ATP-dependent chromatin remodelers - Analysis of expression patterns and impact on gene regulation

ATP-dependent chromatin remodelers are enzymes which use the energy from ATP hydrolysis to alter the chromatin structure. Thus, they play a key role in the transcriptional control of many important cellular processes such as proliferation,senescence and differentiation.The first part of this study focused on CHD5, a novel chromatin remodeling enzyme. To gain further insight into its expression profile and biological function, polyclonal peptide antisera were, together with a commercially available antiserum, successfully established for use in western blot, immunoprecipitation and immunofluorescence staining. ATP-dependent chromatin remodelers are expressed by very different patterns ranging from ubiquitous expression to an expression restricted to specific cell populations. So far, CHD5 expression was thought to be restricted to neural-related tissues. Here, it was shown for the first time that CHD5 expression is not restricted to neural tissues but additionally expressed in rodent testes. Furthermore, several cell lines were tested for CHD5 expression on transcript and protein level. CHD5 expression was not detected in any of the tested neuroblastoma and glioblastoma cell lines. In neuroblastoma cell lines this was expected due to the suggested function as tumor suppressor. Furthermore, a primary astrocyte culture was established. No CHD5 could be detected in protein lysates from astrocytes but was found in those from murine neural stem cells. This suggests that CHD5 expression in brain is restricted to neural stem cells and probably also to neurons. In the second part of this thesis, the impact of the chromatin remodelers CHD4 and BRG1 on a TNFα-induced inflammatory response was investigated. BRG1 and CHD4 expression was disrupted by a siRNA-mediated knockdown in HEK293 cells. After stimulations with TNFα for one to four hours, gene induction of typical inflammatory NF-κB target genes was determined by RT-qPCR. BRG1 and CHD4 were both required for the efficient induction of all tested target genes exclusively after one hour but not after four hours of TNFα treatment. Expression of the housekeeping gene ß-actin was unaffected. Surprisingly, although CHD4 is mainly known to be involved in transcriptional repression, a requirement for CHD4 in active transcription during this inflammatory response was revealed. To gain further insight into the mechanism of involvement, co-immunoprecipitations of the NF-κB subunit p65 with BRG1 and CHD4,respectively, were performed upon TNFα stimulation. No robust interactions between these proteins could be observed. Furthermore, ChIP experiments with the NF-κB subunit p50, histone H3 and CHD4 were carried out on the model target gene CXCL2. p50 was recruited to the promoter of CXCL2 rapidly after TNFα was added. Histone H3 binding to the promoter and the open reading frame of the gene strongly decreased within the first hour of TNFα stimulation, indicating that chromatin remodeling took place during the early period of gene induction. Recruitment of CHD4 was nonconclusive. Thus, the mechanism of how BRG1 and CHD4 act during TNFα response needs to be investigated further. In summary, a major influence of the chromatin remodelers BRG1 and CHD4 on the TNFα-induced gene activation during its early phase in HEK293 cells was demonstrated

Aspirin use and bleeding events during thrombocytopenia after autologous stem-cell transplantation for multiple myeloma

BackgroundIn patients with cardiovascular (CV) comorbidities that necessitate antiplatelet therapy (APT), its optimal management during chemotherapy-induced thrombocytopenia remains elusive, as the risk of bleeding has to be balanced against the risk of CV events. The purpose of this study was to assess the risk for bleeding with APT during thrombocytopenia in patients with multiple myeloma undergoing high-dose chemotherapy and subsequent autologous stem-cell transplantation (ASCT) with and without acetylsalicylic acid (ASA) as comedication.MethodsWe assessed patients who underwent ASCT at the Heidelberg University Hospital between 2011 and 2020 for bleeding events, management strategies for ASA intake during thrombocytopenia, transfusion requirements, and the occurrence of CV events.ResultsThere were 57/1,113 patients who continued ASA until at least 1 day after ASCT; thus, a continuous platelet inhibition during thrombocytopenia was assumed. Most of the patients (41/57) continued ASA until they had a platelet count of 20–50/nl. This range reflects the kinetics of thrombocytopenia and nondaily measurements of platelets during ASCT. A tendency toward a higher risk for bleeding events in the ASA group was demonstrated (1.9% (control group) vs. 5.3% (ASA), p = 0.082). The risk factors for bleeding in multivariate analysis were the duration of thrombocytopenia < 50/nl, a history of gastrointestinal bleeding, and diarrhea. The factors predicting the duration of thrombocytopenia were age >60 years, a hematopoietic stem-cell transplantation comorbidity index ≥3, and an impaired bone marrow reserve at admission. CV events occurred in three patients; none of them took ASA or had an indication for APT.ConclusionsThe intake of ASA until thrombocytopenia with a platelet count of 20–50/nl appears safe, although an elevated risk cannot be excluded. If ASA is indicated for the secondary prevention of CV events, the evaluation of risk factors for bleeding and a prolonged time of thrombocytopenia before conditioning is crucial to adapt the strategy for ASA intake during thrombocytopenia

