Exploiting Cell-Based Assays to Accelerate Drug Development for G Protein-Coupled Receptors.

G protein-coupled receptors (GPCRs) are relevant targets for health and disease as they regulate various aspects of metabolism, proliferation, differentiation, and immune pathways. They are implicated in several disease areas, including cancer, diabetes, cardiovascular diseases, and mental disorders. It is worth noting that about a third of all marketed drugs target GPCRs, making them prime pharmacological targets for drug discovery. Numerous functional assays have been developed to assess GPCR activity and GPCR signaling in living cells. Here, we review the current literature of genetically encoded cell-based assays to measure GPCR activation and downstream signaling at different hierarchical levels of signaling, from the receptor to transcription, via transducers, effectors, and second messengers. Singleplex assay formats provide one data point per experimental condition. Typical examples are bioluminescence resonance energy transfer (BRET) assays and protease cleavage assays (e.g., Tango or split TEV). By contrast, multiplex assay formats allow for the parallel measurement of multiple receptors and pathways and typically use molecular barcodes as transcriptional reporters in barcoded assays. This enables the efficient identification of desired on-target and on-pathway effects as well as detrimental off-target and off-pathway effects. Multiplex assays are anticipated to accelerate drug discovery for GPCRs as they provide a comprehensive and broad identification of compound effects.

STAR® total ankle replacements with BONIT® coating: A prospective 2-17 year follow-up study of 474 ankles from a single center.

BACKGROUND: Different coatings of the metal implants of STAR prostheses have been used since 1999. In Europe metal implants with a double calcium-phosphate coating (BONIT) on a titanium sprayed surface have been available since 1999. METHODS: We present a 2-17 year follow-up of a consecutive series from a single center with 474 STAR ankle replacements where the BONIT type of coating has been used. RESULTS: 55 prostheses (12%) have been revised, the majority of them due to fracture of the mobile bearing. 22 prostheses (5%) have been converted to an arthrodesis. Analysis of survival of the specific components showed an estimated 10-year survival rate of the tibia component, talus component and polyethylene mobile bearing of 99%, 98% and 84%, respectively. The corresponding estimated 15-year survival was 98%, 98% and 74%, respectively. CONCLUSION: This study showed an extraordinary high survival rate of the metal implants. LEVEL OF EVIDENCE: Level III, prospective cohort series.

Effectiveness of a bandage to prevent re-dislocation after total hip arthroplasty in patients with a previous hip dislocation. A randomized controlled trial with 12-week follow-up.

OBJECTIVE: To investigate if using a hip bandage is more effective than standard care in the prevention of total hip arthroplasty re-dislocation in patients with a previous total hip arthroplasty dislocation. DESIGN: randomized controlled trial. SETTING: Holstebro Regional Hospital and Viborg Regional Hospital. SUBJECTS: A total of 99 patients, 51 women, mean 70.7 (SD 9.9) years were enrolled in an un-blinded, clinical randomized controlled trial. INTERVENTIONS: Participants with at least one previous total hip arthroplasty dislocation were randomized to either wearing a bandage reducing flexion, adduction, and internal rotation of the hip (intervention group) or to standard care (control group). The participants were followed for 12 weeks. Main follow-up measures were as follows: number of re-dislocations (primary outcome), hip disability measured with the Oxford Hip Score (0-48, 48 best), quality of life measured with the 36-Item Short Form Survey (0-100, 100 best), satisfaction with treatment and serious adverse events. Statistical analyses followed the intention-to-treat principle. RESULTS: No significant group differences were observed for the primary outcome re-dislocations (9 versus 15, P = 0.143) or for disability (11.3 versus 14.4, P = 0.161), quality of life (57.7 versus 48.3, P = 0.050) or satisfaction with treatment (P = 0.562). There were 3 serious adverse events leading to total hip arthroplasty revision in the intervention group and 4 in the control group. CONCLUSION: We found that a hip bandage is not superior to standard care in the prevention of total hip arthroplasty re-dislocation in those with a previous total hip arthroplasty dislocation.

Bringing discrete-time Langevin splitting methods into agreement with thermodynamics.

