A Robust Protocol to Increase NimbleGen SeqCap EZ Multiplexing Capacity to 96 Samples

Contemporary genetic studies frequently involve sequencing of a targeted gene panel, for instance consisting of a set of genes associated with a specific disease. The NimbleGen SeqCap EZ Choice kit is commonly used for the targeted enrichment of sequencing libraries comprising a target size up to 7 Mb. A major drawback of this commercially available method is the exclusive use of single-indexing, meaning that at most 24 samples can be multiplexed in a single reaction. In case of relatively small target sizes, this will lead to excessive amounts of data per sample. We present an extended version of the NimbleGen SeqCap EZ protocol which allows to robustly multiplex up to 96 samples. We achieved this by incorporating Illumina dual-indexing based custom adapters into the original protocol. To further extend the optimization of cost-efficient sequencing of custom target panels, we studied the effect of higher pre-enrichment pooling factors and show that pre-enrichment pooling of up to 12 samples does not affect the quality of the data. To facilitate evaluation of capture efficiency in custom design panels, we also provide a detailed reporting tool.     

Electroporation-mediated DNA delivery to seedling tissues ofPhaseolus vulgaris L. (common bean)

Summary DNA was delivered to intact embryonic axes of the legumePhaseolus vulgaris L. through electroporation. Expression of the ß-glucuronidase reporter gene was observed in hypocotyl and epicotyl tissue in a spot-like manner. Transgene expression was high when a single pulse of 260 ms at a field strength of 225 V·cm^–1 was applied but could be achieved within a wide range of electrical conditions. Linearization of plasmid DNA greatly enhanced transient expression levels. The procedure was successful for embryonic axes of all testedP. vulgaris cultivars, for similar explants of several large-seeded leguminous species, as well as for some other tissues ofP. vulgaris.     

Differentiating Orienting and Defensive Responses to Concealed Information: The Role of Verbalization

Using physiological measures, concealed information can be validly assessed. Orienting theory has been proposed to account for concealed information testing. As orienting is characterized by heart rate deceleration, one would expect this type of heart rate response to concealed information. However, with some exceptions, an initial heart rate acceleration to concealed information is typically observed. In the present paper, we examine the role of verbalization to explain the mixed pattern of heart rate changes. Using a within-subjects design, 30 participants were asked to either remain silent or to give an overt verbal response (“yes”/”no”) to concealed autobiographical and control information. The results indicate that verbalization accounts for the initial heart rate acceleration. In line with the orienting theory, initial heart rate deceleration is observed when participants remained silent. Bruno Verschuere is a postdoctoral fellow of the Scientific Research Foundation (FWO).     

Environmental certification

Environmental certification of companies is a promising tool for steering the economy in a more environmentally sound direction. It can also be put in a chain perspective so that companies which constitute a production-consumption chain will conclude agreements between each other with respect to their environmental performance. A legal basis is formed by the EMS of ISO 14001 and the EMAS directive of the EU. However, apart from procedural aspects, substantive aspects should also be included. The major steering factor consists of the image of a company. This in contrast to product policy, where the major steering factor lies in the purchase behaviour of consumers. The two types of life cycle approaches should complement each other.     

Experimental and genetic analysis of root development inArabidopsis thaliana

The cellular organisation of theArabidopsis thaliana root is remarkably regular. A fate map of the primary root and root meristem that predicts the developmental destinies of cells within the embryonic root primordium has been constructed. Nevertheless, laser ablation experiments demonstrate that root meristem cells develop according to position and not according to lineage. Mutational analysis has identified genes required for cell specification in the radial as well as in the apical-basal dimension. The corresponding gene functions appear to be necessary during embryogenesis for the formation of a correctly patterned primary root. H Lambers Section editor     

Perfusion flow rate substantially contributes to the performance of the HepaRG‐AMC‐bioartificial liver

Bioartificial livers (BALs) are bioreactors containing liver cells that provide extracorporeal liver support to liver‐failure patients. Theoretically, the plasma perfusion flow rate through a BAL is an important determinant of its functionality. Low flow rates can limit functionality due to limited substrate availability, and high flow rates can induce cell damage. This hypothesis was tested by perfusing the AMC‐BAL loaded with the liver cell line HepaRG at four different medium flow rates (0.3, 1.5, 5, and 10 mL/min). Hepatic functions ammonia elimination, urea production, lactate consumption, and 6β‐hydroxylation of testosterone showed 2–20‐fold higher rates at 5 mL/min compared to 0.3 mL/min, while cell damage remained stable. However, at 10 mL/min cell damage was twofold higher, and maximal hepatic functionality was not changed, except for an increase in lactate elimination. On the other hand, only a low flow rate of 0.3 mL/min allowed for an accurate measurement of the ammonia and lactate mass balance across the bioreactor, which is useful for monitoring the BAL's condition during treatment. These results show that (1) the functionality of a BAL highly depends on the perfusion rate; (2) there is a universal optimal flow rate based on various function and cell damage parameters (5 mL/min for HepaRG‐BAL); and (3) in the current set‐up the mass balance of substrate, metabolite, or cell damage markers between in‐and out‐flow of the bioreactor can only be determined at a suboptimal, low, perfusion rate (0.3 mL/min for HepaRG‐BAL). Biotechnol. Bioeng. 2012; 109: 3182–3188. © 2012 Wiley Periodicals, Inc.