Appropriateness of exercise therapy delivery in chronic low back pain management: cross-sectional online survey of physiotherapy practice in Germany.

BACKGROUND: In Germany, exercise therapy represents the most commonly prescribed physiotherapy service for non-specific, chronic low back pain (NSCLBP). So far, little is known about current practice patterns of German physiotherapists in delivering this intervention. Thus, the aim of this study was to investigate the appropriateness of exercise therapy delivered to NSCLBP patients in German physiotherapy care and to identify practitioner-related drivers of appropriate exercise delivery. METHODS: We used a vignette-based, exploratory, cross-sectional, online-survey study design (76-items; data collection between May and July 2023). Eligible participants were required to hold a professional degree in physiotherapy and were required to be practicing in Germany. Access links to anonymous online surveys were spread via established German physiotherapy networks, educational platforms, social media, e-mail lists, and snowball sampling. Appropriateness of exercise therapy was calculated by an equally weighted total score (400 points) including scales on shared-decision-making, exercise dose selection, pain knowledge and self-management promotion. "Appropriate exercise delivery" was determined by a relative total score achievement of > 80%. "Partly appropriate exercise delivery" was determined by a relative total score achievement of 50-79%, and "inappropriate exercise delivery" by a score achievement of < 50%. Practitioner-related drivers of exercise appropriateness were calculated by bivariate and multiple linear regression analyses. RESULTS: 11.9% (N = 35) of 298 physiotherapists' exercise delivery was considered "appropriate", 83.3% (N = 245) was "partly appropriate", and 4.8% (N = 14) was "inappropriate". In the final multiple regression model, most robust parameters positively influencing appropriate delivery of exercise therapy were increased scientific literacy (B = 10.540; 95% CI [0.837; 20.243]), increased average clinical assessment time (B = 0.461; 95% CI [0.134; 0.789]), increased self-perceived treatment competence (B = 7.180; 95% CI [3.058; 11.302], and short work experience (B = - 0.520; 95% CI [-0.959; - 0.081]). CONCLUSION: Appropriate exercise delivery in NSCLBP management was achieved by only 11.9% of respondents. However, the vast majority of 95.2% of respondents was classified to deliver exercise therapy partly appropriate. Long work experience seemed to negatively affect appropriate exercise delivery. Positive influences were attributed to scientific literacy, the average clinical assessment time per patient as well as the perceived treatment competence in NSCLBP management. REGISTRATION: Open science framework: https://doi.org/10.17605/OSF.IO/S76MF .

[Determinants of use of rehabilitation services before entering disability pension: Time series analysis of insured members of the German Pension Insurance Federation in Berlin and Brandenburg]. / Determinanten zur Inanspruchnahme von Rehabilitationsleistungen vor Eintritt in die Erwerbsminderung: Zeitreihenanalyse von Versicherten der Deutschen Rentenversicherung Bund in Berlin und Brandenburg.

BACKGROUND: The age-standardized application rate for medical rehabilitation services of the German Pension Insurance Association has been declining since 2009. Two of the most frequent reasons for applying for rehabilitation services represent musculoskeletal disorders and mental disorders. The aim of this analysis was to identify factors influencing the utilization of rehabilitation services in the federal states of Berlin and Brandenburg. METHODS: The explorative time series analysis is based on a research dataset of the German Pension Insurance Federation. Insured persons of the German Pension Insurance Federation from the federal states of Berlin and Brandenburg with a diagnosis of musculoskeletal disorders or mental disorders were included. Descriptive differences in targeted and prognostic parameters were calculated using chi-square and t-test statistics. Predictive parameters for the utilization of medical rehabilitation services were calculated using binary, logistic regression analyses. RESULTS: A total of 11,257 insured cases were examined. For the population of insured persons from Berlin, the use of medical rehabilitation services showed significant prognostic variables for gender, age at retirement, marital status, level of education, occupational requirement level, total accumulated earning points, gross pension level, status of a temporary pension, health insurance status, and diagnosis group. For Brandenburg, significant prognostic variables were found for age at retirement, marital status, nationality, education level, occupational requirement level, total accumulated earning points, status of a temporary pension, occupational sector, and diagnosis group. CONCLUSION: In Berlin and Brandenburg mental disorders and sociodemographic parameters are associated with an increased probability of not claiming medical rehabilitation services before the onset of reduced earning capacity. It remains to be investigated which mechanisms cause people with mental disorders to make no use of medical rehabilitation services. Future analyses should examine isolated interaction mechanisms for the utilization of medical rehabilitation services, especially in the case of existing F-diagnoses. In addition, explanations for different influencing variables between states remain to be explored.