In light of the recently published complete set of statistically correct Grønbech-Jensen (GJ) methods for discrete-time thermodynamics, we revise a differential operator splitting method for the Langevin equation in order to comply with the basic GJ thermodynamic sampling features, namely, the Boltzmann distribution and Einstein diffusion, in linear systems. This revision, which is based on the introduction of time scaling along with flexibility of a discrete-time velocity attenuation parameter, provides a direct link between the ABO splitting formalism and the GJ methods. This link brings about the conclusion that any GJ method has at least weak second order accuracy in the applied time step. It further helps identify a novel half-step velocity, which simultaneously produces both correct kinetic statistics and correct transport measures for any of the statistically sound GJ methods. Explicit algorithmic expressions are given for the integration of the new half-step velocity into the GJ set of methods. Numerical simulations, including quantum-based molecular dynamics (QMD) using the QMD suite Los Alamos Transferable Tight-Binding for Energetics, highlight the discussed properties of the algorithms as well as exhibit the direct application of robust, time-step-independent stochastic integrators to QMD.

Dissipation-Dependent Thermal Escape from a Potential Well.

Langevin simulations are conducted to investigate the Josephson escape statistics over a large set of parameter values for damping and temperature. The results are compared to both Kramers and Büttiker-Harris-Landauer (BHL) models, and good agreement is found with the Kramers model for high to moderate damping, while the BHL model provides further good agreement down to lower damping values. However, for extremely low damping, even the BHL model fails to reproduce the progression of the escape statistics. In order to explain this discrepancy, we develop a new model which shows that the bias sweep effectively cools the system below the thermodynamic value as the potential well broadens due to the increasing bias. A simple expression for the temperature is derived, and the model is validated against direct Langevin simulations for extremely low damping values.

The challenge of stochastic Størmer-Verlet thermostats generating correct statistics.

In light of the recently developed complete GJ set of single random variable stochastic, discrete-time Størmer-Verlet algorithms for statistically accurate simulations of Langevin equations [N. Grønbech-Jensen, Mol. Phys. 118, e1662506 (2020)], we investigate two outstanding questions: (1) Are there any algorithmic or statistical benefits from including multiple random variables per time step and (2) are there objective reasons for using one or more methods from the available set of statistically correct algorithms? To address the first question, we assume a general form for the discrete-time equations with two random variables and then follow the systematic, brute-force GJ methodology by enforcing correct thermodynamics in linear systems. It is concluded that correct configurational Boltzmann sampling of a particle in a harmonic potential implies correct configurational free-particle diffusion and that these requirements only can be accomplished if the two random variables per time step are identical. We consequently submit that the GJ set represents all possible stochastic Størmer-Verlet methods that can reproduce time step-independent statistics of linear systems. The second question is thus addressed within the GJ set. Based on numerical simulations of complex molecular systems, as well as on analytic considerations, we analyze apparent friction-induced differences in the stability of the methods. We attribute these differences to an inherent, friction-dependent discrete-time scaling, which depends on the specific method. We suggest that the method with the simplest interpretation of temporal scaling, the GJ-I/GJF-2GJ method, be preferred for statistical applications.

Odontological identification dental charts based upon postmortem computed tomography compared to dental charts based upon postmortem clinical examinations.

Since the beginning of this century, the use of postmortem computed tomography (PMCT) in forensic autopsies has increased. In this study we examined how similar dental charts created using PMCT as a solitary examination mode were to dental charts created using the conventional method of a clinical inspection including intraoral radiographs. A total of 100 previously performed dental identification cases were retrospectively included in the study. For each case, a dental chart was created solely based upon PMCT. The PMCT based dental chart was subsequently compared with the chart created from the previous conventional identification examination. Based upon the accuracy, sensitivity and specificity values PMCT performed very well compared to the conventional method in the identification concerning presence or absence of teeth, the presence of crowns, bridges and endodontic treatments as well as the presence and types of fillings. PMCT performed poorly concerning the extension of fillings and identification of small, tooth-colored fillings. The use of PMCT is a valuable supplement to the conventional methods available for forensic odontologists and may be of great value for initial screening in mass fatalities.

Biosynthesis of bioactive diterpenoids in the medicinal plant Vitex agnus-castus.