Differential expression and sex chromosome association of CHD3/4 and CHD5 during spermatogenesis

ATP-dependent nucleosome remodelers of the CHD family play important roles in chromatin regulation during development and differentiation. The ubiquitously expressed CHD3 and CHD4 proteins are essential for stem cell function and serve to orchestrate gene expression in different developmental settings. By contrast, the closely related CHD5 is predominantly expressed in neural tissue and its role is believed to be restricted to neural differentiation. Indeed, loss of CHD5 contributes to neuroblastoma. In this study, we first demonstrate that CHD5 is a nucleosome-stimulated ATPase. We then compare CHD3/4 and CHD5 expression in mouse brain and show that CHD5 expression is restricted to a subset of cortical and hippocampal neurons whereas CHD3/4 expression is more widespread. We also uncover high levels of CHD5 expression in testis. CHD5 is transiently expressed in differentiating germ cells. Expression is first detected in nuclei of postmeiotic round spermatids, reaches a maximum in stage VIII spermatids and then falls to undetectable levels in stage IX spermatids. Surprisingly, CHD3/4 and CHD5 show complementary expression patterns during spermatogenesis with CHD3/ 4 levels progressively decreasing as CHD5 expression increases. In spermatocytes, CHD3/4 localizes to the pseudoautosomal region, the X centromeric region and then spreads into the XY body chromatin. In postmeiotic cells, CHD5 colocalises with macroH2A1.2 in association with centromeres and part of the Y chromosome. The subnuclear localisations of CHD4 and CHD5 suggest specif

Validation of a proxy‐reported SARC‐F questionnaire for current and retrospective screening of sarcopenia‐related functional impairments

BACKGROUND: The strength, assistance walking, rise from a chair, climb stairs, and falls (SARC‐F) questionnaire is a well‐established instrument for screening of sarcopenia and sarcopenia‐related functional impairments. As it is based on self‐reporting, its use precludes patients who are unable to answer the questionnaire as a consequence of severe acute diseases or cognitive impairment. Therefore, we aimed to validate a proxy‐reported version of the SARC‐F for both ad‐hoc as well as retrospective screening for severe sarcopenia‐related functional impairments. METHODS: Patients aged ≥60 years completed the SARC‐F and performed the short physical performance battery (SPPB) at baseline (T1). Proxies in Cohort A gave a simultaneous assessment of the patients' functional status with the proxy‐reported SARC‐F at T1 and again, retrospectively, after 3 months (T2). Proxies in Cohort B only completed the SARC‐F retrospectively at T2. The questionnaires' performances were assessed through sensitivity/specificity analyses and receiver operating characteristic (ROC) curves. For non‐inferiority analyses, results of both the patient‐reported and proxy‐reported SARC‐F were correlated with the SPPB total score as well as the results of the chair‐rise test subcategory; the respective correlation coefficients were tested against each other. RESULTS: One hundred and four patients and 135 proxies participated. Using a SPPB score < 9 points as the reference standard, the proxy‐reported SARC‐F identified patients at high risk for sarcopenia‐related functional impairment with a sensitivity of 0.81 (ad‐hoc), 0.88 (retrospective Cohort A), and 0.87 (retrospective Cohort B) as well as a specificity of 0.89 (ad‐hoc), 0.78 (retrospective Cohort A), and 0.64 (retrospective Cohort B). Areas under the ROC curves were ≥ 0.9 for the ad‐hoc proxy‐reported SARC‐F and the retrospective proxy‐reported SARC‐F in both cohorts. The proxy‐reported SARC‐F showed a non‐inferior correlation with the SPPB compared with the patient‐reported SARC‐F for ad‐hoc (P = <0.001) as well as retrospective screening for severe sarcopenia‐related functional impairment in both Cohorts A (P = 0.007) and B (P = 0.026). CONCLUSIONS: Proxy‐reported SARC‐F is a valid instrument for both ad‐hoc as well as retrospective screening for sarcopenia‐related functional impairment and could become the standard tool for evaluating this risk in older adults with severe acute disease, for example, in patients with quickly evolving haematological conditions

