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Thermographic flow visualization is already an established imaging method to localize the laminar-turbulent flow transition on the rotor blades of operating wind turbines, while a steady flow state is assumed. To understand the potential of thermographic flow visualization for the investigation of unsteady flow phenomena, its capability to detect the change of the flow transition position due to a wind gust is studied. Previously laminar flow regions become turbulent with the gust, which means a sudden increase of heat transfer between surface and fluid and, thus, a decrease of surface temperature. The latter is detected by evaluating the difference of thermographic images before and during the wind gust. The achievable sensitivity and the temporal resolution are limited by the thermodynamic properties of the rotor blade and the fluid flow, as well as by the natural rotor blade heating with the sun’s radiation. As a result of theory and experiments on real wind turbines, the feasibility to detect flow state changes in the order of seconds is proven. This opens upthe analysis of unsteady flow phenomena on wind turbines by means of thermographic flow visualization.

Continuous processing is the future production method for monoclonal antibodies (mAbs). A fully continuous, fully automated downstream process based on disposable equipment was developed and implemented inside the MoBiDiK pilot plant. However, a study evaluating the comparability between batch and continuous processing based on product quality attributes was not conducted before. The work presented fills this gap comparing both process modes experimentally by purifying the same harvest material (side-by-side comparability). Samples were drawn at different time points and positions in the process for batch and continuous mode. Product quality attributes, product-related impurities, as well as process-related impurities were determined. The resulting polished material was processed to drug substance and further evaluated regarding storage stability and degradation behavior. The in-process control data from the continuous process showed the high degree of accuracy in providing relevant process parameters such as pH, conductivity, and protein concentration during the entire process duration. Minor differences between batch and continuous samples are expected as different processing conditions are unavoidable due to the different nature of batch and continuous processing. All tests revealed no significant differences in the intermediates and comparability in the drug substance between the samples of both process modes. The stability study of the final product also showed no differences in the stability profile during storage and forced degradation. Finally, online data analysis is presented as a powerful tool for online-monitoring of chromatography columns during continuous processing.

Model-inspired signal processing approaches with an enhanced detectability of flow separation on thermographic images are presented. Flow separation causes performance loss, structural loads and increasing acoustic emissions on wind turbine rotor blades. However, due to the low thermal contrast between turbulent and separated flow regions, the non-invasive thermographic visualisation of flow separation is currently only possible for wind tunnel measurements, which are characterised by a high thermal contrast and a small measuring distance. The state-of-the-art signal processing approaches evaluate the surface temperature fluctuation of thermographic image series. However, understanding of the signal measurement chain with a distinct consideration of the influences on the dynamic surface temperature is incomplete. Therefore, designing model-inspired signal processing approaches which provide a high interpretability and a maximum contrast is an open task. The proposed signal processing approaches evaluate the surface response selectively, by using the amplitude information of the surface temperature response to an oscillating input signal or gradient-based for a transient input signal. The approaches are applied to wind tunnel measurements on a rotor blade profile at a near thermodynamic steady state and a transient thermodynamic behaviour at Reynolds numbers that are representative for operational wind turbines. The gradient-based evaluation shows an improved contrast for the detection of flow separation, but is only applicable to profiles with transient thermodynamic behaviour. The amplitude evaluation provides a high degree of interpretability of the processed images based on flow-dependent features and enables for an unambiguous identification of flow separation by a global amplitude minimum close to the separation point. Additionally, an increased spatial resolution for surface modifications is shown, while the contrast between flow regions is significantly decreased. Hence, the proposed approaches allow for an improved identifiability of flow separation with regard to future applications on wind turbines in operation.

