Witchcraft Beliefs and Witch Hunts

This paper proposes an interdisciplinary explanation of the cross-cultural similarities and evolutionary patterns of witchcraft beliefs. It argues that human social dilemmas have led to the evolution of a fear system that is sensitive to signs of deceit and envy. This was adapted in the evolutionary environment of small foraging bands but became overstimulated by the consequences of the Agricultural Revolution, leading to witch paranoia. State formation, civilization, and economic development abated the fear of witches and replaced it in part with more collectivist forms of social paranoia. However, demographic-economic crises could rekindle fear of witches—resulting, for example, in the witch craze of early modern Europe. The Industrial Revolution broke the Malthusian shackles, but modern economic growth requires agricultural development as a starting point. In sub-Saharan Africa, witch paranoia has resurged because the conditions for agricultural development are lacking, leading to fighting for opportunities and an erosion of intergenerational reciprocity.     

Three-dimensional fluorescence recovery after photobleaching with the confocal scanning laser microscope

Confocal scanning laser microscopes (CSLMs) are equipped with the feature to photobleach user-defined regions. This makes them a handy tool to perform fluorescence recovery after photobleaching (FRAP) measurements. To allow quantification of such FRAP experiments, a three-dimensional model has been developed that describes the fluorescence recovery process for a disk-shaped geometry that is photobleached by the scanning beam of a CSLM. First the general mathematical basis is outlined describing the bleaching process for an arbitrary geometry bleached by a scanning laser beam. Next, these general expressions are applied to the bleaching by a CSLM of a disk-shaped geometry and an analytical solution is derived that describes three-dimensional fluorescence recovery in the bleached area as observed by the CSLM. The FRAP model is validated through both the Stokes-Einstein relation and the comparison of the measured diffusion coefficients with their theoretical estimates. Finally, the FRAP model is used to characterize the transport of FITC-dextrans through bulk three-dimensional biological materials: vitreous body isolated from bovine eyes, and lung sputum expectorated by cystic fibrosis patients. The decrease in the diffusion coefficient relative to its value in solution was dependent on the size of the FITC-dextrans in vitreous, whereas it was size-independent in cystic fibrosis sputum.     

G-protein coupled receptor solubilization and purification for biophysical analysis and functional studies,in the total absence of detergent

G-protein coupled receptors (GPCRs) constitute the largest class of membrane proteins and are a major drug target. A serious obstacle to studying GPCR structure/function characteristics is the requirement to extract the receptors from their native environment in the plasma membrane, coupled with the inherent instability of GPCRs in the detergents required for their solubilization. In the present study, we report the first solubilization and purification of a functional GPCR [human adenosine A_2A receptor (A_2AR)], in the total absence of detergent at any stage, by exploiting spontaneous encapsulation by styrene maleic acid (SMA) co-polymer direct from the membrane into a nanoscale SMA lipid particle (SMALP). Furthermore, the A_2AR–SMALP, generated from yeast (Pichia pastoris) or mammalian cells, exhibited increased thermostability (∼5°C) compared with detergent [DDM (n-dodecyl-β-D-maltopyranoside)]-solubilized A_2AR controls. The A_2AR–SMALP was also stable when stored for prolonged periods at 4°C and was resistant to multiple freeze-thaw cycles, in marked contrast with the detergent-solubilized receptor. These properties establish the potential for using GPCR–SMALP in receptor-based drug discovery assays. Moreover, in contrast with nanodiscs stabilized by scaffold proteins, the non-proteinaceous nature of the SMA polymer allowed unobscured biophysical characterization of the embedded receptor. Consequently, CD spectroscopy was used to relate changes in secondary structure to loss of ligand binding ([³H]ZM241385) capability. SMALP-solubilization of GPCRs, retaining the annular lipid environment, will enable a wide range of therapeutic targets to be prepared in native-like state to aid drug discovery and understanding of GPCR molecular mechanisms.     

