Bifurcations of mixed-mode oscillations in three-timescale systems: An extended prototypical example.

We study a class of multi-parameter three-dimensional systems of ordinary differential equations that exhibit dynamics on three distinct timescales. We apply geometric singular perturbation theory to explore the dependence of the geometry of these systems on their parameters, with a focus on mixed-mode oscillations (MMOs) and their bifurcations. In particular, we uncover a novel geometric mechanism that encodes the transition from MMOs with single epochs of small-amplitude oscillations (SAOs) to those with double-epoch SAOs; the former feature SAOs or pseudo-plateau bursting either "below" or "above" in their time series, while in the latter, SAOs or pseudo-plateau bursting occur both "below" and "above." We identify a relatively simple prototypical three-timescale system that realizes our mechanism, featuring a one-dimensional S-shaped 2-critical manifold that is embedded into a two-dimensional S-shaped critical manifold in a symmetric fashion. We show that the Koper model from chemical kinetics is merely a particular realization of that prototypical system for a specific choice of parameters; in particular, we explain the robust occurrence of mixed-mode dynamics with double epochs of SAOs therein. Finally, we argue that our geometric mechanism can elucidate the mixed-mode dynamics of more complicated systems with a similar underlying geometry, such as a three-dimensional, three-timescale reduction of the Hodgkin-Huxley equations from mathematical neuroscience.

Hydrated Nucleus Pulposus Extrusion in Dogs: Thoracolumbar Compared to Cervical Cases.

OBJECTIVE: The aim of this study was to review and describe cases of thoracolumbar (TL) hydrated nucleus pulposus extrusion (HNPE) diagnosed with magnetic resonance imaging and surgery, and compare them to cases of cervical (C) HNPE. STUDY DESIGN: Retrospective, single-center study. RESULTS: Thirty-six dogs met the inclusion criteria. Fifteen cases were C and 21 TL. Thirteen dogs were chondrodystrophic breeds, mean body weight was 13 kg, median age was 7.5 years, and 30/36 were male. Fewer dogs were chondrodystrophic in the C group compared with the TL group (p = 0.022). More than 90% had an acute onset, and strong activity was more often reported in the TL group. TL HNPE was more often painful, and extruded disc material more often lateralized (p = 0.017). Median Modified Frankel Score at presentation was 3 and 72.2% were non-ambulatory. More TL HNPE (11/21) were treated surgically compared with C HNPE (4/15). Treatment choice was correlated with spinal cord compression (p = 0.0075). Median Modified Frankel Score improved during hospitalization (p = 0.002) and there was no difference in outcome between C and TL HNPE or conservative and surgical treatment. Mean follow-up time was 33 days. All patients were ambulatory at follow-up. CONCLUSION: This study suggests that the HNPE is not limited to the C vertebral column of dogs and can occur in the TL vertebral column as well. Dogs with TL HNPE show spinal hyperesthesia more often and extruded nucleus material is more often lateralized. Outcome is similar to what has previously been described for C HNPE.

Higher vs lower doses of dexamethasone in patients with COVID-19 and severe hypoxia (COVID STEROID 2) trial: Protocol and statistical analysis plan.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has resulted in millions of deaths and overburdened healthcare systems worldwide. Systemic low-dose corticosteroids have proven clinical benefit in patients with severe COVID-19. Higher doses of corticosteroids are used in other inflammatory lung diseases and may offer additional clinical benefits in COVID-19. At present, the balance between benefits and harms of higher vs. lower doses of corticosteroids for patients with COVID-19 is unclear. METHODS: The COVID STEROID 2 trial is an investigator-initiated, international, parallel-grouped, blinded, centrally randomised and stratified clinical trial assessing higher (12 mg) vs. lower (6 mg) doses of dexamethasone for adults with COVID-19 and severe hypoxia. We plan to enrol 1,000 patients in Denmark, Sweden, Switzerland and India. The primary outcome is days alive without life support (invasive mechanical ventilation, circulatory support or renal replacement therapy) at day 28. Secondary outcomes include serious adverse reactions at day 28; all-cause mortality at day 28, 90 and 180; days alive without life support at day 90; days alive and out of hospital at day 90; and health-related quality of life at day 180. The primary outcome will be analysed using the Kryger Jensen and Lange test adjusted for stratification variables and reported as adjusted mean differences and median differences. The full statistical analysis plan is outlined in this protocol. DISCUSSION: The COVID STEROID 2 trial will provide evidence on the optimal dosing of systemic corticosteroids for COVID-19 patients with severe hypoxia with important implications for patients, their relatives and society.

