs is to sequester linear telomeric DNA.
Assuntos
Estruturas do Núcleo Celular/metabolismo , Dano ao DNA/efeitos dos fármacos , Corpos de Inclusão Intranuclear/metabolismo , Telômero/metabolismo , Proteínas de Ciclo Celular/metabolismo , Estruturas do Núcleo Celular/genética , Cromossomos Humanos , Reparo do DNA , Eletroforese em Gel Bidimensional , Humanos , Peróxido de Hidrogênio/farmacologia , Corpos de Inclusão Intranuclear/genética , Corpos de Inclusão Intranuclear/ultraestrutura , Metilnitronitrosoguanidina/farmacologiaAssuntos
Serviços Contratados/tendências , Prestação Integrada de Cuidados de Saúde/tendências , Cirurgia Geral/tendências , Programas Nacionais de Saúde/tendências , Organizações Patrocinadas pelo Prestador/tendências , Análise Custo-Benefício/tendências , Previsões , Alemanha , Reforma dos Serviços de Saúde/tendências , Humanos , Mecanismo de Reembolso/tendênciasRESUMO
Approximately 10% of cancers overall use alternative lengthening of telomeres (ALT) instead of telomerase to prevent telomere shortening, and ALT is especially common in astrocytomas and various types of sarcomas. The hallmarks of ALT in telomerase-negative cancer cells include a unique pattern of telomere length heterogeneity, rapid changes in individual telomere lengths, and the presence of ALT-associated promyelocytic leukemia bodies (APBs) containing telomeric DNA and proteins involved in telomere binding, DNA replication, and recombination. The ALT mechanism appears to involve recombination-mediated DNA replication, but the molecular details are largely unknown. In telomerase-null Saccharomyces cerevisiae, an analogous survivor mechanism is dependent on the RAD50 gene. We demonstrate here that overexpression of Sp100, a constituent of promyelocytic leukemia nuclear bodies, sequestered the MRE11, RAD50, and NBS1 recombination proteins away from APBs. This resulted in repression of the ALT mechanism, as evidenced by progressive telomere shortening at 121 bp per population doubling, a rate within the range found in telomerase-negative normal cells, suppression of rapid telomere length changes, and suppression of APB formation. Spontaneously generated C-terminally truncated Sp100 that did not sequester the MRE11, RAD50, and NBS1 proteins failed to inhibit ALT. These findings identify for the first time proteins that are required for the ALT mechanism.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Telômero/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Mutação/genética , Proteínas Nucleares/genética , Ligação Proteica , RNA Interferente Pequeno , Telômero/genética , Transgenes/genéticaRESUMO
Exogenous expression of the catalytic subunit of telomerase, hTERT, in a normal human foreskin fibroblast cell strain resulted in telomerase activity and an extended proliferative lifespan prior to a period of crisis. Three immortalized cell lines with stably maintained telomere lengths were established from cells that escaped crisis. Each of these cultures underwent a significant downregulation of p16(INK4a) expression due to gene deletion events. One cell line also acquired mutations in both alleles of the p53 tumor suppressor gene. Downregulation of p16(INK4a) and loss of wild-type p53 expression occurred after escape from crisis, so these mutations are most likely not required for immortalization of these cells but rather were selected for during continuous growth in vitro. These findings emphasize the need for caution in the use of hTERT-immortalized cells in studies of normal cell biology or in tissue engineering and the need to monitor for genetic instability and the accumulation of mutations in both the p16(INK4a)/pRb and p53 pathways.