Attitudes towards sex workers: a nationwide cross-sectional survey among German healthcare providers

BackgroundWorldwide, sex workers face stigmatization and discrimination, also within healthcare. Only few studies on healthcare providers’ attitudes towards care of sex workers have been performed. This study assessed attitudes and knowledge of healthcare providers in Germany towards sex workers and their specific health risks.MethodsGerman healthcare professionals and medical students were invited to participate in a nationwide cross-sectional study in 2022. The online survey used a German translation of the “Attitudes towards Prostitutes and Prostitution Scale” by Levin and Peled for assessment of attitudes towards sex work and workers, together with prevalence estimates of common mental and physical disorders.ResultsA total of 469 questionnaires were included into analysis. Older participants tended to regard sex work as less of a choice (p &lt; 0.004) and sex workers as more victimized (p &lt; 0.001). The frequency of professional contact to sex workers neither affected the perception of sex workers’ status as victims vs. independent individuals, nor the perceived moral status. Moreover, healthcare professionals overestimated the prevalence of various disorders which was influenced by participants’ attitudes towards sex workers.DiscussionA comparison to a recent Allensbach survey demonstrated similar attitudes of healthcare providers and the general population towards sex workers. Our results suggest that German healthcare professionals are not free of prejudices against sex workers, as has been shown for other marginalized groups in society. Instead, they seem to be influenced by personal opinion rather than by objective facts which they should have acquired during their professional education. Future interventions (e.g., better training regarding marginal societal groups) are necessary to encounter these issues in order to improve healthcare for sex workers

Acute myeloid leukemia: negative prognostic impact of early blast persistence can be in part overcome by a later remission prior to post-induction therapy

In acute myeloid leukemia, there is an ongoing debate on the prognostic value of the early bone marrow assessment in patients receiving intensive therapy. In this retrospective study, we analyzed the prognostic impact of the early response in 1,008 patients with newly diagnosed acute myeloid leukemia, who were treated at our institution with intensive chemotherapy followed by consolidation chemotherapy and/or allogeneic hematopoietic stem cell transplantation (HSCT). We found that early blast persistence has an independent negative prognostic impact on overall survival, eventfree survival and relapse-free survival. This negative prognostic impact may only be overcome in patients showing at least a partial remission at the early bone marrow assessment and who subsequently achieve blast clearance by additional induction chemotherapy prior to consolidation therapy with allogeneic HSCT. In accordance, we propose that the time slope of remission is an additional leukemia-related dynamic parameter that reflects chemosensitivity and thus may inform post-induction therapy decision-making. In addition to patient-related factors, European LeukemiaNet risk group, measurable residual disease monitoring and donor availability, this may particularly apply to European LeukemiaNet intermediate-risk patients, for whom a decision between consolidation chemotherapy and allogeneic HSCT remains challenging in many cases

ATP-dependent chromatin remodelers - Analysis of expression patterns and impact on gene regulation

