Comparison of a laboratory and a production coating spray gun with respect to scale-up

A laboratory spray gun and a production spray gun were investigated in a scale-up study. Two Schlick spray guns, which are equipped with a new antibearding cap, were used in this study. The influence of the atomization air pressure, spray gun-to tablet bed distance, polymer solution viscosity, and spray rate were analyzed in a statistical design of experiments. The 2 spray guns were compared with respect to the spray width and height, droplet size, droplet velocity, and spray density. The droplet size, velocity, and spray density were measured with a Phase Doppler Particle Analyzer. A successful scale-up of the atomization is accomplished if similar droplet sizes, droplet velocities, and spray densities are achieved in the production scale as in the laboratory scale. This study gives basic information for the scale-up of the settings from the laboratory spray gun to the production spray gun. Both spray guns are highly comparable with respect to the droplet size and velocity. The scale-up of the droplet size should be performed by an adjustment of the atomization air pressure. The scale-up of the droplet velocity should be performed by an adjustment of the spray gun to tablet bed distance. The presented statistical model and surface plots are convenient and powerful tools for scaling up the spray settings if the spray gun is changed from laboratory spray gun to the production spray gun. Published: January 19, 2007     

A functional link between hyphal maintenance and quorum sensing in Candida albicans

Morphogenesis in Candida albicans requires hyphal initiation and maintenance, and both processes are regulated by the fungal quorum sensing molecule (QSM) farnesol. We show that deletion of C. albicans EED1, which is crucial for hyphal extension and maintenance, led to a dramatically increased sensitivity to farnesol, and thus identified the first mutant hypersensitive to farnesol. Furthermore, farnesol decreased the transient filamentation of an eed1Δ strain without inducing cell death, indicating that two separate mechanisms mediate quorum sensing and cell lysis by farnesol. To analyze the cause of farnesol hypersensitivity we constructed either hyperactive or deletion mutants of factors involved in farnesol signaling, by introducing the hyperactive RAS1^G13V or pADH1‐CYR1^CAT allele, or deleting CZF1 or NRG1 respectively. Neither of the constructs nor the exogenous addition of dB‐cAMP was able to rescue the farnesol hypersensitivity, highlighting that farnesol mediates its effects not only via the cAMP pathway. Interestingly, the eed1Δ strain also displayed increased farnesol production. When eed1Δ was grown under continuous medium flow conditions, to remove accumulating QSMs from the supernatant, maintenance of eed1Δ filamentation, although not restored, was significantly prolonged, indicating a link between farnesol sensitivity, production, and the hyphal maintenance‐defect in the eed1Δ mutant strain.     

Genetic mapping and QTL analysis of Botrytis resistance in <Emphasis Type="Italic">Gerbera hybrida</Emphasis>

Gerbera hybrida is an economically important cut flower. In the production and transportation of gerbera with unavoidable periods of high relative humidity, grey mould occurs and results in losses in quality and quantity of flowers. Considering the limitations of chemical use in greenhouses and the impossibility to use these chemicals in auction or after sale, breeding for resistant gerbera cultivars is considered as the best practical approach. In this study, we developed two segregating F1 populations (called S and F). Four parental linkage maps were constructed using common and parental specific SNP markers developed from expressed sequence tag sequencing. Parental genetic maps, containing 30, 29, 27 and 28 linkage groups and a consensus map covering 24 of the 25 expected chromosomes, could be constructed. After evaluation of Botrytis disease severity using three different tests, whole inflorescence, bottom (of disc florets) and ray floret, quantitative trait locus (QTL) mapping was performed using the four individual parental maps. A total of 20 QTLs (including one identical QTL for whole inflorescence and bottom tests) were identified in the parental maps of the two populations. The number of QTLs found and the explained variance of most QTLs detected reflect the complex mechanism of Botrytis disease response.     

Surface charge engineering of a Bacillus gibsonii subtilisin protease

In proteins, a posttranslational deamidation process converts asparagine (Asn) and glutamine (Gln) residues into negatively charged aspartic (Asp) and glutamic acid (Glu), respectively. This process changes the protein net charge affecting enzyme activity, pH optimum, and stability. Understanding the principles which affect these enzyme properties would be valuable for protein engineering in general. In this work, three criteria for selecting amino acid substitutions of the deamidation type in the Bacillus gibsonii alkaline protease (BgAP) are proposed and systematically studied in their influence on pH-dependent activity and thermal resistance. Out of 113 possible surface amino acids, 18 (11 Asn and 7 Gln) residues of BgAP were selected and evaluated based on three proposed criteria: (1) The Asn or Gln residues should not be conserved, (2) should be surface exposed, and (3) neighbored by glycine. “Deamidation” in five (N97, N253, Q37, Q200, and Q256) out of eight (N97, N154, N250, N253, Q37, Q107, Q200, and Q256) amino acids meeting all criteria resulted in increased proteolytic activity. In addition, pH activity profiles of the variants N253D and Q256E and the combined variant N253DQ256E were dramatically shifted towards higher activity at lower pH (range of 8.5–10). Variant N253DQ256E showed twice the specific activity of wild-type BgAP and its thermal resistance increased by 2.4 °C at pH?8.5. These property changes suggest that mimicking surface deamidation by substituting Gln by Glu and/or Asn by Asp might be a simple and fast protein reengineering approach for modulating enzyme properties such as activity, pH optimum, and thermal resistance.     