Research designs and instruments to detect physiotherapy overuse of low-value care services in low back pain management: a scoping review.

BACKGROUND: The provision of low-value physiotherapy services in low back pain management is a known but complex phenomenon. Thus, this scoping review aims to systematically map existing research designs and instruments of the field in order to discuss the current state of research methodologies and contextualize results to domains and perspectives of a referred low-value care typology. Ultimately, results will be illustrated and transferred to conditions of the German health care setting as care delivery conditions of physiotherapy in Germany face unique particularities. METHODS: The development of this review is guided by the analysis framework of Arksey and O'Malley. A two-stage, audited search strategy was performed in Medline (PubMed), Web of Science, and google scholar. All types of observational studies were included. Identified articles needed to address a pre-determined population, concept, and context framework and had to be published in English or German language. The publication date of included articles was not subject to any limitation. The applied framework to assess the phenomenon of low-value physiotherapy services incorporated three domains (care effectiveness; care efficiency; patient alignment of care) and perspectives (provider; patient; society) of care. RESULTS: Thirty-three articles met the inclusion criteria. Seventy-nine percent of articles focused on the appropriateness of physiotherapeutic treatments, followed by education and information (30%), the diagnostic process (15%), and goal-setting practice (12%). Study designs were predominantly cross-sectional (58%). Data sources were mainly survey instruments (67%) of which 50% were self-developed. Most studies addressed the effectiveness domain of care (73%) and the provider perspective (88%). The perspective of patient alignment was assessed by 6% of included articles. None of included articles assessed the society perspective. Four methodical approaches of included articles were rated to be transferrable to Germany. CONCLUSION: Identified research on low-value physiotherapy care in low back pain management was widely unidimensional. Most articles focused on the effectiveness domain of care and investigated the provider perspective. Most measures were indirectly and did not monitor low-value care trends over a set period of time. Research on low-value physiotherapy care in secondary care conditions, such as Germany, was scarce. REGISTRATION: This review has been registered on open science framework ( https://osf.io/vzq7k https://doi.org/10.17605/OSF.IO/PMF2G ).

Implementation fidelity in a multifaceted program to foster rational antibiotics use in primary care: an observational study.

BACKGROUND: The ARena study (Sustainable Reduction of Antimicrobial Resistance in German Ambulatory Care) is a three-arm, cluster randomized trial to evaluate a multifaceted implementation program in a German primary care setting. In the context of a prospective process evaluation conducted alongside ARena, this study aimed to document and explore fidelity of the implementation program. METHODS: This observational study is based on data generated in a three-wave survey of 312 participating physicians in the ARena program and attendance documentation. Measures concerned persistence of participation in the ARena program and adherence to intervention components (thematic quality circles, e-learning, basic expenditure reimbursements, additional bonus payments and a computerized decision support system). Participants' views on five domains of the implementation were also measured. Binary logistic and multiple linear regression analyses were used to explore which views on the implementation were associated with participants' adherence to quality circles and use of additional bonus compensation. RESULTS: The analysis of fidelity showed overall high persistence of participation in the intervention components across the three intervention arms (90,1%; 97,9%; 92,9%). 96.4% of planned quality circles were delivered to study participants and, across waves, 30.4% to 93% of practices participated; 56.1% of physicians attended the maximum of four quality circles. 84% of the practices (n = 158) with a minimum of one index patient received a performance-based additional bonus payment at least once. In total, bonus compensation was triggered for 51.8% of affected patients. Participation rate for e-learning (a prerequisite for reimbursement of project-related expenditure) covered 90.8% of practices across all intervention arms, with the highest rate in arm II (96.5%). Uptake of expenditure reimbursement was heterogeneous across study arms, with a mean rate of 86.5% (89.1% in arm I, 96.4% in arm II and 74.1% in arm III). Participants' views regarding participant responsiveness (OR = 2.298) 95% CI [1.598, 3.305] and Context (OR = 2.146) 95% CI [1.135, 4.055] affected additional bonus payment. Participants' views on participant responsiveness (Beta = 0.718) 95% CI [0.479, 0.957], Context (Beta = 0.323) 95% CI [0.055, 0.590] and Culture of shared decision-making (Beta = -0.334) 95% CI [-0.614, -0.053] affected quality circle attendance. CONCLUSION: This study showed an overall high fidelity to the implementation program. Participants' views on the implementation were associated with degree of intervention fidelity. TRIAL REGISTRATION: ISRCTN, ISRCTN58150046.