Status and perspectives of biomarker validation for diagnosis, stratification, and treatment.

OBJECTIVES: The aim of this study was to discuss the status of and perspective for biomarker validation in view of the challenges imposed on national healthcare systems due to an increasing number of citizens with chronic diseases and new expensive drugs with effects that are sometimes poorly documented. The demand for a paradigm shift toward stratification of patients or even 'personalized medicine' (PM) is rising, and the implementation of such novel strategies has the potential to increase patient outcomes and cost efficiency of treatments. The implementation of PM depends on relevant and reliable biomarkers correlated to disease states, prognosis, or effect of treatment. Beyond biomarkers of disease, personalized prevention strategies (such as individualized nutrition guidance) are likely to depend on novel biomarkers. STUDY DESIGN: We discuss the current status of the use of biomarkers and the need for standardization and integration of biomarkers based on multi-omics approaches. METHODS: We present representative cases from laboratory medicine, oncology, and nutrition, where present and emerging biomarkers have or may present opportunities for PM or prevention. RESULTS: Biomarkers vary greatly in complexity, from single genomic mutations to metagenomic analyses of the composition of the gut microbiota and comprehensive analyses of metabolites, metabolomics. Using biomarkers for decision-making has previously often relied on measurements of single biomolecules. The current development now moves toward the use of multiple biomarkers requiring the use of machine learning or artificial intelligence. Still, the usefulness of biomarkers is often challenged by suboptimal validation, and the discovery of new biomarkers moves much faster than standardization efforts. To reap the potential benefits of personalization of treatment and prevention, healthcare systems and regulatory authorities need to focus on validation and standardization of biomarkers. CONCLUSION: There is a great public health need for better understanding of the usefulness, but also limitations, of biomarkers among policy makers, clinicians, and scientists, and efforts securing effective validation are key to the future use of novel sets of complex biomarkers.

In situ nano- to microscopic imaging and growth mechanism of electrochemical dissolution (e.g., corrosion) of a confined metal surface.

Reactivity in confinement is central to a wide range of applications and systems, yet it is notoriously difficult to probe reactions in confined spaces in real time. Using a modified electrochemical surface forces apparatus (EC-SFA) on confined metallic surfaces, we observe in situ nano- to microscale dissolution and pit formation (qualitatively similar to previous observation on nonmetallic surfaces, e.g., silica) in well-defined geometries in environments relevant to corrosion processes. We follow "crevice corrosion" processes in real time in different pH-neutral NaCl solutions and applied surface potentials of nickel (vs. Ag|AgCl electrode in solution) for the mica-nickel confined interface of total area ∼0.03 mm2 The initial corrosion proceeds as self-catalyzed pitting, visualized by the sudden appearance of circular pits with uniform diameters of 6-7 µm and depth ∼2-3 nm. At concentrations above 10 mM NaCl, pitting is initiated at the outer rim of the confined zone, while below 10 mM NaCl, pitting is initiated inside the confined zone. We compare statistical analysis of growth kinetics and shape evolution of individual nanoscale deep pits with estimates from macroscopic experiments to study initial pit growth and propagation. Our data and experimental techniques reveal a mechanism that suggests initial corrosion results in formation of an aggressive interfacial electrolyte that rapidly accelerates pitting, similar to crack initiation and propagation within the confined area. These results support a general mechanism for nanoscale material degradation and dissolution (e.g., crevice corrosion) of polycrystalline nonnoble metals, alloys, and inorganic materials within confined interfaces.