Vitex agnus-castus L. (Lamiaceae) is a medicinal plant historically used throughout the Mediterranean region to treat menstrual cycle disorders, and is still used today as a clinically effective treatment for premenstrual syndrome. The pharmaceutical activity of the plant extract is linked to its ability to lower prolactin levels. This feature has been attributed to the presence of dopaminergic diterpenoids that can bind to dopamine receptors in the pituitary gland. Phytochemical analyses of V. agnus-castus show that it contains an enormous array of structurally related diterpenoids and, as such, holds potential as a rich source of new dopaminergic drugs. The present work investigated the localisation and biosynthesis of diterpenoids in V. agnus-castus. With the assistance of matrix-assisted laser desorption ionisation-mass spectrometry imaging (MALDI-MSI), diterpenoids were localised to trichomes on the surface of fruit and leaves. Analysis of a trichome-specific transcriptome database, coupled with expression studies, identified seven candidate genes involved in diterpenoid biosynthesis: three class II diterpene synthases (diTPSs); three class I diTPSs; and a cytochrome P450 (CYP). Combinatorial assays of the diTPSs resulted in the formation of a range of different diterpenes that can account for several of the backbones of bioactive diterpenoids observed in V. agnus-castus. The identified CYP, VacCYP76BK1, was found to catalyse 16-hydroxylation of the diol-diterpene, peregrinol, to labd-13Z-ene-9,15,16-triol when expressed in Saccharomyces cerevisiae. Notably, this product is a potential intermediate in the biosynthetic pathway towards bioactive furan- and lactone-containing diterpenoids that are present in this species.

Post-mortem computed tomography as part of dental identification - a proposed guideline.

PURPOSE: This paper presents a proposed guideline for the use of post-mortem computed tomography (PMCT) during forensic dental identification. Currently, whole-body PMCT is widely used prior to autopsies for the diagnosis of fractures, organ changes, hemorrhages, and for the localization of foreign bodies, but it may also facilitate the odontological identification process in single cases and in cases involving multiple fatalities. Several studies have described the use of PMCT in forensic odontological work, but we have not found any comprehensive set of guidelines on how to perform a forensic odontological examination using PMCT. The aim was to develop guidelines for creating post-mortem dental charts during forensic odontological identification examinations using the standard functions of PMCT. METHODS: A proposed guideline was developed from 15 selected cases examined at the Section of Forensic Pathology, Department of Forensic Medicine at the University of Copenhagen in Denmark from October 2011 to May 2012. Using the functionalities and three-dimensional (3D) reconstructions of OsiriX DICOM-viewer software (Pixmeo Sarl, Bernex, Geneva, Switzerland) we adjusted the contrast and brightness settings and developed a proposed guideline for creating PMCT-based dental charts. A four-step guideline was produced. CONCLUSION: In our casework, we are currently using the guidelines proposed herein. The use of PMCT has allowed us to target our clinical examinations, greatly improving their efficiency. Furthermore, PMCT allows the storage of data for later documentation and research. Further research is needed to validate the proposed guideline.

Engineering of CYP76AH15 can improve activity and specificity towards forskolin biosynthesis in yeast.

BACKGROUND: Forskolin is a high-value diterpenoid produced exclusively by the Lamiaceae plant Coleus forskohlii. Today forskolin is used pharmaceutically for its adenyl-cyclase activating properties. The limited availability of pure  forskolin is currently hindering its full utilization, thus a new environmentally friendly, scalable and sustainable strategy is needed for forskolin production. Recently, the entire biosynthetic pathway leading to forskolin was elucidated. The key steps of the pathway are catalyzed by cytochrome P450 enzymes (CYPs), which have been shown to be the limiting steps of the pathway. Here we study whether protein engineering of the substrate recognition sites (SRSs) of CYPs can improve their efficiency towards forskolin biosynthesis in yeast. RESULTS: As a proof of concept, we engineered the enzyme responsible for the first putative oxygenation step of the forskolin pathway: the conversion of 13R-manoyl oxide to 11-oxo-13R-manoyl oxide, catalyzed by the CYP76AH15. Four CYP76AH15 variants-engineered in the SRS regions-yielded at least a twofold increase of 11-oxo-13R-manoyl oxide when expressed in yeast cells grown in microtiter plates. The highest titers (5.6-fold increase) were observed with the variant A99I, mutated in the SRS1 region. Double or triple CYP76AH15 mutant variants resulted in additional enzymes with optimized performances. Moreover, in planta CYP76AH15 can synthesize ferruginol from miltiradiene. In this work, we showed that the mutants affecting 11-oxo-13R-manoyl oxide synthesis, do not affect ferruginol production, and vice versa. The best performing variant, A99I, was utilized to reconstruct the forskolin biosynthetic pathway in yeast cells. Although these strains showed increased 11-oxo-manoyl oxide production and higher accumulation of other pathway intermediates compared to the native CYP76AH15, lower production of forskolin was observed. CONCLUSIONS: As demonstrated for CYP76AH15, site-directed mutagenesis of SRS regions of plant CYPs may be an efficient and targeted approach to increase the performance of these enzymes. Although in this work we have managed to achieve higher efficiency and specificity of the first CYP of the pathway, further work is necessary in order to increase the overall production of forskolin in yeast cells.