Rural-urban differences in workplace health promotion among employees of small and medium-sized enterprises in Germany.

BACKGROUND: Rural and urban areas hold different health challenges and resources for resident small and medium-sized enterprises (SMEs) and their employees. Additionally, residents of urban and rural areas differ in individual characteristics. This study aims at investigating potential rural-urban differences (1) in the participation rate in workplace health promotion (WHP) and (2) in the relationship of WHP and health relevant outcomes in residents living in rural or urban German areas and working in SMEs. METHODS: Data of a large German Employee Survey in 2018 were used and analyzed by chi-square and t-tests and regression analyses regarding job satisfaction, sick days, and psychosomatic complaints. A total of 10,763 SME employees was included in analyses (23.9% living in rural, 76.1% living in urban areas). RESULTS: Analyses revealed higher participation rates for SME employees living in rural areas. SME employees living in urban areas reported more often the existence of WHP. Results showed (a) significance of existence of WHP for psychosomatic complaints and (b) significance of participation in WHP for job satisfaction in SME employees living in urban but not for those living in rural areas. CONCLUSION: The revealed disparities of (1) higher participation rates in SME employees living in rural areas and in (2) the relationship of WHP aspects with health relevant outcomes are of special interest for practitioners (, e.g. human resource managers), politicians, and researchers by providing new indications for planning and evaluating WHP measures.

Biomedical Dogmas Still Influence the Delivery of Exercise Therapy in Chronic Low Back Pain Management: Mixed-Methods Study.

Purpose: Non-specific, chronic low back pain (NSCLBP) is a leading cause of disability, prompting long-term rehabilitation. Positive patient beliefs and expectations towards lasting exercise engagement play a crucial role for a successful management of this condition. The aim was to investigate beliefs, unmet needs and expectations of NSCLBP patients for exercise-related health behaviour change in the context of rehabilitative care. Patients and Methods: In a mono-centric mixed-methods study, we conducted semi-structured interviews with NSCLBP patients and care providers. We recruited in a rehabilitation clinic which is specialized in orthopaedics and internal medicine. Interviews were analysed deductively by combining health behaviour change theories with Donabedian's quality model of care. In a patient survey, disability (RMDQ), exercise behaviours, fear avoidance beliefs (FABQ), self-efficacy in chronic disease management (SES6G), process- and outcome-expectations (OEE-2) were queried and analysed descriptively. Results: Twenty-two interviews were conducted and 40 questionnaires completed. Qualitative results revealed that NSCLBP patients had persistent biomedical perspectives on their health condition, marked by strong preferences for biomedical diagnostics and beliefs in the superiority of specific exercise regimes. Based on met expectations and positive movement experiences, patients' motivation was successfully fostered in the motivational phase of health behavior change. In the volitional phase, the postulated desire to receive self-management strategies was largely unmet. Psychosocial aspects of care were not widely accepted. The survey study sample showed a disability score (RMDQ) of M = 6.8 (±4.6). Mean scores of validated scales reflecting on attitudes, beliefs and expectations of chronic NSCLBP management were at FABQ-pa M = 15.4 (±6.0), FABQ-w M = 24.0 (±12.1), SES6G M = 6.4 (±2.3), and OEE-2 M = 2.7 (±0.5). Conclusion: In this sample, patients' understanding of NSCLBP was still dominated by biomedical dogmas and perspectives. Inpatient rehabilitation predominantly addressed expectations towards the motivational phase of exercise-related health behaviour change.

Light chain mutations contribute to defining the fibril morphology in systemic AL amyloidosis.

Systemic AL amyloidosis is one of the most frequently diagnosed forms of systemic amyloidosis. It arises from mutational changes in immunoglobulin light chains. To explore whether these mutations may affect the structure of the formed fibrils, we determine and compare the fibril structures from several patients with cardiac AL amyloidosis. All patients are affected by light chains that contain an IGLV3-19 gene segment, and the deposited fibrils differ by the mutations within this common germ line background. Using cryo-electron microscopy, we here find different fibril structures in each patient. These data establish that the mutations of amyloidogenic light chains contribute to defining the fibril architecture and hence the structure of the pathogenic agent.