Thermographic flow visualization is a contactless, non-invasive technique to visualize the boundary layer flow on wind turbine rotor blades, to assess the aerodynamic condition and consequently the efficiency of the entire wind turbine. In applications on wind turbines in operation, the distinguishability between the laminar and turbulent flow regime cannot be easily increased artificially and solely depends on the energy input from the sun. State-of-the-art image processing methods are able to increase the contrast slightly but are not able to reduce systematic gradients in the image or need excessive a priori knowledge. In order to cope with a low-contrast measurement condition and to increase the distinguishability between the flow regimes, an enhanced image processing by means of the feature extraction method, principal component analysis, is introduced. The image processing is applied to an image series of thermographic flow visualizations of a steady flow situation in a wind tunnel experiment on a cylinder and DU96W180 airfoil measurement object without artificially increasing the thermal contrast between the flow regimes. The resulting feature images, based on the temporal temperature fluctuations in the images, are evaluated with regard to the global distinguishability between the laminar and turbulent flow regime as well as the achievable measurement error of an automatic localization of the local flow transition between the flow regimes. By applying the principal component analysis, systematic temperature gradients within the flow regimes as well as image artefacts such as reflections are reduced, leading to an increased contrast-to-noise ratio by a factor of 7.5. Additionally, the gradient between the laminar and turbulent flow regime is increased, leading to a minimal measurement error of the laminar-turbulent transition localization. The systematic error was reduced by 4% and the random error by 5.3% of the chord length. As a result, the principal component analysis is proven to be a valuable complementary tool to the classical image processing method in flow visualizations. After noise-reducing methods such as the temporal averaging and subsequent assessment of the spatial expansion of the boundary layer flow surface, the PCA is able to increase the laminar-turbulent flow regime distinguishability and reduce the systematic and random error of the flow transition localization in applications where no artificial increase in the contrast is possible. The enhancement of contrast increases the independence from the amount of solar energy input required for a flow evaluation, and the reduced errors of the flow transition localization enables a more precise assessment of the aerodynamic condition of the rotor blade.

For the first time, a thermographic detection and localization of turbulent flow separation on an operating wind turbine is presented and verified. Flow separation on wind turbine rotor blades causes power reduction, structural loads and increased noise emissions. In contrast to established methods for stall detection, the presented infrared thermographic measurement approach is non-invasive, in-process capable and provides a high spatial resolution. With respect to the state of the art for thermographic stall detection in wind tunnel experiments, the thermal surface response to unsteady inflow conditions is evaluated for measurements on an operating wind turbine, in order to achieve unambiguous thermographic features for the detection of flow separation. The evaluation of the thermodynamic response behavior shows a clear detection of flow separation by means of temperature fluctuation maxima in the regions of flow transition as well as an increasing temperature fluctuation within the separated flow region. In addition, a geometric assignment is conducted which enables a localization of the separation point with an uncertainty of 0.6% of the chord length. The detection and localization of flow separation is verified by means of tufts visualization.

Small-molecule inhibitors of hypoxia-inducible factor prolyl hydroxylases (HIF-PHs) are currently under clinical development as novel treatment options for chronic kidney disease (CKD) associated anemia. Inhibition of HIF-PH mimics hypoxia and leads to increased erythropoietin (EPO) expression and subsequently increased erythropoiesis. Herein we describe the discovery, synthesis, structure-activity relationship (SAR), and proposed binding mode of novel 2,4-diheteroaryl-1,2-dihydro-3H-pyrazol-3-ones as orally bioavailable HIF-PH inhibitors for the treatment of anemia. High-throughput screening of our corporate compound library identified BAY-908 as a promising hit. The lead optimization program then resulted in the identification of molidustat (BAY 85-3934), a novel small-molecule oral HIF-PH inhibitor. Molidustat is currently being investigated in clinical phase III trials as molidustat sodium for the treatment of anemia in patients with CKD.

The activation of the transcription factor hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor development, tumor progression, and resistance to chemo- and radiotherapy. In order to identify compounds targeting the HIF pathway, a small molecule library was screened using a luciferase-driven HIF-1 reporter cell line under hypoxia. The high-throughput screening led to the identification of a class of aminoalkyl-substituted compounds that inhibited hypoxia-induced HIF-1 target gene expression in human lung cancer cell lines at low nanomolar concentrations. Lead structure BAY 87-2243 was found to inhibit HIF-1α and HIF-2α protein accumulation under hypoxic conditions in non-small cell lung cancer (NSCLC) cell line H460 but had no effect on HIF-1α protein levels induced by the hypoxia mimetics desferrioxamine or cobalt chloride. BAY 87-2243 had no effect on HIF target gene expression levels in RCC4 cells lacking Von Hippel-Lindau (VHL) activity nor did the compound affect the activity of HIF prolyl hydroxylase-2. Antitumor activity of BAY 87-2243, suppression of HIF-1α protein levels, and reduction of HIF-1 target gene expression in vivo were demonstrated in a H460 xenograft model. BAY 87-2243 did not inhibit cell proliferation under standard conditions. However under glucose depletion, a condition favoring mitochondrial ATP generation as energy source, BAY 87-2243 inhibited cell proliferation in the nanomolar range. Further experiments revealed that BAY 87-2243 inhibits mitochondrial complex I activity but has no effect on complex III activity. Interference with mitochondrial function to reduce hypoxia-induced HIF-1 activity in tumors might be an interesting therapeutic approach to overcome chemo- and radiotherapy-resistance of hypoxic tumors.