A randomized phase II trial of interleukin-2 and interferon-α plus bevacizumab versus interleukin-2 and interferon-α in metastatic renal-cell carcinoma (mRCC): results from the Danish Renal Cancer Group (DaRenCa) study-1.

BACKGROUND: Interleukin-2 (IL2)-based immunotherapy is curative for a small subset of patients with metastatic renal-cell carcinoma (mRCC). Preclinical data suggests that bevacizumab (BEV), a humanized anti-VEGF monoclonal antibody, has potential immunomodulatory effects by permitting efficient natural killer (NK) cell-mediated killing and by reverting immune suppression. PATIENT AND METHODS: We performed a randomized phase II study comparing IL2/IFN (interferon)/BEV with IL2/IFN in favourable/intermediate-risk mRCC patients. One hundred and eighteen patients received IFN 3 MIU subcutaneously (sc) daily and IL2 2.4 MIU/m2 sc twice daily, 5 days per week for two consecutive weeks every 28-day-cycle, for 9 months; or supplemented with BEV 10 mg/kg, every 2 weeks intravenously (iv) until progression, unacceptable toxicity, or 1 year following no evidence of disease (NED). Primary end point was progression-free survival (PFS). RESULTS: Baseline characteristics were well-balanced between the two arms; metastasis-free interval <1 year (75 versus 76%); prior nephrectomy (85 versus 86%); MSKCC favourable/intermediate-risk group (51/49 versus 52%/48%); three or more disease sites (41 versus 44%), respectively. The median PFS was 8.0 mo (95% CI, 4.2-11.9) with IL2/IFN/BEV and 8.1 mo (95% CI, 5.1-11.0) with IL2/IFN, p = .73. There was no difference in secondary endpoints, IL2/IFN/BEV versus IL2/IFN; median time-to-treatment failure (7.4 versus 5.6 mo, p = .54), response rate (44.1 versus 28.8%, p = .13), surgery of residual disease (17.0 versus 17.0%, p = 1.0), patients achieving NED (3.4 versus 8.5%, p = .44), and median overall survival (30.3 versus 34.1 mo, p = .39), respectively. TKI post progression was well-balanced (85 versus 78%). No new/unexpected toxicity was observed. Most common Grade 3/4 adverse events for IL2/IFN/BEV and IL2/IFN were fatigue (64 versus 61%), flu-like symptoms (37 versus 41%) and thrombosis (6.8 versus 18.6%, p = .01), respectively. CONCLUSIONS: The addition of BEV to IL-2/IFN did not add efficacy in mRCC. (ClinicalTrials.gov, NCT01274273.).

Cementless One-Stage Revision in Chronic Periprosthetic Hip Joint Infection. Ninety-One Percent Infection Free Survival in 56 Patients at Minimum 2-Year Follow-Up.

BACKGROUND: Cementless 1-stage revision in chronic periprosthetic hip joint infections is limited evaluated. The purpose of this study was to evaluate a specific treatment protocol in this patient group. METHODS: The study was performed as a multicenter, proof-of-concept, observational study with prospective data collection. Patients were treated with a cementless 1-stage revision according to the CORIHA protocol between 2009 and 2014. Fifty-six patients, McPherson type III-A/B-1/2, were enrolled with a mean follow-up time from the CORIHA procedure of 4 years (minimum of 2 years). The primary outcome was re-revision performed due to infection and was evaluated by competing risk analysis, with death and aseptic revision as competing events. All-cause mortality was evaluated by Kaplan-Meier survival analysis. Oxford Hip Score (OHS) was used as disease-specific patient-reported outcome measure. RESULTS: The cumulative incidence of re-revision due to infection was 8.9% (confidence interval [CI] 3.2%-18.1%). The 1-year and 5-year survival incidence was 96% (CI 86%-99%) and 89% (CI 75%-95%). OHS at baseline was 19.9 (CI 17.3-22.6) and at 24-month follow-up 35.1 (CI 31.7-38.5). The mean change in OHS from baseline to 24-month follow-up was 11.8 points (CI 7.3; 16.3). Three patients had aseptic revision performed: two suffered periprosthetic fractures and one had stem subsidence. Failure analysis of the 5 reinfections did not detect a clear pattern as to the cause of failure. CONCLUSION: We found that cementless 1-stage revision in chronic periprosthetic hip joint infections has low reinfection rates in selected patients and may be applicable as a first-line treatment.