On the regularization of impact without collision: the Painlevé paradox and compliance.

We consider the problem of a rigid body, subject to a unilateral constraint, in the presence of Coulomb friction. We regularize the problem by assuming compliance (with both stiffness and damping) at the point of contact, for a general class of normal reaction forces. Using a rigorous mathematical approach, we recover impact without collision (IWC) in both the inconsistent and the indeterminate Painlevé paradoxes, in the latter case giving an exact formula for conditions that separate IWC and lift-off. We solve the problem for arbitrary values of the compliance damping and give explicit asymptotic expressions in the limiting cases of small and large damping, all for a large class of rigid bodies.

Induction of lipogenesis in white fat during cold exposure in mice: link to lean phenotype.

This corrects the article DOI: 10.1038/ijo.2016.228.

Increased microvascular permeability in mice lacking Epac1 (Rapgef3).

AIM: Maintenance of the blood and extracellular volume requires tight control of endothelial macromolecule permeability, which is regulated by cAMP signalling. This study probes the role of the cAMP mediators rap guanine nucleotide exchange factor 3 and 4 (Epac1 and Epac2) for in vivo control of microvascular macromolecule permeability under basal conditions. METHODS: Epac1-/- and Epac2-/- C57BL/6J mice were produced and compared with wild-type mice for transvascular flux of radio-labelled albumin in skin, adipose tissue, intestine, heart and skeletal muscle. The transvascular leakage was also studied by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) using the MRI contrast agent Gadomer-17 as probe. RESULTS: Epac1-/- mice had constitutively increased transvascular macromolecule transport, indicating Epac1-dependent restriction of baseline permeability. In addition, Epac1-/- mice showed little or no enhancement of vascular permeability in response to atrial natriuretic peptide (ANP), whether probed with labelled albumin or Gadomer-17. Epac2-/- and wild-type mice had similar basal and ANP-stimulated clearances. Ultrastructure analysis revealed that Epac1-/- microvascular interendothelial junctions had constitutively less junctional complex. CONCLUSION: Epac1 exerts a tonic inhibition of in vivo basal microvascular permeability. The loss of this tonic action increases baseline permeability, presumably by reducing the interendothelial permeability resistance. Part of the action of ANP to increase permeability in wild-type microvessels may involve inhibition of the basal Epac1-dependent activity.

Induction of lipogenesis in white fat during cold exposure in mice: link to lean phenotype.

BACKGROUND/OBJECTIVE: Futile substrate cycling based on lipolytic release of fatty acids (FA) from intracellular triacylglycerols (TAG) and their re-esterification (TAG/FA cycling), as well as de novo FA synthesis (de novo lipogenesis (DNL)), represent the core energy-consuming biochemical activities of white adipose tissue (WAT). We aimed to characterize their roles in cold-induced thermogenesis and energy homeostasis. METHODS: Male obesity-resistant A/J and obesity-prone C57BL/6J mice maintained at 30 °C were exposed to 6 °C for 2 or 7 days. In epididymal WAT (eWAT), TAG synthesis and DNL were determined using in vivo 2H incorporation from 2H2O into tissue TAG and nuclear magnetic resonance spectroscopy. Quantitative real-time-PCR and/or immunohistochemistry and western blotting were used to determine the expression of selected genes and proteins in WAT and liver. RESULTS: The mass of WAT depots declined during cold exposure (CE). Plasma levels of TAG and non-esterified FA were decreased by day 2 but tended to normalize by day 7 of CE. TAG synthesis (reflecting TAG/FA cycle activity) gradually increased during CE. DNL decreased by day 2 of CE but increased several fold over the control values by day 7. Expression of genes involved in lipolysis, glyceroneogenesis, FA re-esterification, FA oxidation and mitochondrial biogenesis in eWAT was induced during CE. All these changes were more pronounced in obesity-resistant A/J than in B6 mice and occurred in the absence of uncoupling protein 1 in eWAT. Expression of markers of glyceroneogenesis in eWAT correlated negatively with hepatic FA synthesis by day 7 in both strains. Leptin and fibroblast growth factor 21 plasma levels were differentially affected by CE in the two mouse strains. CONCLUSIONS: Our results indicate integrated involvement of (i) TAG/FA cycling and DNL in WAT, and (ii) hepatic very-low-density lipoprotein-TAG synthesis in the control of blood lipid levels and provision of FA fuels for thermogenesis in cold. They suggest that lipogenesis in WAT contributes to a lean phenotype.

