SIAH3 is frequently epigenetically silenced in cancer and regulates mitochondrial metabolism.

Of the seven in absentia homologue (SIAH) family, three members have been identified in the human genome. In contrast to the E3 ubiquitin ligase encoding SIAH1 and SIAH2, little is known on the regulation and function of SIAH3 in tumorigenesis. In this study, we reveal that SIAH3 is frequently epigenetically silenced in different cancer entities, including cutaneous melanoma, lung adenocarcinoma and head and neck cancer. Low SIAH3 levels correlate with an impaired survival of cancer patients. Additionally, induced expression of SIAH3 reduces cell proliferation and induces cell death. Functionally, SIAH3 negatively affects cellular metabolism by shifting cells form aerobic oxidative phosphorylation to glycolysis. SIAH3 is localized in the mitochondrion and interacts with proteins involved in mitochondrial ribosome biogenesis and translation. We also report that SIAH3 interacts with ubiquitin ligases, including SIAH1 or SIAH2, and is degraded by them. These results suggest that SIAH3 acts as an epigenetically controlled tumor suppressor by regulating cellular metabolism through the inhibition of oxidative phosphorylation.

DYRK1B regulates Hedgehog-induced microtubule acetylation.

The posttranslational modification (PTM) of tubulin subunits is important for the physiological functions of the microtubule (MT) cytoskeleton. Although major advances have been made in the identification of enzymes carrying out MT-PTMs, little knowledge is available on how intercellular signaling molecules and their associated pathways regulate MT-PTM-dependent processes inside signal-receiving cells. Here we show that Hedgehog (Hh) signaling, a paradigmatic intercellular signaling system, affects the MT acetylation state in mammalian cells. Mechanistically, Hh pathway activity increases the levels of the MT-associated DYRK1B kinase, resulting in the inhibition of GSK3ß through phosphorylation of Serine 9 and the subsequent suppression of HDAC6 enzyme activity. Since HDAC6 represents a major tubulin deacetylase, its inhibition increases the levels of acetylated MTs. Through the activation of DYRK1B, Hh signaling facilitates MT-dependent processes such as intracellular mitochondrial transport, mesenchymal cell polarization or directed cell migration. Taken together, we provide evidence that intercellular communication through Hh signals can regulate the MT cytoskeleton and contribute to MT-dependent processes by affecting the level of tubulin acetylation.

The inverse agonist DG172 triggers a PPARß/δ-independent myeloid lineage shift and promotes GM-CSF/IL-4-induced dendritic cell differentiation.

The stilbene derivative (Z)-2-(2-bromophenyl)-3-{[4-(1-methylpiperazine)amino]phenyl}acrylonitrile (DG172) was developed as a highly selective inhibitory peroxisome proliferator-activated receptor (PPAR)ß/δ ligand. Here, we describe a novel PPARß/δ-independent, yet highly specific, effect of DG172 on the differentiation of bone marrow cells (BMCs). DG172 strongly augmented granulocyte-macrophage-colony-stimulating factor (GM-CSF)-induced differentiation of primary BMCs from Ppard null mice into two specific populations, characterized as mature (CD11c(hi)MHCII(hi)) and immature (CD11c(hi)MHCII(lo)) dendritic cells (DCs). IL-4 synergized with DG172 to shift the differentiation from MHCII(lo) cells to mature DCs in vitro. The promotion of DC differentiation occurred at the expense of differentiation to granulocytic Gr1(+)Ly6B(+) cells. In agreement with these findings, transcriptome analyses showed a strong DG172-mediated repression of genes encoding neutrophilic markers in both differentiating wild-type and Ppard null cells, while macrophage/DC marker genes were up-regulated. DG172 also inhibited the expression of transcription factors driving granulocytic differentiation (Cebpe, Gfi1, and Klf5), and increased the levels of transcription factors promoting macrophage/DC differentiation (Irf4, Irf8, Spib, and Spic). DG172 exerted these effects only at an early stage of BMC differentiation induced by GM-CSF, did not affect macrophage-colony-stimulating factor-triggered differentiation to macrophages and had no detectable PPARß/δ-independent effect on other cell types tested. Structure-function analyses demonstrated that the 4-methylpiperazine moiety in DG172 is required for its effect on DC differentiation, but is dispensable for PPARß/δ binding. Based on these data we developed a new compound, (Z)-2-(4-chlorophenyl)-3-[4-(4-methylpiperazine-1-yl)phenyl]acrylonitrile (DG228), which enhances DC differentiation in the absence of significant PPARß/δ binding.