ATP-dependent chromatin remodelers are enzymes which use the energy from ATP hydrolysis to alter the chromatin structure. Thus, they play a key role in the transcriptional control of many important cellular processes such as proliferation,senescence and differentiation.The first part of this study focused on CHD5, a novel chromatin remodeling enzyme. To gain further insight into its expression profile and biological function, polyclonal peptide antisera were, together with a commercially available antiserum, successfully established for use in western blot, immunoprecipitation and immunofluorescence staining. ATP-dependent chromatin remodelers are expressed by very different patterns ranging from ubiquitous expression to an expression restricted to specific cell populations. So far, CHD5 expression was thought to be restricted to neural-related tissues. Here, it was shown for the first time that CHD5 expression is not restricted to neural tissues but additionally expressed in rodent testes. Furthermore, several cell lines were tested for CHD5 expression on transcript and protein level. CHD5 expression was not detected in any of the tested neuroblastoma and glioblastoma cell lines. In neuroblastoma cell lines this was expected due to the suggested function as tumor suppressor. Furthermore, a primary astrocyte culture was established. No CHD5 could be detected in protein lysates from astrocytes but was found in those from murine neural stem cells. This suggests that CHD5 expression in brain is restricted to neural stem cells and probably also to neurons. In the second part of this thesis, the impact of the chromatin remodelers CHD4 and BRG1 on a TNFα-induced inflammatory response was investigated. BRG1 and CHD4 expression was disrupted by a siRNA-mediated knockdown in HEK293 cells. After stimulations with TNFα for one to four hours, gene induction of typical inflammatory NF-κB target genes was determined by RT-qPCR. BRG1 and CHD4 were both required for the efficient induction of all tested target genes exclusively after one hour but not after four hours of TNFα treatment. Expression of the housekeeping gene ß-actin was unaffected. Surprisingly, although CHD4 is mainly known to be involved in transcriptional repression, a requirement for CHD4 in active transcription during this inflammatory response was revealed. To gain further insight into the mechanism of involvement, co-immunoprecipitations of the NF-κB subunit p65 with BRG1 and CHD4,respectively, were performed upon TNFα stimulation. No robust interactions between these proteins could be observed. Furthermore, ChIP experiments with the NF-κB subunit p50, histone H3 and CHD4 were carried out on the model target gene CXCL2. p50 was recruited to the promoter of CXCL2 rapidly after TNFα was added. Histone H3 binding to the promoter and the open reading frame of the gene strongly decreased within the first hour of TNFα stimulation, indicating that chromatin remodeling took place during the early period of gene induction. Recruitment of CHD4 was nonconclusive. Thus, the mechanism of how BRG1 and CHD4 act during TNFα response needs to be investigated further. In summary, a major influence of the chromatin remodelers BRG1 and CHD4 on the TNFα-induced gene activation during its early phase in HEK293 cells was demonstrated

Three-Year Mortality of Older Hospitalized Patients with Osteosarcopenia: Data from the OsteoSys Study

Osteosarcopenia, the concurrent presence of sarcopenia and osteopenia/osteoporosis, poses a significant health risk to older adults, yet its impact on clinical outcomes is not fully understood. The aim of this prospective, longitudinal multicentre study was to examine the impact of osteosarcopenia on 3-year mortality and unplanned hospitalizations among 572 older hospitalized patients (mean age 75.1 ± 10.8 years, 78% female). Sarcopenia and low bone mineral density (BMD) were evaluated using Dual Energy X-ray Absorptiometry and the European Working Group on Sarcopenia in Older People (EWGSOP2) and WHO criteria, respectively. Among participants, 76% had low BMD, 9% were sarcopenic, and 8% had osteosarcopenia. Individuals with osteosarcopenia experienced a significantly higher rate of mortality (46%, p < 001) and unplanned hospitalization (86%, p < 001) compared to those without this condition. Moreover, “healthy” subjects—those without sarcopenia or low BMD—showed markedly lower 3-year mortality (9%, p < 001) and less unplanned hospitalization (53%, p < 001). The presence of osteosarcopenia (p = 0.009) increased the 3-year mortality risk by 30% over sarcopenia alone and by 8% over low BMD alone, underscoring the severe health implications of concurrent muscle and bone deterioration. This study highlights the substantial impact of osteosarcopenia on mortality among older adults, emphasizing the need for targeted diagnostic and therapeutic strategies