Cellular oxygen partial pressure is sensed by a family of prolyl-4-hydroxylase domain (PHD) enzymes that modify hypoxia-inducible factor (HIF)α subunits. Upon hydroxylation under normoxic conditions, HIFα is bound by the von Hippel-Lindau tumor suppressor protein and targeted for proteasomal destruction. Since PHD activity is dependent on oxygen and ferrous iron, HIF-1 mediates not only oxygen- but also iron-regulated transcriptional gene expression. Here we show that copper (CuCl2) stabilizes nuclear HIF-1α under normoxic conditions, resulting in hypoxia-response element (HRE)-dependent reporter gene expression. In in vitro hydroxylation assays CuCl2 inhibited prolyl-4-hydroxylation independently of the iron concentration. Ceruloplasmin, the main copper transport protein in the plasma and a known HIF-1 target in vitro, was also induced in vivo in the liver of hypoxic mice. Both hypoxia and CuCl2 increased ceruloplasmin (as well as vascular endothelial growth factor [VEGF] and glucose transporter 1 [Glut-1]) mRNA levels in hepatoma cells, which was due to transcriptional induction of the ceruloplasmin gene (CP) promoter. In conclusion, our data suggest that PHD/HIF/HRE-dependent gene regulation can serve as a sensory system not only for oxygen and iron but also for copper metabolism, regulating the oxygen-, iron- and copper-binding transport proteins hemoglobin, transferrin, and ceruloplasmin, respectively. (Blood. 2005;105:4613-4619)

Oxygen sensing by hypoxia-inducible factor prolyl hydroxylases (HIF-PHs) is the dominant regulatory mechanism of erythropoietin (EPO) expression. In chronic kidney disease (CKD), impaired EPO expression causes anemia, which can be treated by supplementation with recombinant human EPO (rhEPO). However, treatment can result in rhEPO levels greatly exceeding the normal physiological range for endogenous EPO, and there is evidence that this contributes to hypertension in patients with CKD. Mimicking hypoxia by inhibiting HIF-PHs, thereby stabilizing HIF, is a novel treatment concept for restoring endogenous EPO production. HIF stabilization by oral administration of the HIF-PH inhibitor BAY 85-3934 (molidustat) resulted in dose-dependent production of EPO in healthy Wistar rats and cynomolgus monkeys. In repeat oral dosing of BAY 85-3934, hemoglobin levels were increased compared with animals that received vehicle, while endogenous EPO remained within the normal physiological range. BAY 85-3934 therapy was also effective in the treatment of renal anemia in rats with impaired kidney function and, unlike treatment with rhEPO, resulted in normalization of hypertensive blood pressure in a rat model of CKD. Notably, unlike treatment with the antihypertensive enalapril, the blood pressure normalization was achieved without a compensatory activation of the renin–angiotensin system. Thus, BAY 85-3934 may provide an approach to the treatment of anemia in patients with CKD, without the increased risk of adverse cardiovascular effects seen for patients treated with rhEPO. Clinical studies are ongoing to investigate the effects of BAY 85-3934 therapy in patients with renal anemia.

DokA, a homolog of bacterial hybrid histidine kinases, is essential for hyperosmotic stress resistance in Dictyostelium: We show that a transient intracellular cAMP signal, dependent on the presence of DokA, is generated in response to an osmotic shock. This variation of cAMP levels contributes to survival under hypertonic conditions. In contrast to the low cAMP levels observed in dokA(-) strains, overexpression of the receiver domain of DokA causes an increase in cAMP levels, resulting in a rapidly developing phenotype. We present biochemical and cell biological data indicating that the DokA receiver domain is a dominant-negative regulator of a phosphorelay, which controls the intracellular cAMP phosphodiesterase RegA. The activity of the DokA receiver domain depends on a conserved aspartate, mutation of which reverses the developmental phenotype, as well as the deregulation of cAMP metabolism.

We have used PCR to identify a Dictyostelium homolog of the bacterial two-component system. The gene dokA codes for a member of the hybrid histidine kinase family which is defined by the presence of conserved amino acid sequence motifs corresponding to an N-terminal receptor domain, a central kinase and a C-terminal response regulator moiety. Potential function of the regulator domain was demonstrated by phosphorylation in vitro. dokA mutants are deficient in the osmoregulatory pathway, resulting in premature cell death under high osmotic stress. Under less stringent osmotic conditions, cells grow at a normal rate, but development at the multicellular stage is altered. dokA is a member of a family of histidine kinase-like genes that play regulatory roles in eukaryotic cell function.