Mixed-polarization phenotype of ascites-associated macrophages in human ovarian carcinoma: correlation of CD163 expression, cytokine levels and early relapse.

Ovarian cancer is typically accompanied by the occurrence of malignant ascites containing large number of macrophages. It has been suggested that these tumor-associated macrophages (TAMs) are skewed to alternative polarization (M2) and thereby play an essential role in therapy resistance and metastatic spread. In our study, we have investigated the nature, regulation and clinical correlations of TAM polarization in serous ovarian cancer. Macrophage polarization markers on TAMs and ascites cytokine levels were analyzed for 30 patients and associated with relapse-free survival (RFS) in a prospective study with 20 evaluable patients. Surface expression of the M2 marker CD163 on TAMs was inversely associated with RFS (p < 0.01). However, global gene expression profiles determined for 17 of these patients revealed a mixed-polarization phenotype unrelated to the M1/M2 classification. CD163 surface expression also correlated with the ascites levels of IL-6 and IL-10 (p < 0.05), both cytokines induced CD163 expression, and their ascites levels showed a clear inverse association with RFS (p < 0.01). These findings define a subgroup of patients with high CD163 expression, high IL-6 and/or IL-10 levels and poor clinical outcome.

Quality of care in patients with hypertension: a retrospective cohort study of primary care routine data in Germany.

BACKGROUND: Hypertension is a leading cause of morbidity and mortality if not properly managed. Primary care has a major impact on these outcomes if its strengths, such as continuity of care, are deployed wisely. The analysis aimed to evaluate the quality of care for newly diagnosed hypertension in routine primary care data. METHODS: In the retrospective cohort study, routine data (from 2016 to 2022) from eight primary care practices in Germany were exported in anonymized form directly from the electronic health record (EHR) systems and processed for this analysis. The analysis focused on five established quality indicators for the care of patients who have been recently diagnosed with hypertension. RESULTS: A total of 30,691 patients were treated in the participating practices, 2,507 of whom have recently been diagnosed with hypertension. Prior to the pandemic outbreak, 19% of hypertensive patients had blood pressure above 140/90 mmHg and 68% received drug therapy (n = 1,372). After the pandemic outbreak, the proportion of patients with measured blood pressure increased from 63 to 87%, while the other four indicators remained relatively stable. Up to 80% of the total variation of the quality indicators could be explained by individual practices. CONCLUSION: For the majority of patients, diagnostic procedures are not used to the extent recommended by guidelines. The analysis showed that quality indicators for outpatient care could be mapped onto the basis of routine data. The results could easily be reported to the practices in order to optimize the quality of care.

Sex differences in symptoms following the administration of BNT162b2 mRNA COVID-19 vaccine in children below 5 years of age in Germany (CoVacU5): a retrospective cohort study.

