Jan Maes

Evaluation of 14 organic solvents and carriers for screening applications in zebrafish embryos and larvae.

Zebrafish are rapidly growing in popularity as an in vivo model system for chemical genetics, drug discovery, and toxicology, and more recently also for natural product discovery. Experiments involving the pharmacological evaluation of small molecules or natural product extracts in zebrafish bioassays require the effective delivery of these compounds to embryos and larvae. While most samples to be screened are first solubilized in dimethyl sulfoxide (DMSO), which is then diluted in the embryo medium, often this method is not sufficient to prevent the immediate or eventual precipitation of the sample. Certain compounds and extracts are also not highly soluble in DMSO. In such instances the use of carriers and/or other solvents might offer an alternative means to achieve the required sample concentration. Towards this end, we determined the maximum tolerated concentration (MTC) of several commonly used solvents and carriers in zebrafish embryos and larvae at various developmental stages. Solvents evaluated for this study included acetone, acetonitrile, butanone, dimethyl formamide, DMSO, ethanol, glycerol, isopropanol, methanol, polyethylene glycol (PEG-400), propylene glycol, and solketal, and carriers included albumin (BSA) and cyclodextrin (2-hydroxypropyl-beta-cyclodextrin, or HPBCD). This study resulted in the identification of polyethylene glycol (PEG400), propylene glycol, and methanol as solvents that were relatively well-tolerated over a range of developmental stages. In addition, our results showed that acetone was well-tolerated by embryos but not by larvae, and 1% cyclodextrin (HPBCD) was well-tolerated by both embryos and larvae, indicating the utility of this carrier for compound screening in zebrafish. However, given the relatively small differences (2–3 fold) between concentrations that are apparently safe and those that are clearly toxic, further studies – e.g. omics analyses –should be carried out to determine which cellular processes and signalling pathways are affected by any solvents and carriers that are used for small-molecule screens in zebrafish.

Zebrafish Bioassay-Guided Natural Product Discovery: Isolation of Angiogenesis Inhibitors from East African Medicinal Plants

Natural products represent a significant reservoir of unexplored chemical diversity for early-stage drug discovery. The identification of lead compounds of natural origin would benefit from therapeutically relevant bioassays capable of facilitating the isolation of bioactive molecules from multi-constituent extracts. Towards this end, we developed an in vivo bioassay-guided isolation approach for natural product discovery that combines bioactivity screening in zebrafish embryos with rapid fractionation by analytical thin-layer chromatography (TLC) and initial structural elucidation by high-resolution electrospray mass spectrometry (HRESIMS). Bioactivity screening of East African medicinal plant extracts using fli-1:EGFP transgenic zebrafish embryos identified Oxygonum sinuatum and Plectranthus barbatus as inhibiting vascular development. Zebrafish bioassay-guided fractionation identified the active components of these plants as emodin, an inhibitor of the protein kinase CK2, and coleon A lactone, a rare abietane diterpenoid with no previously described bioactivity. Both emodin and coleon A lactone inhibited mammalian endothelial cell proliferation, migration, and tube formation in vitro, as well as angiogenesis in the chick chorioallantoic membrane (CAM) assay. These results suggest that the combination of zebrafish bioassays with analytical chromatography methods is an effective strategy for the rapid identification of bioactive natural products.

Anticonvulsant activity of bisabolene sesquiterpenoids of Curcuma longa in zebrafish and mouse seizure models

Turmeric, obtained from the rhizomes of Curcuma longa, is used in South Asia as a traditional medicine for the treatment of epilepsy. To date, in vivo studies on the anticonvulsant activity of turmeric have focused on its principal curcuminoid, curcumin. However, poor absorption and rapid metabolism have limited the therapeutic application of curcumin in humans. To explore the therapeutic potential of turmeric for epilepsy further, we analyzed its anticonvulsant activity in a larval zebrafish seizure assay. Initial experiments revealed that the anticonvulsant activity of turmeric in zebrafish larvae cannot be explained solely by the effects of curcumin. Zebrafish bioassay-guided fractionation of turmeric identified bisabolene sesquiterpenoids as additional anticonvulsants that inhibit PTZ-induced seizures in both zebrafish and mice. Here, we present the first report of the anticonvulsant properties of bisabolene sesquiterpenoids and provide evidence which warrants further investigation toward the mechanistic understanding of their neuromodulatory activity.