SARS-CoV-2-Related Adaptation Mechanisms of Rehabilitation Clinics Affecting Patient-Centered Care: Qualitative Study of Online Patient Reports.

BACKGROUND: The SARS-CoV-2 pandemic impacted access to inpatient rehabilitation services. At the current state of research, it is unclear to what extent the adaptation of rehabilitation services to infection-protective standards affected patient-centered care in Germany. OBJECTIVE: The aim of this study was to determine the most relevant aspects of patient-centered care for patients in inpatient rehabilitation clinics under early phase pandemic conditions. METHODS: A deductive-inductive framework analysis of online patient reports posted on a leading German hospital rating website, Klinikbewertungen (Clinic Reviews), was performed. This website is a third-party, patient-centered commercial platform that operates independently of governmental entities. Following a theoretical sampling approach, online reports of rehabilitation stays in two federal states of Germany (Brandenburg and Saarland) uploaded between March 2020 and September 2021 were included. Independent of medical specialty groups, all reports were included. Keywords addressing framework domains were analyzed descriptively. RESULTS: In total, 649 online reports reflecting inpatient rehabilitation services of 31 clinics (Brandenburg, n=23; Saarland, n=8) were analyzed. Keywords addressing the care environment were most frequently reported (59.9%), followed by staff prerequisites (33.0%), patient-centered processes (4.5%), and expected outcomes (2.6%). Qualitative in-depth analysis revealed SARS-CoV-2-related reports to be associated with domains of patient-centered processes and staff prerequisites. Discontinuous communication of infection protection standards was perceived to threaten patient autonomy. This was amplified by a tangible gratification crisis of medical staff. Established and emotional supportive relationships to clinicians and peer groups offered the potential to mitigate the adverse effects of infection protection standards. CONCLUSIONS: Patients predominantly reported feedback associated with the care environment. SARS-CoV-2-related reports were strongly affected by increased staff workloads as well as patient-centered processes addressing discontinuous communication and organizationally demanding implementation of infection protection standards, which were perceived to threaten patient autonomy. Peer relationships formed during inpatient rehabilitation had the potential to mitigate these mechanisms.

Research designs and instruments to detect physiotherapy overuse of low-value care services in low back pain management: a scoping review protocol.

BACKGROUND: The provision of low-value care services in low back pain management is a problem of global scope. Inappropriate imaging, overmedication, and overused invasive therapies are prevalent in physician services. Yet, little is known about overused low-value physiotherapy services. Most studies addressing physiotherapy overuse in low back pain management arose from countries in which physiotherapy is established in primary care. However, measures and instruments addressing physiotherapy overuse limitedly fit legislative conditions of health systems in which physiotherapy is a service of secondary care. Thus, this scoping review's purpose is to map existing research designs and instruments aiming to detect overused low-value physiotherapy services taking specific healthcare settings and aspects of medical overuse into account. METHODS: The development of this scoping review is guided by the Arksey and O'Malley framework. A two-step, peer-reviewed search strategy in accordance with the PRESS checklist will be conducted on MEDLINE (PubMed), Web of Science, and CINHAL. Additionally, gray literature will be searched on Google Scholar. Preprints of empirical studies will be included. Initially, two reviewers will independently screen articles for eligibility by title and abstract. A third reviewer will mediate discrepancies. Uncertainties will be eliminated by a full-text analysis or by contacting the corresponding authors. A four-step analytical process will guide result reporting focusing on major research questions outlined in this protocol. Numerical and narrative tables, graphics, and narrative summaries will be the methods to summarize and collate results. In the final step, the German health system will serve as an exemplary setting and frame to practically apply results. DISCUSSION: Results of this scoping review will help researchers to systematically select overuse measures referring to aspects of the overuse typology, specific healthcare settings, and physiotherapy services. It will further provide information on the limitations of present studies and will give advice on how to address them. Moreover, this review will illustrate to what degree existing studies succeed to comprehensively cover the concept of the overuse typology. SYSTEMATIC REVIEW REGISTRATION: This protocol has been registered on the open science framework ( https://doi.org/10.17605/OSF.IO/PMF2G ).

Organizational Health Literacy in the Context of Employee Health: An Expert-Panel-Guided Scoping Review Protocol.