BACKGROUND: Sex differences exist not only in the efficacy but also in adverse event rates of many vaccines. Here we compared the safety of BNT162b2 vaccine administered off-label in female and male children younger than 5 years in Germany. METHODS: This is a retrospective cohort study, in which we performed a post-hoc analysis of a dataset collected through an authentication-based survey of individuals having registered children aged 0-<5 years for vaccination against SARS-CoV-2 in six private practices and/or two lay person-initiated vaccination campaigns. We analyzed the safety profiles of the first 3 doses of 3-10 µg BNT162b2. Primary outcome was comparison in frequencies of 4 common post-vaccination symptom categories such as local, general, musculoskeletal symptoms and fever. Data were analyzed according to sex in bivariate analyses and regression models adjusting for age, weight, and dosage. Interaction between sex and BNT162b2 dosage was assessed. An active-comparator analysis was applied to compare post-vaccination symptoms after BNT162b2 versus non-SARS-CoV-2 vaccines. RESULTS: The dataset for the present analysis consisted of 7801 participants including 3842 females (49%) and 3977 males (51%) with an age of 3 years (median, interquartile: 2 years). Among individuals receiving 3 µg BNT162b2, no sex differences were noted, but after a first dose of 5-10 µg BNT162b2, local injection-site symptoms were more prevalent in girls compared to boys. In logistic regression, female sex was associated with higher odds of local symptoms, odds ratio (OR) of 1.33 (95% confidence interval [CI]: 1.15-1.55, p < 0.05) and general symptoms with OR 1.21 (95% CI: 1.01-1.44, p < 0.05). Following non-BNT162b2 childhood vaccinations, female sex was associated with a lower odds of post-vaccination musculoskeletal symptoms (OR: 0.29, 95% CI: 0.11-0.82, p < 0.05). An active comparator analysis between BNT162b2 and non-SARS-CoV-2 vaccinations revealed that female sex positively influenced the association between BNT162b2 vaccine type and musculoskeletal symptoms. CONCLUSIONS: Sex differences exist in post-vaccination symptoms after BNT162b2 administration even in young children. These are of importance for the conception of approval studies, for post-vaccination monitoring and for future vaccination strategies (German Clinical Trials Register ID: DRKS00028759).

For many childhood vaccines, the immune responses are different between the sexes. For the COVID-19 vaccine BNT162b2, the symptoms occurring after vaccination in children have not sufficiently been investigated. In this study, we performed an analysis of a dataset of retrospectively collected information on symptoms occurring after vaccinations with BNT162b2 and regular childhood vaccines in 7801 children under the age of 5 years in Germany, with a focus on the sex differences. Among the four categories assessed, local injection-site and general symptoms were more frequent in girls compared to boys. In contrast, following the administration of other childhood vaccines, the caregivers more frequently reported muscle-related symptoms in boys than in girls. In conclusion, sex differences exist in symptoms occurring after vaccinations even in young children, which is important to consider for future studies evaluating vaccine safety.

Reverse crosstalk of TGFß and PPARß/δ signaling identified by transcriptional profiling.

Previous work has provided strong evidence for a role of peroxisome proliferator-activated receptor ß/δ (PPARß/δ) and transforming growth factor-ß (TGFß) in inflammation and tumor stroma function, raising the possibility that both signaling pathways are interconnected. We have addressed this hypothesis by microarray analyses of human diploid fibroblasts induced to myofibroblastic differentiation, which revealed a substantial, mostly reverse crosstalk of both pathways and identified distinct classes of genes. A major class encompasses classical PPAR target genes, including ANGPTL4, CPT1A, ADRP and PDK4. These genes are repressed by TGFß, which is counteracted by PPARß/δ activation. This is mediated, at least in part, by the TGFß-induced recruitment of the corepressor SMRT to PPAR response elements, and its release by PPARß/δ ligands, indicating that TGFß and PPARß/δ signals are integrated by chromatin-associated complexes. A second class represents TGFß-induced genes that are downregulated by PPARß/δ agonists, exemplified by CD274 and IL6, which is consistent with the anti-inflammatory properties of PPARß/δ ligands. Finally, cooperative regulation by both ligands was observed for a minor group of genes, including several regulators of cell proliferation. These observations indicate that PPARß/δ is able to influence the expression of distinct sets of both TGFß-repressed and TGFß-activated genes in both directions.

The ambulatory care of patients with post-acute sequelae of COVID-19.