Lysine residue 185 of Rad1 is a topological but not a functional counterpart of lysine residue 164 of PCNA

Monoubiquitylation of the homotrimeric DNA sliding clamp PCNA at lysine residue 164 (PCNA(K164)) is a highly conserved, DNA damage-inducible process that is mediated by the E2/E3 complex Rad6/Rad18. This ubiquitylation event recruits translesion synthesis (TLS) polymerases capable of replicating across damaged DNA templates. Besides PCNA, the Rad6/Rad18 complex was recently shown in yeast to ubiquitylate also 9-1-1, a heterotrimeric DNA sliding clamp composed of Rad9, Rad1, and Hus1 in a DNA damage-inducible manner. Based on the highly similar crystal structures of PCNA and 9-1-1, K185 of Rad1 (Rad1(K185)) was identified as the only topological equivalent of PCNA(K164). To investigate a potential role of posttranslational modifications of Rad1(K185) in DNA damage management, we here generated a mouse model with a conditional deletable Rad1(K185R) allele. The Rad1(K185) residue was found to be dispensable for Chk1 activation, DNA damage survival, and class switch recombination of immunoglobulin genes as well as recruitment of TLS polymerases during somatic hypermutation of immunoglobulin genes. Our data indicate that Rad1(K185) is not a functional counterpart of PCNA(K164).     

In situ analysis of single-stranded and duplex siRNA integrity in living cells

To attain the full therapeutic promise of short interfering RNA (siRNA), it is believed that improvements such as increased biostability are critical. Regrettably, thus far, insufficient in situ data are on hand regarding the intracellular stability of siRNAs. We report on the use of an advanced fluorescence-based method to probe the nucleolytic decay of double labeled siRNAs, which are subject to fluorescence resonance energy transfer (FRET). In vitro measurements with RNAse A and cellular extracts demonstrate that the ratio of acceptor (5'-Cy5) to donor (3'-rhodamine green) fluorescence can be used to study the degradation of the labeled siRNA substrates upon donor excitation. Intracellular FRET analysis showed substantial degradation of single-stranded siRNA, whereas duplex siRNA stayed intact during the measured time period. These data underline the high intrinsic nuclease resistance of unmodified duplex siRNA and prove that cellular persistence is much more critical for the single-stranded structure. For the first time, the stability of siRNA is investigated in real-time inside living cells. The fluorescence-based method presented here is a straightforward technique to gain direct information on siRNA integrity inside living cells and provides a bright outlook to learn more about the intracellular fate of siRNA therapeutics.     

Thermofluor-based high-throughput stability optimization of proteins for structural studies

Production of proteins well suited for structural studies is inherently difficult and time-consuming. Protein sample homogeneity, stability, and solubility are strongly correlated with the proteins' probability of yielding crystals, and optimization of these properties will improve success rates of crystallization. In the current study, we applied the thermofluor method as a high-throughput approach for identifying optimal protein formulation for crystallization. The method also allowed optimal stabilizing buffer compositions to be rapidly identified for each protein. Furthermore, the method allowed the identification of potential ligands, physiological or non-physiological, that can be used in subsequent crystallization trials. For this study, the thermally induced melting points were determined in different buffers as well as with additives for a total of 25 Escherichia coli proteins. Crystallization trials were set up together with stabilizing and destabilizing additives identified using thermofluor screening. A twofold increase in the number of crystallization leads was observed when the proteins were cocrystallized with stabilizing additives as compared with experiments without these additives. This suggests that thermofluor constitutes an efficient generic high-throughput method for identification of protein properties predictive of crystallizability.     

In-vivo range of motion of the subtalar joint using computed tomography

Understanding in vivo subtalar joint kinematics is important for evaluation of subtalar joint instability, the design of a subtalar prosthesis and for analysing surgical procedures of the ankle and hindfoot. No accurate data are available on the normal range of subtalar joint motion. The purpose of this study was to introduce a method that enables the quantification of the extremes of the range of motion of the subtalar joint in a loaded state using multidetector computed tomography (CT) imaging. In 20 subjects, an external load was applied to a footplate and forced the otherwise unconstrained foot in eight extreme positions. These extreme positions were foot dorsiflexion, plantarflexion, eversion, inversion and four extreme positions in between the before mentioned positions. CT images were acquired in a neutral foot position and each extreme position separately. After bone segmentation and contour matching of the CT data sets, the helical axes were determined for the motion of the calcaneus relative to the talus between four pairs of opposite extreme foot positions. The helical axis was represented in a coordinate system based on the geometric principal axes of the subjects’ talus. The greatest relative motion between the calcaneus and the talus was calculated for foot motion from extreme eversion to extreme inversion (mean rotation about the helical axis of 37.3±5.9°, mean translation of 2.3±1.1 mm). A consistent pattern of range of subtalar joint motion was found for motion of the foot with a considerable eversion and inversion component.