Reciprocal Regulation as a Source of Ultrasensitivity in Two-Component Systems with a Bifunctional Sensor Kinase

Two-component signal transduction systems, where the phosphorylation state of a regulator protein is modulated by a sensor kinase, are common in bacteria and other microbes. In many of these systems, the sensor kinase is bifunctional catalyzing both, the phosphorylation and the dephosphorylation of the regulator protein in response to input signals. Previous studies have shown that systems with a bifunctional enzyme can adjust the phosphorylation level of the regulator protein independently of the total protein concentrations – a property known as concentration robustness. Here, I argue that two-component systems with a bifunctional enzyme may also exhibit ultrasensitivity if the input signal reciprocally affects multiple activities of the sensor kinase. To this end, I consider the case where an allosteric effector inhibits autophosphorylation and, concomitantly, activates the enzyme''s phosphatase activity, as observed experimentally in the PhoQ/PhoP and NRII/NRI systems. A theoretical analysis reveals two operating regimes under steady state conditions depending on the effector affinity: If the affinity is low the system produces a graded response with respect to input signals and exhibits stimulus-dependent concentration robustness – consistent with previous experiments. In contrast, a high-affinity effector may generate ultrasensitivity by a similar mechanism as phosphorylation-dephosphorylation cycles with distinct converter enzymes. The occurrence of ultrasensitivity requires saturation of the sensor kinase''s phosphatase activity, but is restricted to low effector concentrations, which suggests that this mode of operation might be employed for the detection and amplification of low abundant input signals. Interestingly, the same mechanism also applies to covalent modification cycles with a bifunctional converter enzyme, which suggests that reciprocal regulation, as a mechanism to generate ultrasensitivity, is not restricted to two-component systems, but may apply more generally to bifunctional enzyme systems.     

Hydrological Response to Climate Change for Gilgel Abay River,in the Lake Tana Basin - Upper Blue Nile Basin of Ethiopia

Climate change is likely to have severe effects on water availability in Ethiopia. The aim of the present study was to assess the impact of climate change on the Gilgel Abay River, Upper Blue Nile Basin. The Statistical Downscaling Tool (SDSM) was used to downscale the HadCM3 (Hadley centre Climate Model 3) Global Circulation Model (GCM) scenario data into finer scale resolution. The Soil and Water Assessment Tool (SWAT) was set up, calibrated, and validated. SDSM downscaled climate outputs were used as an input to the SWAT model. The climate projection analysis was done by dividing the period 2010-2100 into three time windows with each 30 years of data. The period 1990-2001 was taken as the baseline period against which comparison was made. Results showed that annual mean precipitation may decrease in the first 30-year period but increase in the following two 30-year periods. The decrease in mean monthly precipitation may be as much as about -30% during 2010-2040 but the increase may be more than +30% in 2070-2100. The impact of climate change may cause a decrease in mean monthly flow volume between -40% to -50% during 2010-2040 but may increase by more than the double during 2070-2100. Climate change appears to have negligible effect on low flow conditions of the river. Seasonal mean flow volume, however, may increase by more than the double and +30% to +40% for the Belg (small rainy season) and Kiremit (main rainy season) periods, respectively. Overall, it appears that climate change will result in an annual increase in flow volume for the Gilgel Abay River. The increase in flow is likely to have considerable importance for local small scale irrigation activities. Moreover, it will help harnessing a significant amount of water for ongoing dam projects in the Gilgel Abay River Basin.     

The Paternal Landscape along the Bight of Benin – Testing Regional Representativeness of West-African Population Samples Using Y-Chromosomal Markers

Patterns of genetic variation in human populations across the African continent are still not well studied in comparison with Eurasia and America, despite the high genetic and cultural diversity among African populations. In population and forensic genetic studies a single sample is often used to represent a complete African region. In such a scenario, inappropriate sampling strategies and/or the use of local, isolated populations may bias interpretations and pose questions of representativeness at a macrogeographic-scale. The non-recombining region of the Y-chromosome (NRY) has great potential to reveal the regional representation of a sample due to its powerful phylogeographic information content. An area poorly characterized for Y-chromosomal data is the West-African region along the Bight of Benin, despite its important history in the trans-Atlantic slave trade and its large number of ethnic groups, languages and lifestyles. In this study, Y-chromosomal haplotypes from four Beninese populations were determined and a global meta-analysis with available Y-SNP and Y-STR data from populations along the Bight of Benin and surrounding areas was performed. A thorough methodology was developed allowing comparison of population samples using Y-chromosomal lineage data based on different Y-SNP panels and phylogenies. Geographic proximity turned out to be the best predictor of genetic affinity between populations along the Bight of Benin. Nevertheless, based on Y-chromosomal data from the literature two population samples differed strongly from others from the same or neighbouring areas and are not regionally representative within large-scale studies. Furthermore, the analysis of the HapMap sample YRI of a Yoruban population from South-western Nigeria based on Y-SNPs and Y-STR data showed for the first time its regional representativeness, a result which is important for standard population and forensic genetic applications using the YRI sample. Therefore, the uniquely and powerful geographical information carried by the Y-chromosome makes it an important locus to test the representativeness of a certain sample even in the genomic era, especially in poorly investigated areas like Africa.