BACKGROUND: There is an increasing number of patients that do not make a rapid or full recovery from a SARS-CoV-2 infection, the Coronavirus Disease 2019 (COVID-19) and suffer from the so-called "long-COVID" or post-acute sequelae of COVID-19 (PASC). The long-term implications for health services are expected to be substantial. The objective of this analysis was to estimate the utilization of outpatient services from primary and secondary care. Further, we evaluated the multidisciplinary ambulatory care management of PASC patients in Germany. METHODS: All members of the Physician network "MEDI Baden-Württemberg e.V.", i.e., 1,263 primary care physicians (PCPs) and 1,772 specialists working in secondary care were invited to participate in a questionnaire surveying routine data regarding the general care situation at the physician practice level of patients suffering from PASC. Bivariate analyses were applied to consider potential differences between primary and secondary care. RESULTS: In total, 194 physicians participated in this survey (response rates of 9.6% (primary care) and 4.1% (secondary care). On average, each physician treated 31.9 PASC patients. Most PASC patients (61.2%) had three or more long-COVID symptoms. On average, 10.6 PASC patients visited a physicians' practice per quarter. The additional consulting effort for treating PASC patients was 34.1 min (median: 20 min) and higher in primary care. Most PCPs (71.1%) integrated secondary care in the treatment of their PASC patients. Less than half of the PASC patients (42.0%) sought secondary care with a referral from primary care. 5.7 patients visited the physicians' practices per week, who were concerned about suffering from PASC without any following medical confirmation. This caused an average additional effort for the physicians of 17.5 min per visit. There were no differences between rural and urban areas. CONCLUSION: Our results reveal that there is a substantial additional consulting effort for treating PASC patients that is especially high in primary care. The additional consulting effort results from the consultation of a particular high number of patients that are concerned about suffering from PACS without a following medical confirmation. To guarantee a high quality and adequate provision of care for a potentially further increasing number of concerned patients, the ambulatory health services should be strengthened and adequately compensated.

The use of routine data from primary care practices in Germany to analyze the impact of the outbreak of SARS-CoV-2 on the utilization of primary care services for patients with type 2 diabetes mellitus.

BACKGROUND: Routinely collected health data from ambulatory care providers offer a wide range of research opportunities. However, the access is often (e.g., technically) hindered, particularly in Germany. In the following, we describe the development of an infrastructure for the analysis of pseudonymized routine data extracted from primary care practices in Germany. Further, we analyze the impact of the outbreak of SARS-CoV-2 on the utilization of primary care services for patients with type 2 diabetes mellitus (DM type 2). METHODS: In this retrospective cohort study, routine data were extracted from nine private primary care practices before and since the outbreak of SARS-CoV-2 in Germany. The sample consisted of patients who were treated between 2016 and 2022 in one of the participating practices. The effects of the outbreak on the frequency of practice visits and the disease course of DM type 2 patients were analyzed by means of bivariate and multivariate analyses. RESULTS: The developed infrastructure offers an analysis of routine data from outpatient care within 24 h. In total, routine data of 30,734 patients could be processed for the analyses with 4182 (13.6%) patients having a diagnosed DM type 2 and 59.0% of these patients were enrolled in a disease management program (DMP). In the multivariate analysis, there was a significant negative effect of the SARS-CoV-2 outbreak on utilization of outpatient services of patients with DM type 2 disease. This decrease was less pronounced among DMP patients. The glycated haemoglobin level (HbA1c) has not changed significantly. CONCLUSIONS: The study showed that the analysis of routine data from outpatient care in Germany is possible in a timely manner using a special developed electronic health record system and corresponding software. The significantly negative effect of the SARS-CoV-2 outbreak on utilization of outpatient services of patients with DM type 2 disease was less pronounced among DMP patients. Two years after the start of the Covid pandemic a significantly worsened course of illness cannot be observed. However, it must be taken into account that the observation period for clinically relevant outcomes is still relatively short.

Comparative Safety of the BNT162b2 Messenger RNA COVID-19 Vaccine vs Other Approved Vaccines in Children Younger Than 5 Years.