Health literacy (HL) is an interplay of individual and organizational health literacy (OHL). While individual HL has been intensively studied, the importance of OHL has become a greater focus of research attention. The National Action Plan Health Literacy in Germany emphasizes the promotion of HL in all areas of everyday life, including occupation and the workplace. The proposed scoping review aims at identifying and evaluating definitions, empirical studies and instruments on OHL targeting employee recipients. The search will be conducted in two consecutive steps and guided by expert-panel discussions in accordance to the method of Consensus Development Panels. The search will be conducted in Web of Science, PubMed and Google Scholar according to the methodological framework of Arksey and O'Malley and supplemented by the snowball principle and a hand search. All records will be included that were published until the final search date. To define eligibility criteria, the PCC framework of the Joanna Briggs Institute is used. The scoping review will critically discuss whether a new definition of OHL in the context of employee health is of purpose for future research and practice. Nonetheless, it will provide orientation in the context of employee health, also facing the consequences of SARS-CoV-2.

Amyloid fibril structure from the vascular variant of systemic AA amyloidosis.

Systemic AA amyloidosis is a debilitating protein misfolding disease in humans and animals. In humans, it occurs in two variants that are called 'vascular' and 'glomerular', depending on the main amyloid deposition site in the kidneys. Using cryo electron microscopy, we here show the amyloid fibril structure underlying the vascular disease variant. Fibrils purified from the tissue of such patients are mainly left-hand twisted and contain two non-equal stacks of fibril proteins. They contrast in these properties to the fibrils from the glomerular disease variant which are right-hand twisted and consist of two structurally equal stacks of fibril proteins. Our data demonstrate that the different disease variants in systemic AA amyloidosis are associated with different fibril morphologies.

Conditional deletion of ferritin H in mice induces loss of iron storage and liver damage.

UNLABELLED: Ferritin plays a central role in iron metabolism by acting both as iron storage and a detoxifying protein. We generated a ferritin H allele with loxP sites and studied the conditional ferritin H deletion in adult mice. Ten days after Mx-Cre induced deletion, ferritin H messenger RNA (mRNA) was below 5% in the liver, spleen, and bone marrow of deleted mice compared to control littermates. Mice lost their cellular iron stores indicating the requirement of ferritin H in iron deposition. Serum iron and transferrin saturation were slightly increased and correlated with a two-fold increased liver hepcidin 1 mRNA and a reduced duodenal DcytB mRNA level. Under a normal iron regimen, deleted mice survived for 2 years without visible disadvantage. Mice fed on a high iron diet prior to ferritin H deletion suffered from severe liver damage. Similarly, ferritin H deleted mouse embryonic fibroblasts showed rapid cell death after exposure to iron salt in the medium. This was reversed by wild-type ferritin H but not by a ferritin H mutant lacking ferroxidase activity. Cell death was preceded by an increase in cytoplasmic free iron, reactive oxygen species, and mitochondrial depolarization. CONCLUSION: Our results provide evidence that the iron storage function of ferritin plays a major role in preventing iron-mediated cell and tissue damage.

Fostering Appropriate Antibiotic Use in a Complex Intervention: Mixed-Methods Process Evaluation Alongside the Cluster-Randomized Trial ARena.

The cluster randomized trial ARena (sustainable reduction of antibiotic-induced antimicrobial resistance, 2017-2020) promoted appropriate use of antibiotics for acute non-complicated infections in primary care networks (PCNs) in Germany. A process evaluation assessed determinants of practice and explored factors associated with antibiotic prescribing patterns. This work describes its findings on uptake and impacts of the complex intervention program and indicates potential implementation into routine care. In a nested mixed-methods approach, a three-wave study-specific survey for participating physicians and medical assistants assessed potential impacts and uptake of the complex intervention program. Stakeholders received a one-time online questionnaire to reflect on network-related aspects. Semi-structured, open-ended interviews, with a purposive sample of physicians, medical assistants and stakeholders, explored program component acceptance for daily practice and perceived sustainability of intervention component effects. Intervention components were perceived to be smoothly integrable into practice routines. The highest uptake was reported for educational components: feedback reports, background information, e-learning modules and disease-specific quality circles (QCs). Participation in PCNs was seen as the motivational factor for guideline-oriented patient care and adoption of new routines. Future approaches to fostering appropriate antibiotics use by targeting health literacy competencies and clinician's therapy decisions should combine evidence-based information sources, audit and feedback reports and QCs.