Integration of Microfractionation, qNMR and Zebrafish Screening for the In Vivo Bioassay-Guided Isolation and Quantitative Bioactivity Analysis of Natural Products

Natural products (NPs) are an attractive source of chemical diversity for small-molecule drug discovery. Several challenges nevertheless persist with respect to NP discovery, including the time and effort required for bioassay-guided isolation of bioactive NPs, and the limited biomedical relevance to date of in vitro bioassays used in this context. With regard to bioassays, zebrafish have recently emerged as an effective model system for chemical biology, allowing in vivo high-content screens that are compatible with microgram amounts of compound. For the deconvolution of the complex extracts into their individual constituents, recent progress has been achieved on several fronts as analytical techniques now enable the rapid microfractionation of extracts, and microflow NMR methods have developed to the point of allowing the identification of microgram amounts of NPs. Here we combine advanced analytical methods with high-content screening in zebrafish to create an integrated platform for microgram-scale, in vivo NP discovery. We use this platform for the bioassay-guided fractionation of an East African medicinal plant, Rhynchosia viscosa, resulting in the identification of both known and novel isoflavone derivatives with anti-angiogenic and anti-inflammatory activity. Quantitative microflow NMR is used both to determine the structure of bioactive compounds and to quantify them for direct dose-response experiments at the microgram scale. The key advantages of this approach are (1) the microgram scale at which both biological and analytical experiments can be performed, (2) the speed and the rationality of the bioassay-guided fractionation – generic for NP extracts of diverse origin – that requires only limited sample-specific optimization and (3) the use of microflow NMR for quantification, enabling the identification and dose-response experiments with only tens of micrograms of each compound. This study demonstrates that a complete in vivo bioassay-guided fractionation can be performed with only 20 mg of NP extract within a few days.

Impact of Precursor Chemistry and Process Conditions on the Scalability of ALD HfO2 Gate Dielectrics

The downscaling of high-k/metal gate transistor devices requires thin-film deposition processes that deliver not only an outstanding high-k oxide quality, but also a strict interfacial oxide thickness control in the sub-1 nm thickness range. To study the impact of atomic layer deposition (ALD) process conditions and chemistry on the HfO 2 quality and interfacial oxide thickness, we have used tetrakis[ethylmethylamino]hafnium (TEMAH) as a metal precursor and H 2 O and 0 3 as oxidants. The deposition temperature ranged from 285 up to 365 °C, where TEMAH decomposition plays a role in the growth mechanism. Physical characterization and Pt dot capacitor devices have been used to study the impact of the oxidant and process conditions on the equivalent oxide thickness and gate leakage current of 2-4 nm thin HfO 2 films. By combining X-ray reflectometry and ellipsometry, we evaluated the Si/high-k interfacial oxide layer thickness. Time-of-flight secondary-ion mass spectroscopy was used to determine the C impurity levels. Both the interfacial oxide layer thickness and the C impurity level in the Si/SiO 2 /HfO 2 stacks are strongly dependent on the oxidant. The temperature dependence of the C impurity level is opposite for O 3 and H 2 O. Furthermore, SiO 2 regrowth was found for the 0 3 process.

POWELL–SABIN SPLINE WAVELETS

Recently we developed a subdivision scheme for Powell–Sabin splines. It is a triadic scheme and it is general in the sense that it is not restricted to uniform triangles, the vertices must not have valence six and there are no restrictions on the initial triangulation. A sequence of nested spaces or multiresolution analysis can be associated with the base triangulation. In this paper we use the lifting scheme to construct basis functions for the complement space that captures the details that are lost when going to a coarser resolution. The subdivision scheme appears as the first lifting step or prediction step. A second lifting step, the update, is used to achieve certain properties for the complement spaces and the wavelet functions such as orthogonality and vanishing moments. The design of the update step is based on stability considerations. We prove stability for both the scaling functions and the wavelet functions.