Selective inhibitors of Jumonji domain-containing protein (JMJD) histone demethylases are candidate anticancer agents as well as potential tools for elucidating the biological functions of JMJDs. On the basis of the crystal structure of JMJD2A and a homology model of JMJD2C, we designed and prepared a series of hydroxamate analogues bearing a tertiary amine. Enzyme assays using JMJD2C, JMJD2A, and prolyl hydroxylases revealed that hydroxamate analogue 8 is a potent and selective JMJD2 inhibitor, showing 500-fold greater JMJD2C-inhibitory activity and more than 9100-fold greater JMJD2C-selectivity compared with the lead compound N-oxalylglycine 2. Compounds 17 and 18, prodrugs of compound 8, each showed synergistic growth inhibition of cancer cells in combination with an inhibitor of lysine-specific demethylase 1 (LSD1). These findings suggest that combination treatment with JMJD2 inhibitors and LSD1 inhibitors may represent a novel strategy for anticancer chemotherapy.

The abundance of the transcription factor hypoxia-inducible factor is regulated through hydroxylation of its alpha-subunits by a family of prolyl-hydroxylases (PHD1-3). Enzymatic activity of these PHDs is O2-dependent, which enables PHDs to act as cellular O2 sensor enzymes. Herein we studied endogenous PHD activity that was induced in cells grown under hypoxia or in the presence of nitric oxide. Under such conditions nuclear extracts contained much higher PHD activity than the respective cytoplasmic extracts. Although PHD1-3 were abundant in both compartments, knockdown experiments for each isoenzyme revealed that nuclear PHD activity was only due to PHD2. Maximal PHD2 activity was found between 120 and 210 microm O2. PHD2 activity was strongly decreased below 100 microm O2 with a half-maximum activity at 53 +/- 13 microm O2 for the cytosolic and 54 +/- 10 microm O2 for nuclear PHD2 matching the physiological O2 concentration within most cells. Our data suggest a role for PHD2 as a decisive oxygen sensor of the hypoxia-inducible factor degradation pathway within the cell nucleus.

N-nitrosomelatonin (NOMela) is well-known for its capabilities of transnitrosating nucleophiles such as thiols and ascorbate, thereby generating nitric oxide (NO)-releasing compounds. It is unknown, however, whether NOMela can be successfully applied as a precursor of NO in a complex biological environment like a cell culture system. NO donors may be useful to induce the transcription factor hypoxia inducible factor 1 (HIF-1), which coordinates the protection of cells and tissues from the lack of oxygen (hypoxia). In this study, the effects of NOMela in an in vitro cell-free assay [NO-release, inhibition of prolylhydroxylase1 (PHD1)] and in living cells (upregulation of HIF-1, reduction of HIF-1 hydroxylation, upregulation of the HIF-1-target gene PHD2) were compared with those of the frequently applied NO donor S-nitrosoglutathione (GSNO) under normoxic and hypoxic conditions. In contrast to GSNO, NOMela released NO in a predictable manner and this release in vitro was found to be independent of the composition of the buffer system. The NOMela-mediated effects in oxygenated cells were in all cases comparable to the hypoxic response, whereas unphysiological strong effects were observed with GSNO. Probably, because of the antioxidative power of the NOMela-dependent formation of melatonin, cells were completely protected against the attack of reactive nitrogen oxygen species, which are generated by autoxidation of NO. In conclusion, NOMela had to be an excellent NO precursor for cells in culture and potentially tissues.

The prolyl-4-hydroxylase domain (PHD) oxygen sensor proteins hydroxylate hypoxia-inducible transcription factor (HIF)-alpha (alpha) subunits, leading to their subsequent ubiquitinylation and degradation. Since oxygen is a necessary cosubstrate, a reduction in oxygen availability (hypoxia) decreases PHD activity and, subsequently, HIF-alpha hydroxylation. Non-hydroxylated HIF-alpha cannot be bound by the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL), and HIF-alpha proteins thus become stabilized. HIF-alpha then heterodimerizes with HIF-beta (beta) to form the functionally active HIF transcription factor complex, which targets approximately 200 genes involved in adaptation to hypoxia. The three HIF-alpha PHDs are of a different nature compared with the prototype collagen prolyl-4-hydroxylase, which hydroxylates a mass protein rather than a rare transcription factor. Thus, novel assays had to be developed to express and purify functionally active PHDs and to measure PHD activity in vitro. A need also exists for such assays to functionally distinguish the three different PHDs in terms of substrate specificity and drug function. We provide a detailed description of the expression and purification of the PHDs as well as of an HIF-alpha-dependent and a HIF-alpha-independent PHD assay.