Destabilization of interleukin-6 mRNA requires a putative RNA stem-loop structure, an AU-rich element, and the RNA-binding protein AUF1.

Interleukin-6 mRNA is unstable and degraded with a half-life of 30 min. Instability determinants can entirely be attributed to the 3' untranslated region. By grafting segments of this region to stable green fluorescent protein mRNA and subsequent scanning mutagenesis, we have identified two conserved elements, which together account for most of the instability. The first corresponds to a short noncanonical AU-rich element. The other, 80 nucleotides further 5', comprises a sequence predicted to form a stem-loop structure. Neither element alone was sufficient to confer full instability, suggesting that they might cooperate. Overexpression of myc-tagged AUF1 p37 and p42 isoforms as well as suppression of endogenous AUF1 by RNA interference stabilized interleukin-6 mRNA. Both effects required the AU-rich instability element. Similarly, the proteasome inhibitor MG132 stabilized interleukin-6 mRNA probably through an increase of AUF1 levels. The mRNA coimmunoprecipitated specifically with myc-tagged AUF1 p37 and p42 in cell extracts but only when the AU-rich instability element was present. These results indicate that AUF1 binds to the AU-rich element in vivo and promotes IL-6 mRNA degradation.

Differential translational regulation of IRE-containing mRNAs in Drosophila melanogaster by endogenous IRP and a constitutive human IRP1 mutant.

Insects, like vertebrates, express iron regulatory proteins (IRPs) that may regulate proteins in cellular iron storage and energy metabolism. Two mRNAs, an unspliced form of ferritin H mRNA and succinate dehydrogenase subunit b (SDHb) mRNA, are known to comprise an iron responsive element (IRE) in their 5'-untranslated region making them susceptible to translational repression by IRPs at low iron levels. We have investigated the effect of wild-type human IRP1 (hIRP1) and the constitutively active mutant hIRP1-S437 in transgenic Drosophila melanogaster. Endogenous Drosophila IRE-binding activity was readily detected in gel retardation assays. However, translational repression assessed by polysome gradients was only visible for unspliced IRE-containing ferritin H mRNA, but not for SDHb mRNA. Upon expression of exogenous hIRP1-S437 both mRNAs were strongly repressed. This correlated with a diminished survival rate of adult flies with hIRP1 and complete lethality with hIRP1-S437. We conclude that constitutive IRP1 expression is deleterious to fly survival, probably due to the essential function of SDHb or proteins encoded by yet unidentified target mRNAs.

Short-lived AUF1 p42-binding mRNAs of RANKL and BCL6 have two distinct instability elements each.

Regulation of mRNA stability by RNA-protein interactions contributes significantly to quantitative aspects of gene expression. We have identified potential mRNA targets of the AU-rich element binding protein AUF1. Myc-tagged AUF1 p42 was induced in mouse NIH/3T3 cells and RNA-protein complexes isolated using anti-myc tag antibody beads. Bound mRNAs were analyzed with Affymetrix microarrays. We have identified 508 potential target mRNAs that were at least 3-fold enriched compared to control cells without myc-AUF1. 22.3% of the enriched mRNAs had an AU-rich cluster in the ARED Organism database, against 16.3% of non-enriched control mRNAs. The enrichment towards AU-rich elements was also visible by AREScore with an average value of 5.2 in the enriched mRNAs versus 4.2 in the control group. Yet, numerous mRNAs were enriched without a high ARE score. The enrichment of tetrameric and pentameric sequences suggests a broad AUF1 p42-binding spectrum at short U-rich sequences flanked by A or G. Still, some enriched mRNAs were highly unstable, as those of TNFSF11 (known as RANKL), KLF10, HES1, CCNT2, SMAD6, and BCL6. We have mapped some of the instability determinants. HES1 mRNA appeared to have a coding region determinant. Detailed analysis of the RANKL and BCL6 3'UTR revealed for both that full instability required two elements, which are conserved in evolution. In RANKL mRNA both elements are AU-rich and separated by 30 bases, while in BCL6 mRNA one is AU-rich and 60 bases from a non AU-rich element that potentially forms a stem-loop structure.

Role of Ets-1 in transcriptional regulation of transferrin receptor and erythroid differentiation.