Tanshinone IIA exhibits anticonvulsant activity in zebrafish and mouse seizure models

Danshen or Chinese red sage (Salvia miltiorrhiza, Bunge) is used by traditional Chinese medicine (TCM) practitioners to treat neurological, cardiovascular, and cerebrovascular disorders and is included in some TCM formulations to control epileptic seizures. In this study, acetonic crude extracts of danshen inhibited pentylenetetrazol (PTZ)-induced seizure activity in zebrafish larvae. Subsequent zebrafish bioassay-guided fractionation of the extract resulted in the isolation of four major tanshinones, which suppressed PTZ-induced activity to varying degrees. One of the active tanshinones, tanshinone IIA, also reduced c-fos expression in the brains of PTZ-exposed zebrafish larvae. In rodent seizure models, tanshinone IIA showed anticonvulsive activity in the mouse 6-Hz psychomotor seizure test in a biphasic manner and modified seizure thresholds in a complex manner for the mouse i.v. PTZ seizure assay. Interestingly, tanshinone IIA is used as a prescription drug in China to address cerebral ischemia in patients. Here, we provide the first in vivo evidence demonstrating that tanshinone IIA has anticonvulsant properties as well.

Atomic layer deposition of hafnium silicate gate dielectric layers

Downscaling equivalent oxide thickness (EOT) by decreasing the physical thickness or increasing the permittivity of the gate dielectric is required to reach the complementary metal-oxide semiconductor (CMOS) (sub) 45nm node performance specifications. In this work, we study the atomic layer deposition (ALD) of hafnium silicate gate dielectrics from HfCl4, SiCl4, and H2O both experimentally and theoretically. Hafnium silicate is characterized by Rutherford backscattering, time-of-flight secondary ion mass spectroscopy, and x-ray photoelectron spectroscopy. The interaction of the precursors with the surface sites is investigated by first-principles calculations. The electrical properties are evaluated on TaN gated capacitors. The HfCl4∕H2O reactions create surface sites that enable the chemisorption of SiCl4 in doses much smaller than the doses required for SiO2 ALD from SiCl4∕H2O. The hydrolysis of Si–Cl is slower than the hydrolysis of Hf–Cl. Optimization of the hafnium silicate deposition results in a le...

The direct impact of landslides on household income in tropical regions: a case study from the Rwenzori Mountains in Uganda

Landslides affect millions of people worldwide, but theoretical and empirical studies on the impact of landslides remain scarce, especially in Sub-Saharan Africa. This study proposes and applies a method to estimate the direct impact of landslides on household income and to investigate the presence of specific risk sharing and mitigation strategies towards landslides in a tropical and rural environment. An original cross-sectional household survey is used in combination with geographical data to acquire detailed information on livelihoods and on hazards in the Rwenzori mountains, Uganda. Ordinary least square regressions and probit estimations with village fixed effects are used to estimate the impact of landslides and the presence of mitigation strategies. Geographical information at household level allows to disentangle the direct impact from the indirect effects of landslides. We show that the income of affected households is substantially reduced during the first years after a landslide has occurred. We find that members of recently affected households participate more in wage-employment or in self-employed activities, presumably to address income losses following a landslide. Yet, we see that these jobs do not provide sufficient revenue to compensate for the loss of income from agriculture. Given that landslides cause localized shocks, finding a significant direct impact in our study indicates that no adequate risk sharing mechanisms are in place in the Rwenzori sub-region. These insights are used to derive policy recommendations for alleviating the impact of landslides in the region. By quantifying the direct impact of landslides on household income in an agricultural context in Africa this study draws the attention towards a problem that has been broadly underestimated so far and provides a sound scientific base for disaster risk reduction in the region. Both the methodology and the findings of this research are applicable to other tropical regions with high landslide densities.

ALD of Ta(Si)N Thin Films Using TDMAS as a Reducing Agent and as a Si Precursor

Ta(Si)N thin films were deposited by atomic layer deposition (ALD) from tantalum chloride, ammonia, and tris(dimethylamino)silane (TDMAS). TDMAS was used as a reducing agent and as a silicon precursor. The pulsing order and the length of the TDMAS pulse were optimized. The deposition temperature was varied between 300 and 500°C. The film properties were analyzed by time-of-flight elastic recoil detection analysis, energy dispersive X-ray spectroscopy, X-ray diffraction, and the standard four-point probe method. Additionally work function values were measured by depositing 50 nm thick Ta(Si)N films on different thicknesses of hafnium oxide layers on silicon.