IVT-seq reveals extreme bias in RNA sequencing

Mapping-by-sequencing has emerged as a powerful technique for genetic mapping in several plant and animal species. As this resequencing-based method requires a reference genome, its application to complex plant genomes with incomplete and fragmented sequence resources remains challenging. We perform exome sequencing of phenotypic bulks of a mapping population of barley segregating for a mutant phenotype that increases the rate of leaf initiation. Read depth analysis identifies a candidate gene, which is confirmed by the analysis of independent mutant alleles. Our method illustrates how the genomic resources of barley together with exome resequencing can underpin mapping-by-sequencing.     

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation,Barrier Function,and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.     

Promising high monetary rewards for future task performance increases intermediate task performance

In everyday life contexts and work settings, monetary rewards are often contingent on future performance. Based on research showing that the anticipation of rewards causes improved task performance through enhanced task preparation, the present study tested the hypothesis that the promise of monetary rewards for future performance would not only increase future performance, but also performance on an unrewarded intermediate task. Participants performed an auditory Simon task in which they responded to two consecutive tones. While participants could earn high vs. low monetary rewards for fast responses to every second tone, their responses to the first tone were not rewarded. Moreover, we compared performance under conditions in which reward information could prompt strategic performance adjustments (i.e., when reward information was presented for a relatively long duration) to conditions preventing strategic performance adjustments (i.e., when reward information was presented very briefly). Results showed that high (vs. low) rewards sped up both rewarded and intermediate, unrewarded responses, and the effect was independent of the duration of reward presentation. Moreover, long presentation led to a speed-accuracy trade-off for both rewarded and unrewarded tones, whereas short presentation sped up responses to rewarded and unrewarded tones without this trade-off. These results suggest that high rewards for future performance boost intermediate performance due to enhanced task preparation, and they do so regardless whether people respond to rewards in a strategic or non-strategic manner.     

HSPBs: Small proteins with big implications in human disease

Although initially somewhat ignored, recent studies have now clearly established that the diverse members of the human family of small HSPs (HSPB1-HSPB10) play crucial roles in a wide range of cell types to maintain the integrity and function of tissues, in particular that of nervous and muscular tissue. The 10 human HSPBs clearly have overlapping and non-overlapping functional characteristics. Their ability to self- and hetero-oligomerise provides the cells with a large array of potentially different, specific functions. Single HSPB members can have a multitude of functions (moonlighting) and act on different "clients", thus affecting a wide range of different processes or structures that can ultimately affect the rate of aging of tissues and entire organisms. This is underscored by the findings that some inherited diseases involve mutations in several HSPB members that cause premature (mostly muscle and neuronal) tissue degeneration. Inversely, cancer cell resistance to different anticancer therapies is associated with elevated expression of several HSPBs. Still, many unanswered questions exist about the precise functioning of HSPBs, their collaboration with other HSPB members as well as their functions within the entire cellular chaperone network. Also, better insight in the regulation of expression of the various members and how their function is modulated post-translationally is needed. Such may be crucially important to develop means to intervene with their function for therapeutic purposes, which would require functional down-regulation in cancer but up-regulation in, for instance, cardiac or degenerative neuro/neuromuscular diseases. This article is part of a Directed Issue entitled: Small HSPs in physiology and pathology.     

A PCR amplification strategy for unrestricted generation of chimeric genes

For analyzing protein function, protein dynamics, or protein–protein interactions, the use of chimeric proteins has become an indispensable tool. The generation of DNA constructs coding for such fused proteins can, however, be a tedious process. Currently used strategies often make use of available endonuclease sites, leading to limitations in the choice of the site of fusion between two genes and problems in maintaining protein secondary structure. We have developed a cloning strategy to get around these disadvantages that is based on a single round of PCR amplification followed by antibiotic-resistant gene complementation.     

TCR gene therapy of spontaneous prostate carcinoma requires in vivo T cell activation

Analogous to the clinical use of recombinant high-affinity Abs, transfer of TCR genes may be used to create a T cell compartment specific for self-Ags to which the endogenous T cell repertoire is immune tolerant. In this study, we show in a spontaneous prostate carcinoma model that the combination of vaccination with adoptive transfer of small numbers of T cells that are genetically modified with a tumor-specific TCR results in a marked suppression of tumor development, even though both treatments are by themselves without effect. These results demonstrate the value of TCR gene transfer to target otherwise nonimmunogenic tumor-associated self-Ags provided that adoptive transfer occurs under conditions that allow in vivo expansion of the TCR-modified T cells.     

Dynamic shear behavior of mandibular condylar cartilage is dependent on testing direction

Little information is available on the direction-dependency of shear behavior in mandibular condylar cartilage. Therefore, we tested the hypothesis that such a dependency of the dynamic shear properties is present in mandibular condylar cartilage. From each of 17 condyles, two cartilage-bone plugs were dissected and tested in a simple shear sandwich configuration under a compressive strain of 10%. Sinusoidal shear strain (frequency range: 0.01-10 Hz) was applied in the medio-lateral or antero-posterior direction with an amplitude of 1.0%, 2.0%, and 3.0%. The magnitudes of the dynamic shear moduli, as calculated from the resulting shear stress, were found to increase with applied frequency and the shear strain amplitude. The values |G*|, G' and G' for a medio-laterally applied shear were about 20-33% of those in the antero-posterior shear, although the loss tangent (elasticity/viscosity ratio) was almost the same. In conclusion, the present results clearly show the direction-dependent characteristic of the mandibular condylar cartilage in dynamic shear.