Modularity of Conifer Diterpene Resin Acid Biosynthesis: P450 Enzymes of Different CYP720B Clades Use Alternative Substrates and Converge on the Same Products.

Cytochrome P450 enzymes of the CYP720B subfamily play a central role in the biosynthesis of diterpene resin acids (DRAs), which are a major component of the conifer oleoresin defense system. CYP720Bs exist in families of up to a dozen different members in conifer genomes and fall into four different clades (I-IV). Only two CYP720B members, loblolly pine (Pinus taeda) PtCYP720B1 and Sitka spruce (Picea sitchensis) PsCYP720B4, have been characterized previously. Both are multisubstrate and multifunctional clade III enzymes, which catalyze consecutive three-step oxidations in the conversion of diterpene olefins to DRAs. These reactions resemble the sequential diterpene oxidations affording ent-kaurenoic acid from ent-kaurene in gibberellin biosynthesis. Here, we functionally characterized the CYP720B clade I enzymes CYP720B2 and CYP720B12 in three different conifer species, Sitka spruce, lodgepole pine (Pinus contorta), and jack pine (Pinus banksiana), and compared their activities with those of the clade III enzymes CYP720B1 and CYP720B4 of the same species. Unlike the clade III enzymes, clade I enzymes were ultimately found not to be active with diterpene olefins but converted the recently discovered, unstable diterpene synthase product 13-hydroxy-8(14)-abietene. Through alternative routes, CYP720B enzymes of both clades produce some of the same profiles of conifer oleoresin DRAs (abietic acid, neoabietic acid, levopimaric acid, and palustric acid), while clade III enzymes also function in the formation of pimaric acid, isopimaric acid, and sandaracopimaric acid. These results highlight the modularity of the specialized (i.e. secondary) diterpene metabolism, which produces conifer defense metabolites through variable combinations of different diterpene synthase and CYP720B enzymes.

Improving heterologous production of phenylpropanoids in Saccharomyces cerevisiae by tackling an unwanted side reaction of Tsc13, an endogenous double-bond reductase.

Phenylpropanoids, such as flavonoids and stilbenoids, are of great commercial interest, and their production in Saccharomyces cerevisiae is a very promising strategy. However, to achieve commercially viable production, each step of the process must be optimised. We looked at carbon loss, known to occur in the heterologous flavonoid pathway in yeast, and identified an endogenous enzyme, the enoyl reductase Tsc13, which turned out to be responsible for the accumulation of phloretic acid via reduction of p-coumaroyl-CoA. Tsc13 is an essential enzyme involved in fatty acid synthesis and cannot be deleted. Hence, two approaches were adopted in an attempt to reduce the side activity without disrupting the natural function: site saturation mutagenesis identified a number of amino acid changes which slightly increased flavonoid production but without reducing the formation of the side product. Conversely, the complementation of TSC13 by a plant gene homologue essentially eliminated the unwanted side reaction, while retaining the productivity of phenylpropanoids in a simulated fed batch fermentation.

Selection of test-setup parameters to target specific signal-to-noise regions in speech-on-speech intelligibility testing.

OBJECTIVE: To investigate the magnitude of the change in speech-reception threshold (SRT) provided by altering four different test-setup parameters. Furthermore, to determine whether these changes in SRT are of a sufficient magnitude, such that they can be used to design a test-setup in future experiments that target a predefined signal-to-noise ratio (SNR) region. This could be particularly important if the test contrast investigated is confounded with test SNR. DESIGN: The investigated test-setup parameters were: Spatial separation between target (0°) and maskers (±15°, ±30°, ±45°, or ±75°), number of maskers (two, four or six), scoring method (scoring percent-correct words or sentences) and masker gender (same or opposite to target). Twenty SRTs were measured per test subject. STUDY SAMPLE: Twenty hearing-impaired test subjects participated over two visits. RESULTS: Alteration of masker gender, spatial separation between target and masker (±15°, ±30°, ± 45°), and scoring method was shown to offer SRT changes of a sufficient magnitude. The different test setups resulted in average SRTs ranging from -4.0 to 3.3 dB. CONCLUSION: Deliberately selecting test setup parameters can change the overall difficulty of the test by up to 7.3 dB SRT. Thus, a future experiment can, to this extent, be designed to target a specific SNR region.