Importance: SARS-CoV-2 vaccines are authorized for use in most age groups. The safety of SARS-CoV-2 vaccines is unknown in children younger than 5 years. Objective: To retrospectively evaluate the safety of the BNT162b2 vaccine used off-label in children younger than 5 years compared with the safety of non-SARS-CoV-2 vaccines in the same sample. Design, Setting, and Participants: This investigator-initiated retrospective cohort study included parents or caregivers who registered children for SARS-CoV-2 vaccination in outpatient care facilities in Germany. The study was performed as an authenticated online survey. A total of 19 000 email addresses were contacted from vaccination registration databases between April 14 and May 9, 2022. Inclusion criteria were child age younger than 5 years at the first BNT162b2 vaccination and use of a correct authentication code to prove invitation. Exposures: Off-label BNT162b2 vaccination and on-label non-SARS-CoV-2 vaccinations. Main Outcomes and Measures: Reported short-term safety data of 1 to 3 doses of 3 to 10 µg BNT162b2 in children from birth to younger than 60 months are presented. Coprimary outcomes were the frequencies of 11 categories of symptoms after vaccination with bivariate analyses and regression models adjusting for age, sex, weight, and height. Results: The study included 7806 children (median age, 3 years [IQR, 2-4 years]; 3824 [49.0%] female) who were followed up of for a mean (SD) of 91.4 (38.8) days since first BNT162b2 vaccination (survey response rate, 41.1%). A 10-µg dosage was more frequently associated with local injection-site symptoms compared with lower dosages. In the active-comparator analysis, the probability of any symptoms (odds ratio [OR], 1.62; 95% CI, 1.43-1.84), local symptoms (OR, 1.68; 95% CI, 1.38-2.05), musculoskeletal symptoms (OR, 2.55; 95% CI, 1.32-4.94), dermatologic symptoms (OR, 2.18; 95% CI, 10.7-4.45), or otolaryngologic symptoms (OR, 6.37; 95% CI, 1.50-27.09) were modestly elevated after BNT162b2 compared with non-SARS-CoV-2 vaccines, whereas the probabilities of general symptoms (OR, 0.77; 95% CI, 0.63-0.95) and fever (OR, 0.42; 95% CI, 0.32-0.55) were lower after BNT162b2. Symptoms requiring hospitalization (n = 10) were reported only at BNT162b2 dosages above 3 µg. Conclusions and Relevance: In this cohort study, the symptoms reported after BNT162b2 administration were comparable overall to those for on-label non-SARS-CoV-2 vaccines in this cohort of children younger than 5 years. The present data may be used together with prospective licensure studies of BNT162b2 efficacy and safety and could help guide expert recommendations about BNT162b2 vaccinations in this age group.

Transcriptional profiling identifies functional interactions of TGF ß and PPAR ß/δ signaling: synergistic induction of ANGPTL4 transcription.

Peroxisome proliferator-activated receptors (PPARs) not only play a key role in regulating metabolic pathways but also modulate inflammatory processes, pointing to a functional interaction between PPAR and cytokine signaling pathways. In this study, we show by genome-wide transcriptional profiling that PPARß/δ and transforming growth factor-ß (TGFß) pathways functionally interact in human myofibroblasts and that a subset of these genes is cooperatively activated by TGFß and PPARß/δ. Using the angiopoietin-like 4 (ANGPTL4) gene as a model, we demonstrate that two enhancer regions cooperate to mediate the observed synergistic response. A TGFß-responsive enhancer located ∼8 kb upstream of the transcriptional start site is regulated by a mechanism involving SMAD3, ETS1, RUNX, and AP-1 transcription factors that interact with multiple contiguous binding sites. A second enhancer (PPAR-E) consisting of three juxtaposed PPAR response elements is located in the third intron ∼3.5 kb downstream of the transcriptional start site. The PPAR-E is strongly activated by all three PPAR subtypes, with a novel type of PPAR response element motif playing a central role. Although the PPAR-E is not regulated by TGFß, it interacts with SMAD3, ETS1, RUNX2, and AP-1 in vivo, providing a possible mechanistic explanation for the observed synergism.

Experiences made by family physicians managing patients with SARS-CoV-2 infection during spring 2020 - a cross-sectional analysis.