Long-term consumption of an obesogenic high fat diet prior to ischemia-reperfusion mediates cardioprotection via Epac1-dependent signaling.

BACKGROUND: Obesity is still considered a risk factor for cardiovascular disease, although more recent knowledge also suggests obesity to be associated with reduced morbidity and mortality - the "obesity paradox". This study explores if long-term feeding of an obesogenic high fat diet renders the myocardium less susceptible to ischemic-reperfusion induced injury via Epac-dependent signaling. METHODS: Wild type (wt), Epac1 (Epac1-/-) and Epac2 (Epac2-/-) deficient mice were fed a high fat (HFD) or normal chow diet (ND) for 33 ± 1 weeks. Six experimental groups were included: (1) control wt ND (wt ND), (2) control wt HFD (wt HFD), (3) Epac1-/- mice on ND (Epac1-/-ND), (4) Epac1-/- mice on HFD (Epac1-/-HFD), (5) Epac2-/- mice on ND (Epac2-/-ND), and (6) Epac2-/- mice on HFD (Epac2-/-HFD). Isolated ex vivo mice hearts were perfused in a constant pressure Langendorff mode, and exposed to 30min of global ischemia (GI) and 60min of reperfusion. Endpoints were infarct size and functional recovery. RESULTS: All groups fed a HFD presented with significantly enhanced body weight, visceral fat content and reduced glucose clearance compared to corresponding ND groups. Although the HFD cohorts presented with an overall comparable systemic capability to clear glucose, the Epac1-/- HFD group presented with glucose levels slightly above the human diabetes criteria at the end of the intraperitoneal glucose tolerance test (ipGTT). Moreover, the HFD significantly reduced infarct size in both wild type (wt HFD 41.3 ± 5.5% vs. wt ND 58.0 ± 9.8%, p < 0.05) and Epac2-/- cohorts (Epac2-/-HFD 34.4 ± 7.2% vs. Epac2-/-ND 56.5 ± 3.8%, p < 0.05). Interestingly, however, the HFD did not reduce infarct size in Epac1-/- deficient mice hearts (Epac1-/-HFD 65.1 ± 5.1% vs. Epac1-/-ND 56.1 ± 3.5%, ns.). CONCLUSION: Epac1-dependent signaling is involved in mediating the cardioprotection afforded by long-term feeding of an obesogenic high fat diet in mice hearts.

MDM2 facilitates adipocyte differentiation through CRTC-mediated activation of STAT3.

The ubiquitin ligase MDM2 is best known for balancing the activity of the tumor suppressor p53. We have previously shown that MDM2 is vital for adipocyte conversion through controlling Cebpd expression in a p53-independent manner. Here, we show that the proadipogenic effect of MDM2 relies on activation of the STAT family of transcription factors. Their activation was required for the cAMP-mediated induction of target genes. Interestingly, rather than influencing all cAMP-stimulated genes, inhibition of the kinases directly responsible for STAT activation, namely JAKs, or ablation of MDM2, each resulted in abolished induction of a subset of cAMP-stimulated genes, with Cebpd being among the most affected. Moreover, STATs were able to interact with the transcriptional cofactors CRTC2 and CRTC3, hitherto only reported to associate with the cAMP-responsive transcription factor CREB. Last but not least, the binding of CRTC2 to a transcriptional enhancer that interacts with the Cebpd promoter was dramatically decreased upon JAK inhibition. Our data reveal the existence of an unusual functional interplay between STATs and CREB at the onset of adipogenesis through shared CRTC cofactors.