A phase II study of Epirubicin in oxaliplatin-resistant patients with metastatic colorectal cancer and TOP2A gene amplification.

UNLABELLED: The overall purpose of this study is to provide proof of concept for introducing the anthracycline epirubicin as an effective, biomarker-guided treatment for metastatic colorectal cancer (mCRC) patients who are refractory to treatment with oxaliplatin-based chemotherapy and have TOP2A gene amplification in their tumor cells. BACKGROUND: Epirubicin is an anthracycline that targets DNA topoisomerase 2-α enzyme encoded by the TOP2A gene. It is used for treatment of several malignancies, but currently not in CRC. TOP2A gene amplifications predict improved efficacy of epirubicin in patients with breast cancer and thus could be an alternative option for patients with CRC and amplified TOP2A gene. We have previously analysed the frequency of TOP2A gene aberrations in CRC and found that 46.6% of these tumors had TOP2A copy gain and 2.0% had loss of TOP2A when compared to adjacent normal tissue. The TOP2A gene is located on chromosome 17 and when the TOP2A/CEN-17 ratio was applied to identify tumors with gene loss or amplifications, 10.5% had a ratio ≥ 1.5 consistent with gene amplification and 2.6% had a ratio ≤ 0.8 suggesting gene deletions. Based on these observations and the knowledge gained from treatment of breast cancer patients, we have initiated a prospective clinical, phase II protocol using epirubicin (90 mg/m2 iv q 3 weeks) in mCRC patients, who are refractory to treatment with oxaliplatin. METHODS/DESIGN: The study is an open label, single arm, phase II study, investigating the efficacy of epirubicin in patients with oxaliplatin refractory mCRC and with a cancer cell TOP2A/CEN-17 ratio ≥ 1.5. TOP2A gene amplification measured by fluorescence in situ hybridization. A total of 25 evaluable patients (15 + 10 in two steps) will be included (Simon's two-stage minimax design). Every nine weeks, response is measured by computed tomography imaging and evaluated according to RECIST 1.1. The primary end-point of the study is progression-free survival. TRIAL REGISTRATION: Eudract no. 2013-001648-79.

Engineering and systems-level analysis of Saccharomyces cerevisiae for production of 3-hydroxypropionic acid via malonyl-CoA reductase-dependent pathway.

BACKGROUND: In the future, oil- and gas-derived polymers may be replaced with bio-based polymers, produced from renewable feedstocks using engineered cell factories. Acrylic acid and acrylic esters with an estimated world annual production of approximately 6 million tons by 2017 can be derived from 3-hydroxypropionic acid (3HP), which can be produced by microbial fermentation. For an economically viable process 3HP must be produced at high titer, rate and yield and preferably at low pH to minimize downstream processing costs. RESULTS: Here we describe the metabolic engineering of baker's yeast Saccharomyces cerevisiae for biosynthesis of 3HP via a malonyl-CoA reductase (MCR)-dependent pathway. Integration of multiple copies of MCR from Chloroflexus aurantiacus and of phosphorylation-deficient acetyl-CoA carboxylase ACC1 genes into the genome of yeast increased 3HP titer fivefold in comparison with single integration. Furthermore we optimized the supply of acetyl-CoA by overexpressing native pyruvate decarboxylase PDC1, aldehyde dehydrogenase ALD6, and acetyl-CoA synthase from Salmonella enterica SEacs (L641P). Finally we engineered the cofactor specificity of the glyceraldehyde-3-phosphate dehydrogenase to increase the intracellular production of NADPH at the expense of NADH and thus improve 3HP production and reduce formation of glycerol as by-product. The final strain produced 9.8 ± 0.4 g L(-1) 3HP with a yield of 13% C-mol C-mol(-1) glucose after 100 h in carbon-limited fed-batch cultivation at pH 5. The 3HP-producing strain was characterized by (13)C metabolic flux analysis and by transcriptome analysis, which revealed some unexpected consequences of the undertaken metabolic engineering strategy, and based on this data, future metabolic engineering directions are proposed. CONCLUSIONS: In this study, S. cerevisiae was engineered for high-level production of 3HP by increasing the copy numbers of biosynthetic genes and improving flux towards precursors and redox cofactors. This strain represents a good platform for further optimization of 3HP production and hence an important step towards potential commercial bio-based production of 3HP.