BACKGROUND: In Germany, family physicians care for about 85% of the patients infected with SARS-CoV-2. The geographic distribution of the first wave in 2020 was heterogeneous, and each federal state experienced different percentages of patients that died from COVID-19. Each of the 16 federal states implemented its own regulation about medical care for SARS-CoV-2 infected patients. Against this background, the objective of this analysis was to gather experiences made by primary care physicians managing SARS-CoV-2 infected patients during the first wave in March 2020 and to clinically characterize these patients. METHODS: In total, 5,632 physicians were invited to participate in an online questionnaire surveying routine data regarding the general care situation at the physician practice level and the care for patients infected with SARS-CoV-2. Bivariate and multivariate analyses were applied to characterize treatment experiences and to identify patient characteristics predicting the course of disease. RESULTS: 132 family physicians from all German federal states (except from Berlin) participated in this analysis (response rate 2.3%) and provided routine care data for 1,085 patients. Information from 373 of these patients were provided in greater detail. On average, each physician treated 8.5 patients infected with SARS-CoV-2. About 15% of the physicians used video consultations to communicate with their infected patients. More than 82% made positive experiences with the exceptional regulation to provide a certificate of incapacity to work by telephone. Half of the physicians faced equipment insufficiencies due to a lack of protective gear, and in 10% of the practices, the staff themselves acquired SARS-CoV-2 infection. Greater numbers of SARS-CoV-2 cases treated in a practice translated into higher odds for members of the practice to get infected (odds ratio (OR) 1.03, 95% CI [1.01;1.06]). Older persons, males and patients in rural areas had higher odds of a severe course of disease. CONCLUSIONS: Our results show that a large percentage of primary care physicians additionally managed their COVID-19 patients remotely by telephone or video during the outbreak, while also being at a higher risk for SARS-CoV-2 infection. Further, the increased severity in rural areas underlines the importance of strong primary health care in order to enable hospitals to concentrate on critically ill patients.

15-hydroxyeicosatetraenoic acid is a preferential peroxisome proliferator-activated receptor beta/delta agonist.

Peroxisome proliferator-activated receptor (PPARs) modulate target gene expression in response to unsaturated fatty acid ligands, such as arachidonic acid (AA). Here, we report that the AA metabolite 15-hydroxyeicosatetraenoic acid (15-HETE) activates the ligand-dependent activation domain (AF2) of PPARbeta/delta in vivo, competes with synthetic agonists in a PPARbeta/delta ligand binding assay in vitro, and triggers the interaction of PPARbeta/delta with coactivator peptides. These agonistic effects were also seen with PPARalpha and PPARgamma, but to a significantly weaker extent. We further show that 15-HETE strongly induces the expression of the bona fide PPAR target gene Angptl4 in a PPARbeta/delta-dependent manner and, conversely, that inhibition of 15-HETE synthesis reduces PPARbeta/delta transcriptional activity. Consistent with its function as an agonistic ligand, 15-HETE triggers profound changes in chromatin-associated PPARbeta/delta complexes in vivo, including the recruitment of the coactivator cAMP response element-binding protein binding protein. Both 15R-HETE and 15S-HETE are similarly potent at inducing PPARbeta/delta coactivator binding and transcriptional activation, indicating that 15-HETE enantiomers generated by different pathways function as PPARbeta/delta agonists.

Ligand-mediated regulation of peroxisome proliferator-activated receptor (PPAR) beta/delta: a comparative analysis of PPAR-selective agonists and all-trans retinoic acid.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that modulate target gene expression in response to natural fatty acid ligands and synthetic agonists. It is noteworthy that all trans-retinoic acid (atRA) has recently been reported to act as a ligand for PPARbeta/delta, to activate its transcriptional activity, and, in contrast to the "classic" function of atRA, to stimulate cell proliferation (Schug et al., 2007). Here, we report that in contrast to synthetic PPARbeta/delta agonists, atRA failed to induce the transcriptional activity of PPARbeta/delta using different types of reporter gene assays. Likewise, atRA did not affect the expression of the bona fide PPARbeta/delta target genes ADRP and ANGPTL4 but strongly increased expression of the retinoic acid target gene CYP26A under the identical experimental conditions. Consistent with these observations, atRA did not compete with established PPARbeta/delta agonists in a ligand binding assay, and atRA did not enable the interaction of PPARbeta/delta with a coactivator peptide in a time-resolved fluorescence resonance energy transfer assay in vitro. These results are in sharp contrast to the effect of established PPARbeta/delta agonists in both in vitro assays. Taken as a whole, these data strongly suggest that atRA does not function as a ligand of PPARbeta/delta in any of the experimental systems tested and that the previously reported atRA effects are more likely to reflect an uncharacterized and less direct mechanism.