In vitro screening of inhibition of PPAR-γ activity as a first step in identification of potential breast carcinogens.

Alcohol consumption and increased estrogen levels are major risk factors for breast cancer, and peroxisome proliferator-activated receptor γ (PPAR-γ) plays an important role in alcohol-induced breast cancer. PPAR-γ activity is inhibited by ethanol, leading to increased aromatase activity and estrogen biosynthesis ultimately leading to breast cancer. If other organic solvents inhibit PPAR-γ activity, they should also lead to increased oestrogen biosynthesis and thus be potential breast carcinogens. Ten commonly used hydrophilic organic solvents were first tested in a cell-based screening assay for inhibitory effects on PPAR-γ transactivation. The chemicals shown to inhibit PPAR-γ were tested with vectors encoding PPAR-γ with deleted AB domains and only the ligand-binding domain to rule out unspecific toxicity. Next, the effects on biosynthesis of estradiol, testosterone and oestrone sulphate were measured in the H295R steroidogenesis assay after incubation with the chemicals. Ethylene glycol, ethyl acetate, and dimethyl sulphoxide inhibited PPAR-γ transactivation in a dose-dependent manner. The inhibitory effect on PPAR-γ was specific for PPAR-γ since the AB domain of PPAR-γ was required for the inhibitory effect. In the second step, ethylene glycol significantly increased production of oestradiol by 19% (p < 0.05) and ethyl acetate inhibited production of testosterone (p < 0.05). We here show that screening of 10 commonly used organic solvents for the ability to inhibit PPAR-γ transactivation followed by a well-established steroidogenesis assay for production of sex hormones in exposed H295 R cells may provide a screening tool for potential breast carcinogens. This initial screening thus identified ethylene glycol and possibly ethyl acetate as potential breast carcinogens.

Molecular aspects of adipoepithelial transdifferentiation in mouse mammary gland.

The circular, reversible conversion of the mammary gland during pregnancy and involution is a paradigm of physiological tissue plasticity. The two most prominent cell types in mammary gland, adipocytes and epithelial cells, interact in an orchestrated way to coordinate this process. Previously, we showed that this conversion is at least partly achieved by reciprocal transdifferentiation between mammary adipocytes and lobulo-alveolar epithelial cells. Here, we aim to shed more light on the regulators of mammary transdifferentiation. Using immunohistochemistry with cell type-specific lipid droplet-coating markers (Perilipin1 and 2), we show that cells with an intermediate adipoepithelial phenotype exist during and after pregnancy. Nuclei of cells with similar transitional structural characteristics are highly positive for Elf5, a master regulator of alveologenesis. In cultured adipocytes, we could show that transient and stable ectopic expression of Elf5 induces expression of the milk component whey acidic protein, although the general adipocyte phenotype is not affected suggesting that additional pioneering factors are necessary. Furthermore, the lack of transdifferentiation of adipocytes during pregnancy after clearing of the epithelial compartment indicates that transdifferentiation signals must emanate from the epithelial part. To explore candidate genes potentially involved in the transdifferentiation process, we devised a high-throughput gene expression study to compare cleared mammary fat pads with developing, contralateral controls at several time points during pregnancy. Incorporation of bioinformatic predictions of secretory proteins provides new insights into possible paracrine signaling pathways and downstream transdifferentiation factors. We discuss a potential role for osteopontin (secreted phosphoprotein 1 [Spp1]) signaling through integrins to induce adipoepithelial transdifferentiation.

