Design and analysis of drug combination experiments

In this paper we present and discuss a novel, simple and easy to implement parametric modeling approach to assess synergy. An extended three parameter log-logistic model is used to analyse the data and calculate confidence intervals of the interaction indices. In addition the model corrects for the bias due to plate-location effects. The analysis is performed with PROC NLMIXED and SAS-code is provided. The approach is illustrated using data coming from an oncology study in which the inhibition effect of a combination of two compounds is studied using 96-well plates and a fixed-ratio design.     

Assay and characterization of an osmolarity inducible promoter newly isolated from Bacillus subtilis

An osmolarity-sensitive promoter fragment, P23423, isolated from Bacillus subtilis was characterized. The expression of β-galactosidase (β-Gal) driven by P23423 was regulated by osmolarity both in Escherichia coli and B. subtilis. The classical conserved region of this prokaryotic promoter was found within the sequence of the cloned fragment, and the putative promoter was identified as the control element of RNA not coding for protein (a RNA molecule that is not translated into a protein). The efficiency and benefit of this promoter was further demonstrated via osmolarity-induced expression of three other heterologous proteins in E. coli. Thus, this approach provided a simple and inexpensive inducible promoter element for the expression of cloned genes.     

Large-scale recording of neurons by movable silicon probes in behaving rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design. Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (http://www.neuronexustech.com/; http://www.sbmicrosystems.com/; http://www.acreo.se/) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.     

Improvement of the recessive genic male sterile lines with a subgenomic background in Brassica napus by molecular marker-assisted selection

Both the pollination control system and genetic distance are major factors in the utilization of crop heterosis. The recessive genic male sterile line (RGMS) 7-7365A (Bnms3ms3ms4ms4) has been widely applied to hybrid seed production because it can generate a completely male sterile population by crossing with the 7-7365C temporary line (Bnms3ms3rfrf). In this study, the sterile genes of 7-7365A were transferred to the new Brassica napus lines 7-749 and 7-750 with a high content of subgenomes by backcross breeding. We used the amplified fragment length polymorphism (AFLP) technique combined with bulk segregant analysis (BSA) to identify markers linked to the BnMs4 gene. Twelve AFLP markers linked to the BnMs4 gene were identified. Of them, SA06MG09 and P08MG16 were the closest makers, which were on either side of the gene at a distance of 0.9 and 0.8?cM, respectively. Twenty AFLP primer combinations were used to screen the F2, BC1F3, and BC2F4 populations from the breeding program, and the markers linked to the BnMs3 and BnMs4 genes were used to screen the BC2F4 populations. As a result, we obtained two types of improved sterile lines, 7-749A and 7-750A, and their indexes of subgenomic components (ISG) were 44.2?C49.8 and 20.2?C26.6%, respectively. The combining ability analyses of seed yield character were conducted in the crosses from the three sterile lines and ten restorers within a random block design in three environments for two successive years. The general combining ability (GCA) of the two improved sterile lines were significantly higher than the GCA of 7-7365A in every environment tested. The two improved sterile lines had stability in seed yield, and they will be used in the future for hybrid seed production.     

Interactions of uranyl ion with cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript> and its His39Ser variant as revealed by molecular simulation in combination with experimental methods

The biological toxicity of uranyl ion (UO₂²⁺) lies in interacting with proteins and disrupting their native functions. The structural and functional consequences of UO₂²⁺ interacting with cytochrome b ₅ (cyt b ₅), a small membrane heme protein, and its heme axial ligand His39Ser variant, cyt b ₅ H39S, were investigated both experimentally and theoretically. In experiments, although cyt b ₅ was only slightly affected, UO₂²⁺ binding to cyt b ₅ H39S with a K _D of 2.5 μM resulted in obvious alteration of the heme active site, and led to a decrease in peroxidase activity. Theoretically, molecular simulation proposed a uranyl ion binding site for cyt b ₅ at surface residues of Glu37 and Glu43, revealing both coordination and hydrogen bonding interactions. The information gained in this study provides insights into the mechanism of uranyl toxicity toward membrane protein at an atomic level.