Expression level and agonist-binding affect the turnover, ubiquitination and complex formation of peroxisome proliferator activated receptor beta.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that modulate target gene expression in response to fatty acid ligands. Their regulation by post-translational modifications has been reported but is poorly understood. In the present study, we investigated whether ligand binding affects the turnover and ubiquitination of the PPARbeta subtype (also known as PPARdelta). Our data show that the ubiquitination and degradation of PPARbeta is not significantly influenced by the synthetic agonist GW501516 under conditions of moderate PPARbeta expression. By contrast, the overexpression of PPARbeta dramatically enhanced its degradation concomitant with its polyubiquitination and the formation of high molecular mass complexes containing multiple, presumably oligomerized PPARbeta molecules that lacked stoichiometical amounts of the obligatory PPARbeta dimerization partner, retinoid X receptor. The formation of these apparently aberrant complexes, as well as the ubiquitination and destabilization of PPARbeta, were strongly inhibited by GW501516. Our findings suggest that PPARbeta is subject to complex post-translational regulatory mechanisms that partly may serve to safeguard the cell against deregulated PPARbeta expression. Furthermore, our data have important implications regarding the widespread use of overexpression systems to evaluate the function and regulation of PPARs.

Induction of PPARbeta and prostacyclin (PGI2) synthesis by Raf signaling: failure of PGI2 to activate PPARbeta.

A role for the nuclear receptor peroxisome proliferator-activated receptor-beta (PPARbeta) in oncogenesis has been suggested by a number of observations but its precise role remains elusive. Prostaglandin I2 (PGI2, prostacyclin), a major arachidonic acid (AA) derived cyclooxygenase (Cox) product, has been proposed as a PPARbeta agonist. Here, we show that the 4-hydroxytamoxifen (4-OHT) mediated activation of a C-Raf-estrogen receptor fusion protein leads to the induction of both the PPARbeta and Cox-2 genes, concomitant with a dramatic increase in PGI2 synthesis. Surprisingly, however, 4-OHT failed to activate PPARbeta transcriptional activity, indicating that PGI2 is insufficient for PPARbeta activation. In agreement with this conclusion, the overexpression of ectopic Cox-2 and PGI2 synthase (PGIS) resulted in massive PGI2 synthesis but did not activate the transcriptional activity of PPARbeta. Conversely, inhibition of PGIS blocked PGI2 synthesis but did not affect the AA mediated activation of PPARbeta. Our data obtained with four different cell types and different experimental strategies do not support the prevailing opinion that PGI2 plays a significant role in the regulation of PPARbeta.

Assembly and isolation of intermediate steps of transcription complexes formed on the human 5S rRNA gene.

By employing purified transcription factors and RNA polymerase III (pol III), we generated active pol III transcription complexes on the human 5S rRNA gene. These large complexes were separated by size exclusion chromatography from non- incorporated proteins. In addition, we succeeded in isolating specific intermediate stages of complex formation. Such isolated partial complexes require complementation with the missing activities for full transcription activity. One central finding is that a 5S DNA-TFIIIA-TFIIIC2-TFIIIBbeta complex could be isolated which had been assembled in the absence of the general pol III transcription factor IIIC1. Thus TFIIIC1 is not an assembly factor for other transcription factors. Although pol III has the potential to bind unspecifically to DNA, such polymerase molecules cannot be rendered initiation competent by direct recruitment to a 5S DNA-TFIIIA-TFIIIC2- TFIIIBbeta complex, but this process strictly requires additional TFIIIC1 activity. This clearly demonstrates that in contrast to yeast cells, hTFIIIB(beta), although required, does not suffice for the functional recruitment of polymerase III. These data document that TFIIIC1 is the second transcription factor required for the recruitment of pol III in mammalian cells.