Weight cycling promotes fat gain and altered clock gene expression in adipose tissue in C57BL/6J mice.

Repeated attempts to lose weight by temporary dieting may result in weight cycling, eventually further gain of body fat, and possible metabolic adaptation. We tested this with a controlled experiment in C57BL/6J mice subjected to four weight cycles (WC), continuous hypercaloric feeding (HF), or low-fat feeding (LF). To search for genes involved in an adaptive mechanism to former weight cycling and avoid acute effects of the last cycle, the last hypercaloric feeding period was prolonged by an additional 2 wk before euthanization. Total energy intake was identical in WC and HF. However, compared with HF, the WC mice gained significantly more total body mass and fat mass and showed increased levels of circulating leptin and lipids in liver. Both the HF and WC groups showed increased adipocyte size and insulin resistance. Despite these effects, we also observed an interesting maintenance of circulating adiponectin and free fatty acid levels after WC, whereas changes in these parameters were observed in HF mice. Global gene expression was analyzed by microarrays. Weight-cycled mice were characterized by a downregulation of several clock genes (Dbp, Tef, Per1, Per2, Per3, and Nr1d2) in adipose tissues, which was confirmed by quantitative PCR. In 3T3-L1 cells, we found reduced expression of Dbp and Tef early in adipogenic differentiation, which was mediated via cAMP-dependent signaling. Our data suggest that clock genes in adipose tissue may play a role in metabolic adaptation to weight cycling.

A clinical study of the cognitive effects of benzodiazepines in psychogeriatric patients.

Previous studies have shown cognitive impairment in long-term benzodiazepine users compared to non-users. However, little is known about such effects in a population of geriatric psychiatry patients. The aim of this study was to identify differences between benzodiazepine users and non-users on standardized tests of the cognitive fields of learning and memory, executive functions and vigilance, at admittance to a department of geriatric psychiatry.Hopkins verbal learning test, Stroop test and digit vigilance test were performed in all patients. Test performances were compared between benzodiazepine users (n=168) and non-users (n=73). A multiple linear regression model was used, adjusting for different baseline characteristics (years of education, dementia and depression).No significant differences in test results were found between benzodiazepine users and non-users on 11 out of 12 cognitive tests results. On one of the 12 test results (time used on the digit vigilance test), benzodiazepine users showed better performance compared to non-users (ß=-0.20, p=0.032). This finding was not statistically significant after Bonferroni correction for multiple testing.This study of geriatric psychiatry benzodiazepine users did not reveal cognitive impairment compared to non-users on the cognitive areas tested. Other possible negative consequences of benzodiazepine use should, however, also be considered when prescribing drugs to older patients.

Exome sequencing-driven discovery of coding polymorphisms associated with common metabolic phenotypes.

AIMS/HYPOTHESIS: Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes. METHODS: The study comprised three stages. We performed medium-depth (8×) whole exome sequencing in 1,000 cases with type 2 diabetes, BMI >27.5 kg/m(2) and hypertension and in 1,000 controls (stage 1). We selected 16,192 polymorphisms nominally associated (p < 0.05) with case-control status, from four selected annotation categories or from loci reported to associate with metabolic traits. These variants were genotyped in 15,989 Danes to search for association with 12 metabolic phenotypes (stage 2). In stage 3, polymorphisms showing potential associations were genotyped in a further 63,896 Europeans. RESULTS: Exome sequencing identified 70,182 polymorphisms with MAF >1%. In stage 2 we identified 51 potential associations with one or more of eight metabolic phenotypes covered by 45 unique polymorphisms. In meta-analyses of stage 2 and stage 3 results, we demonstrated robust associations for coding polymorphisms in CD300LG (fasting HDL-cholesterol: MAF 3.5%, p = 8.5 × 10(-14)), COBLL1 (type 2 diabetes: MAF 12.5%, OR 0.88, p = 1.2 × 10(-11)) and MACF1 (type 2 diabetes: MAF 23.4%, OR 1.10, p = 8.2 × 10(-10)). CONCLUSIONS/INTERPRETATION: We applied exome sequencing as a basis for finding genetic determinants of metabolic traits and show the existence of low-frequency and common coding polymorphisms with impact on common metabolic traits. Based on our study, coding polymorphisms with MAF above 1% do not seem to have particularly high effect sizes on the measured metabolic traits.