The prolyl-4-hydroxylase domain (PHD) oxygen sensor proteins hydroxylate hypoxia-inducible transcription factor (HIF)-alpha (alpha) subunits, leading to their subsequent ubiquitinylation and degradation. Since oxygen is a necessary cosubstrate, a reduction in oxygen availability (hypoxia) decreases PHD activity and, subsequently, HIF-alpha hydroxylation. Non-hydroxylated HIF-alpha cannot be bound by the ubiquitin ligase von Hippel-Lindau tumor suppressor protein (pVHL), and HIF-alpha proteins thus become stabilized. HIF-alpha then heterodimerizes with HIF-beta (beta) to form the functionally active HIF transcription factor complex, which targets approximately 200 genes involved in adaptation to hypoxia. The three HIF-alpha PHDs are of a different nature compared with the prototype collagen prolyl-4-hydroxylase, which hydroxylates a mass protein rather than a rare transcription factor. Thus, novel assays had to be developed to express and purify functionally active PHDs and to measure PHD activity in vitro. A need also exists for such assays to functionally distinguish the three different PHDs in terms of substrate specificity and drug function. We provide a detailed description of the expression and purification of the PHDs as well as of an HIF-alpha-dependent and a HIF-alpha-independent PHD assay.

The transcriptional activation of hypoxia-inducible genes is essential for the adaptation of mammalian tissues to oxygen deficiency. The hypoxia-inducible transcription factor (HIF) is a cellular switch for the up-regulation of these genes during hypoxia. Under normoxia, HIFs are hydroxylated on conserved prolyl residues by a recently discovered family of HIF prolyl hydroxylases (HIF-PHD1-3). Hydroxylated HIF specifically interacts with the von Hippel–Lindau protein–elongin B–elongin C complex (VBC) which leads to ubiquitination and subsequent proteasomal degradation of HIF. We developed a nonradioactive microtiter plate assay based on the interaction of hydroxylated HIF with VBC which enabled us to detect hydroxylated HIF in the nanomolar concentration range. A biotinylated HIF peptide substrate was bound to a streptavidin-coated microtiter plate and hydroxylated with the HIF-PHD3 isoenzyme. Recombinant VBC complex with a thioredoxin (Trx) tag was purified from Escherichia coli and bound to the hydroxylated HIF peptide. The interaction between VBC and hydroxylated HIF was detected by using an anti-thioredoxin antibody.

Hypoxia-inducible transcription factors (HIFs) are important for transcriptional adaptation to hypoxia. Availability of HIFs is regulated via posttranslational modification of their alpha subunits (HIF-1alpha and HIF-2alpha). Under normoxia, two highly conserved proline residues within the oxygen-dependent degradation domain (ODDD) are hydroxylated by oxoglutarate-dependent proline 4-hydroxylases EGLN1-3. Hydroxylated HIF-alpha interacts with the pVHL-E3 ubiquitin ligase complex and, subsequently, is degraded via the proteasomal pathway. We identified a novel putative proline 4-hydroxylase, PH-4, with an aminoterminal EF-hand motif and a carboxyterminal catalytic domain, which was highly expressed in most organs, and-unlike EGLNs which localize to the cytoplasm and nucleus-was associated with the endoplasmic reticulum. Like EGLNs, PH-4 overexpressed in cellular reporter assays suppressed the HIF transactivation activity, dependent on the consensus ODDD proline residues. Suppression of transactivation was correlated with decrease of cellular contents of HIF. Thus, PH-4 might be related to cellular oxygen sensing.

Two-component systems consisting of histidine kinases and their corresponding receivers are widespread in bacterial signal transduction. In the past few years, genes coding for homologues of two-component systems were also discovered in eukaryotic organisms. DokA, a homologue of bacterial histidine kinases, is an element of the osmoregulatory pathway in the amoeba Dictyostelium. The work described here addresses the question whether DokA is phosphorylated in vivo in response to osmotic stress. We have endogenously overexpressed individual domains of DokA to investigate post-translational modification of the protein in response to osmotic shock in vivo. Dictyostelium cells were labeled with [32P]-orthophosphate, exposed to osmotic stress and DokA fragments were subsequently isolated by immunoprecipitation. Thus, a stress-dependent phosphorylation could be demonstrated, with the site of phosphorylation being located in the kinase domain. We demonstrate biochemically that the phosphorylated amino acid is serine, and by mutational analysis that the phosphorylation reaction is not due to an autophosphorylation of DokA. Furthermore, mutation of the conserved histidine did not affect the osmostress-dependent phosphorylation reaction. A stimulus-dependent serine phosphorylation of a eukaryotic histidine kinase homologue was demonstrated for the first time in vivo. That implies that DokA, although showing typical structural features of a bacterial two-component system, might be part of a eukaryotic signal transduction pathway that involves serine/threonine kinases.