High expression of transferrin receptor (TfR) on the membrane of erythroid cells accounts for the high level of iron required to sustain heme synthesis. Several studies indicate that during erythroid differentiation TfR expression is highly dependent on transcriptional regulation. In this study we characterized the minimal region able to confer transcriptional regulation during erythroid differentiation in Friend leukemia cells (FLC). This region of 120 bp, upstream the transcription start site, contains an overlapping consensus recognition sequence for AP1/CREB/ATF transcription factors and for proteins of the Ets family and a GC rich region. Here, we report that both the Ets and the Ap1/CRE like sites are essential for promoter activity during erythroid differentiation. We showed that Ets-1 binds to the EBS-TfR and its binding activity decreases in FLC induced to differentiate and during normal erythroid differentiation. Consistent with this, FLC constitutively expressing Ets-1 show a decrease in TfR gene expression, globin mRNA and hemoglobin synthesis. We conclude that Ets-1 binding activity is modulated during erythroid maturation and that a deregulated expression of this transcription factor interferes with terminal erythroid differentiation.

Analysis of the contribution of changes in mRNA stability to the changes in steady-state levels of cyclin mRNA in the mammalian cell cycle.

Cyclins are the essential regulatory subunits of cyclin-dependent protein kinases. They accumulate and disappear periodically at specific phases of the cell cycle. Here we investigated whether variations in cyclin mRNA levels in exponentially growing cells can be attributed to changes in mRNA stability. Mouse EL4 lymphoma cells and 3T3 fibroblasts were synchronized by elutriation or cell sorting. Steady-state levels and degradation of cyclin mRNAs and some other cell cycle related mRNAs were measured at early G1, late G1, S and G2/M phases. In both cell lines mRNAs of cyclins C, D1 and D3 remained unchanged throughout the cell cycle. In contrast, cyclin A2 and B1 mRNAs accumulated 3.1- and 5.7-fold between early G1 and G2/M phase, whereas cyclin E1 mRNA decreased 1.7-fold. Mouse cyclin A2 and B1 genes, by alternative polyadenylation, gave rise to more than one transcript. In both cases, the longer transcripts were the minor species but accumulated more strongly in G2/M phase. All mRNAs were rather stable with half-lives of 1.5-2 h for cyclin E1 mRNA and 3-4 h for the others. Changes in mRNA stability accounted for the accumulation in G2/M phase of the short cyclin A2 and B1 mRNAs, but contributed only partially to changes in levels of the other mRNAs.

Iron regulatory proteins and their role in controlling iron metabolism.

Cellular iron homeostasis is regulated by post-transcriptional feedback mechanisms, which control the expression of proteins involved in iron uptake, release and storage. Two cytoplasmic proteins with mRNA-binding properties, iron regulatory proteins 1 and 2 (IRP1 and IRP2) play a central role in this regulation. Foremost, IRPs regulate ferritin H and ferritin L translation and thus iron storage, as well as transferrin receptor 1 (TfR1) mRNA stability, thereby adjusting receptor expression and iron uptake via receptor-mediated endocytosis of iron-loaded transferrin. In addition splice variants of iron transporters for import and export at the plasma-membrane, divalent metal transporter 1 (DMT1) and ferroportin are regulated by IRPs. These mechanisms have probably evolved to maintain the cytoplasmic labile iron pool (LIP) at an appropriate level. In certain tissues, the regulation exerted by IRPs influences iron homeostasis and utilization of the entire organism. In intestine, the control of ferritin expression limits intestinal iron absorption and, thus, whole body iron levels. In bone marrow, erythroid heme biosynthesis is coordinated with iron availability through IRP-mediated translational control of erythroid 5-aminolevulinate synthase mRNA. Moreover, the translational control of HIF2α mRNA in kidney by IRP1 coordinates erythropoietin synthesis with iron and oxygen supply. Besides IRPs, body iron absorption is negatively regulated by hepcidin. This peptide hormone, synthesized and secreted by the liver in response to high serum iron, downregulates ferroportin at the protein level and thereby limits iron absorption from the diet. Hepcidin will not be discussed in further detail here.

Ferritin H gene deletion in the choroid plexus and forebrain results in hydrocephalus.

Ferritin H, the major iron storage protein, has essential functions in early embryonic development as well as in adult liver and intestine. To address the question whether ferritin H has similarly essential functions in the brain we used the Cre/loxP system to generate mice with a forebrain-specific inactivation of the ferritin H gene. Ferritin H deficiency in most cells of the forebrain including cells of the choroid plexus caused accumulation of cerebrospinal fluid in the lateral ventricles and the subarachnoid space. Brain tissue iron content was unchanged.