Mdm2 controls CREB-dependent transactivation and initiation of adipocyte differentiation.

The role of the E3 ubiquitin ligase murine double minute 2 (Mdm2) in regulating the stability of the p53 tumor suppressor is well documented. By contrast, relatively little is known about p53-independent activities of Mdm2 and the role of Mdm2 in cellular differentiation. Here we report a novel role for Mdm2 in the initiation of adipocyte differentiation that is independent of its ability to regulate p53. We show that Mdm2 is required for cAMP-mediated induction of CCAAT/enhancer-binding protein δ (C/EBPδ) expression by facilitating recruitment of the cAMP regulatory element-binding protein (CREB) coactivator, CREB-regulated transcription coactivator (Crtc2)/TORC2, to the c/ebpδ promoter. Our findings reveal an unexpected role for Mdm2 in the regulation of CREB-dependent transactivation during the initiation of adipogenesis. As Mdm2 is able to promote adipogenesis in the myoblast cell line C2C12, it is conceivable that Mdm2 acts as a switch in cell fate determination.

First Report of Golovinomyces cichoracearum as the Causal Agent of Powdery Mildew on Symphyotrichum novi-belgii (Synonym Aster novi-belgii) in Denmark.

Symphyotrichum novi-belgii (L.) G.L. Nesom (synonym Aster novi-belgii L.) is an autumn flowering perennial used in gardens and as a cut flower. During the last 20 years, it has been developed as a potted plant, thereby increasing its economic importance. In Denmark, 7 to 8 million S. novi-belgii plants are produced annually, making it one of the 10 most popular potted plant crops ( http://floradania.dk/index.php?id=165 ). In general, S. novi-belgii is a healthy plant, but it can be severely attacked by powdery mildew both in greenhouse production and outdoors, and diseased plants have been observed in most parts of the country. Infected plants show typical symptoms: leaf surfaces become covered with white mycelium and as the disease progresses infected leaves turn yellow and die. Powdery mildew is regarded the main disease problem in S. novi-belgii and it causes problems year round in greenhouse production. Normally, the disease is controlled by fungicides, but once out of the production system, symptom development in the retail trade will reduce the plant's appeal to customers to a degree that prevents sales. The powdery mildew identified in this study was collected in a small research field at Aarslev, Denmark in September 2004. Since collection, the pathogen has been maintained in a greenhouse on S. novi-belgii and it has been used for disease resistance screening. However, lack of proper identification of the causal agent has hindered the development of powdery mildew resistant cultivars. To identify the pathogen, the internal transcribed spacer region (ITS) of the rDNA was amplified using primers ITS1 and ITS4 (2) and sequenced. The resulting sequence was deposited in GenBank (Accession No. HM769725). BLASTn analysis of the 598-bp fragment showed 99% identity to Golovinomyces cichoracearum (DC.) V.P. Heluta from Rudbeckia laciniata L. (Accession No. AB077622). The powdery mildew colonies were slightly pink with barrel-shaped, hyaline conidia borne in chains of three to four. The length of the conidia was 30 ± 4 µm and the width was 13 ± 1 µm (n = 105). Foot cells of the conidiophores were 101 ± 16 µm long and 12 ± 5 µm wide (n = 50) with a slight constriction at the base. Chasmothecia were not observed. These morphological characteristics confirmed the identification as G. cichoracearum (1). To fulfill Koch's postulates, 10 healthy S. novi-belgii 'Victoria Fanny' plants were inoculated in an inoculation tower by shaking infected S. novi-belgii plants over the tower, resulting in a spore density of 47 spores/mm2 on the leaf surface. The infected plants were placed in a growth chamber with 16 h of light (200 µmol·m-2·s-1) and day and night temperatures of 20 and 15°C, respectively. Symptoms developed on all plants after 11 days. Colony morphology on the leaves and the morphological characteristics were as described above. Conidia were washed off the leaves, DNA extracted, and the ITS was amplified by PCR. The resulting PCR product was sequenced and was identical to HM769725. To our knowledge, this is the first report of G. cichoracearum on S. novi-belgii in Denmark. References: (1) U. Braun. The Powdery Mildews (Erysiphales) of Europe. Gustav Fischer Verlag, Jena Germany, 1995. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, New York, 1990.