Patterns of migrating soaring migrants indicate attraction to marine wind farms.

Monitoring of bird migration at marine wind farms has a short history, and unsurprisingly most studies have focused on the potential for collisions. Risk for population impacts may exist to soaring migrants such as raptors with K-strategic life-history characteristics. Soaring migrants display strong dependence on thermals and updrafts and an affinity to land areas and islands during their migration, a behaviour that creates corridors where raptors move across narrow straits and sounds and are attracted to islands. Several migration corridors for soaring birds overlap with the development regions for marine wind farms in NW Europe. However, no empirical data have yet been available on avoidance or attraction rates and behavioural reactions of soaring migrants to marine wind farms. Based on a post-construction monitoring study, we show that all raptor species displayed a significant attraction behaviour towards a wind farm. The modified migratory behaviour was also significantly different from the behaviour at nearby reference sites. The attraction was inversely related to distance to the wind farm and was primarily recorded during periods of adverse wind conditions. The attraction behaviour suggests that migrating raptor species are far more at risk of colliding with wind turbines at sea than hitherto assessed.

A first-in-human, double-blind, randomised, placebo-controlled, dose ascending study of intra-articular rhFGF18 (sprifermin) in patients with advanced knee osteoarthritis.

OBJECTIVES: To evaluate the safety of intra-articular sprifermin (primary), and to evaluate systemic exposure, biomarkers, histology, and other cartilage parameters in patients with advanced osteoarthritis (OA). METHODS: This was a first-in-human, double-blind, randomised, placebo-controlled trial of single and multiple ascending doses of sprifermin from 3-300 µg in knee OA patients scheduled for total knee replacement. Patients were randomised 3:1 to sprifermin or placebo, injected into the target knee once or once weekly for 3 weeks, and followed-up for 24 weeks. RESULTS: Fifty-five patients were treated with sprifermin, 25 with single and 30 with multiple doses, 18 received placebo. There was no clear difference between the active and placebo groups in incidence, severity, and nature of reported treatment emergent adverse events. Acute inflammatory reactions were slightly more common with sprifermin 300 µg, but none led to discontinuation. No clear difference was seen between placebo and sprifermin in physician-assessed local tolerability, pain, or swelling in the knee. No meaningful changes over time, or differences between treatment groups, were observed for safety laboratory parameters or ECG. Although individual abnormalities were observed, no patterns were evident suggesting a relation to treatment or potential safety concern. No systemic sprifermin exposure, anti-FGF18 antibodies, or clear-cut effects on systemic biomarkers were detected. CONCLUSIONS: This first clinical trial of sprifermin revealed no serious safety concerns, although larger studies are needed. The possibility of positive effects of intra-articular sprifermin on histological and other cartilage parameters in knee OA also warrant further investigation.

Trends in cancer of the urinary bladder and urinary tract in elderly in Denmark, 2008-2012.

BACKGROUND: The aim of this study was to examine the trends in incidence, mortality, survival, and prevalence of cancers of the urinary bladder and urinary tract in Denmark from 1980 to 2012 with particular focus on elderly patients over age 70 years. DESIGN: Cancer of the urinary bladder and urinary tract was defined as ICD-10 codes C67.9, D09.0, D41.4. Data were derived from the NORDCAN database with comparable data on cancer incidence, mortality, prevalence and relative survival in the Nordic countries, where the Danish data were delivered from the Danish Cancer Registry and the Danish Cause of Death Registry. RESULTS: The average annual number of bladder cancers increased from 1478 to 1810 (22%) from 1980 to 2012, with close to 60% occurring in the elderly population. The incidence rates were 7-10 times higher in persons aged 70 years or more compared with younger persons. Mortality rates were decreasing with time in all age groups but 90+-year-old men. The one- and five-year relative survival improved significantly with time for all age groups both in men and women. The prevalence increased two times from 6014 in 1980 to 12 359 in 2012 among men and from 1974 to 4454 among women. There was a relatively higher proportional increase in prevalence among elderly men compared to younger patients. CONCLUSION: More prospective data are needed, preferably as randomized clinical trials, for determining the influence of age on the decisions of the surgical approach as well as chemo/radiotherapy for the elderly patients with urothelial cancers compared to younger patients.