[Changing to a career in general practice - a qualitative study reveals motives of specialists]. / Liebe auf den zweiten Blick - qualitative Erhebung von Motiven für den Quereinstieg in die Allgemeinmedizin.

BACKGROUND: In 2011, the national German Medical Association (Bundesärztekammer) published guidelines for a slim-lined training program in general practice (Quereinstieg) for qualified medical specialists in other fields (e. g., surgeons, internists or anesthesiologists). This step is part of a strategy to prevent further shortages of general practitioners in Germany. In the state of Baden-Wuerttemberg, qualified medical specialists are allowed to complete their general practice training in approximately two years instead of five. The aim of this study was to understand the reasons of specialists for changing to a career in general practice. METHODS: The postgraduate training program VerbundweiterbildungplusBaden-Württemberg had 597 trainees at the time of the study in December 2015. Previously qualified specialists in another medical discipline were identified and invited to participate in this study. Qualitative data was gathered using semi-structured interviews with content analysis of the interviews performed by three independent members of the research team. RESULTS: In total, 36 out of 597 trainees were identified as previously qualified specialists in another medical discipline. All 36 were invited to take part and 15 agreed to participate in this study. Overall, 15 interviews were performed, with a mean time of 24.19minutes. Participants with a median age of 40 years (33-59 years) - mainly anesthesiologists (n=7), surgeons (n=3) and internists (n=3) - presented with an average of 6.5 years of professional experience in their specialty. First, the participants' motivation to switch career arose from the wish to intensify the quality of patient contacts with a holistic approach including family and social background and from the infinite variety of general practice. Another reason given for a career change was self-employment opportunities. Finally, feelings of frustration over poor working conditions in hospitals resulted in a job search elsewhere in medicine, taking account of the challenges of ageing and family life. A major finding was that without the slim-lined program, the majority of participants would not have changed their career. DISCUSSION: The slim-lined training program in general practice attracts experienced medical doctors. Specialists decide to change career because of the particular ways of working in general practice and with the intention to improve their daily work as a physician, either to improve individual working conditions and/or to improve their individual curative work profile. In addition, specialists are attracted by the concept of self-employment in general practice. Therefore, appreciation of the specific ways of working in general practice as well as management skills are most important during the reduced 2-year training. Further studies should investigate if facilitating a career switch to general practice is a good way to improve the shortage of general practitioners.

Intracellular route and transcriptional competence of polyethylenimine-DNA complexes.

Polyethylenimine (PEI) is a cationic polymer which can be complexed with DNA. PEI-DNA complexes can be used for in vitro and in vivo gene delivery approaches. The excess of positive surface charges enhances the association of the complex with the plasmamembrane of cells and facilitates their uptake by endocytosis. The intracellular transport pathway from the endosome to the nucleus is not understood. Here we show that PEI-DNA complexes are taken up by all cells which are treated with these complexes, indicating, that the uptake is not the rate limiting step in the final transfection efficiency. We reveal by fluorescent microscopy, cell fractionation studies and electron microscopy, that PEI-DNA complexes accumulate in the lysosomal compartment, from where they are released through small local membrane damages. However, the cytoplasmic pool of PEI-DNA complexes is small and with the applied morphological approaches PEI aggregates could not be detected in the nucleus. This indicates, that only a small fraction of the complexes reach their final destiny. To test whether the association of DNA with PEI might be the critical step for transfection, we performed in vitro transcription assays with PEI-DNA complexes. These experiments revealed, that the transcription is not impaired when PEI is closely attached to the template DNA. Our results thus point to the transfer of PEI-DNA complexes from the lysosomal compartment to the nucleus as the rate limiting step in cell transfection.