Activation of the nuclear receptor PPARγ by metabolites isolated from sage (Salvia officinalis L.).

ETHNOPHARMACOLOGICAL RELEVANCE: Salvia officinalis has been used as a traditional remedy against diabetes in many countries and its glucose-lowering effects have been demonstrated in animal studies. The active compounds and their possible mode of action are still unknown although it has been suggested that diterpenes may be responsible for the anti-diabetic effect of Salvia officinalis. AIM OF THE STUDY: To investigate whether the reported anti-diabetic effects of Salvia officinalis are related to activation of the nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ and to identify the bioactive constituents. MATERIALS AND METHODS: From a dichloromethane extract of Salvia officinalis able to activate PPARγ several major metabolites were isolated by chromatographic techniques. To assess bioactivity of the isolated metabolites a PPARγ transactivation assay was used. RESULTS: Eight diterpenes were isolated and identified including a new abietane diterpene being the epirosmanol ester of 12-O-methyl carnosic acid and 20-hydroxyferruginol, which was isolated from Salvia officinalis for the first time, as well as viridiflorol, oleanolic acid, and α-linolenic acid. 12-O-methyl carnosic acid and α-linolenic acid were able to significantly activate PPARγ whereas the remaining metabolites were either unable to activate PPARγ or yielded insignificant activation. CONCLUSIONS: Selected metabolites from Salvia officinalis were able to activate PPARγ and hence, the anti-diabetic activity of this plant could in part be mediated through this nuclear receptor.

The MAPK-dependent regulation of the Jagged/Notch gene expression by VEGF, bFGF or PPAR gamma mediated angiogenesis in HUVEC.

The Jagged-Notch signalling, plays a crucial role in cell differentiation. Angiogenesis, is regulated by VEGF, bFGF as well as by the free fatty acid metabolites , which are regulators of transcription factors such as peroxisome proliferation activating receptors (PPARs). The study analyzed the signalling pathways involved in the regulation of Jagged-1/Notch-4 expression in endothelial cells (HUVECs) in response to VEGF, bFGF and PPAR-gamma exogenous activator - ciglitazone. HUVECs were incubated with investigated substances for 24 hours, with or without the presence of the MAP-kinases inhibitors were used. Jagged-1 and Notch-4 gene expression was determined using quantitative Real-Time PCR. The Jagged-1/Notch-4 protein expression was compared by flow cytometry, when the phosphorylation-dependent activation of kinases was estimated by Western-blot method. The opposite effect of VEGF, bFGF, or ciglitazone on the Jagged-1/Notch-4 expression on HUVEC was connected with the different activation of MAPKs. Ciglitazone, activated p38 MAPK pathway and simultaneously inhibited phosphorylation of p42/44 MAPK. The pro-angiogenic: bFGF and VEGF, also activated the p38 MAPK, but they did not attenuate the p42/44 MAPK phosphorylation. Maintaining of the Jagged/Notch interactions by VEGF, when down-regulation by bFGF and ciglitazone, seems to be dependent on the different effect on p38 MAPK and p42/44 MAPK pathway regulation.