Forensic entomology, the use of insects and other arthropods in forensic investigations, is becoming increasingly more important in such investigations. To ensure its optimal use by a diverse group of professionals including pathologists, entomologists and police officers, a common frame of guidelines and standards is essential. Therefore, the European Association for Forensic Entomology has developed a protocol document for best practice in forensic entomology, which includes an overview of equipment used for collection of entomological evidence and a detailed description of the methods applied. Together with the definitions of key terms and a short introduction to the most important methods for the estimation of the minimum postmortem interval, the present paper aims to encourage a high level of competency in the field of forensic entomology.

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Channelrhodopsin-2 from Chlamydomonas reinhardtii is a lightgated ion channel. Over recent years, this ion channel has attracted considerable interest because of its unparalleled role in optogenetic applications. However, despite considerable efforts, an understanding of how molecular events during the photocycle, including the retinal trans-cis isomerization and the deprotonation/reprotonation of the Schiff base, are coupled to the channel-opening mechanism remains elusive. To elucidate this question, changes of conformation and configuration of several photocycle and conducting/nonconducting states need to be determined at atomic resolution. Here, we show that such data can be obtained by solid-state NMR enhanced by dynamic nuclear polarization applied to 15 N-labeled channelrhodopsin-2 carrying 14,15-13 C 2 retinal reconstituted into lipid bilayers. In its dark state, a pure all-trans retinal conformation with a stretched C14-C15 bond and a significant out-of-plane twist of the H-C14-C15-H dihedral angle could be observed. Using a combination of illumination, freezing, and thermal relaxation procedures, a number of intermediate states was generated and analyzed by DNP-enhanced solid-state NMR. Three distinct intermediates could be analyzed with high structural resolution: the early P 500 1 K-like state, the slowly decaying late intermediate P 480 4 , and a third intermediate populated only under continuous illumination conditions. Our data provide novel insight into the photoactive site of channelrhodopsin-2 during the photocycle. They further show that DNP-enhanced solid-state NMR fills the gap for challenging membrane proteins between functional studies and X-ray-based structure analysis, which is required for resolving molecular mechanisms.ince their discovery (1), channelrhodopsins (ChRs) have generated enormous interest because of the rapid development of their applications in optogenetics (2-7). Commonly, ChR2 from Chlamydomonas reinhardtii (8) and its variants are used thanks to their favorable expression levels. They are the only proteins known today functioning as light-gated ion channels (Fig. 1A). Like other microbial retinal proteins, they undergo a periodic photocycle. In ChRs, this photocycle is coupled to channel opening and closing as revealed in electrophysiological recordings (8). A chimera of ChR1 and ChR2 has been crystallized to yield a structure at 2.3-Å resolution (9). However, little is known on how this coupling functions on a molecular level, and a large number of studies based on visible (10-13), IR (11,[14][15][16][17][18][19], resonance Raman spectroscopy (20, 21), and EPR spectroscopy (22, 23) has been performed to address this question.The photocycles of microbial rhodopsins are usually compared with bacteriorhodopsin, the first discovered and most studied lightdriven proton pump (24). Without any illumination, microbial retinal proteins thermally equilibrate into a dark state (25). In the case of bacteriorhodopsin, for example, this state contains a mixture of two species terme...

The 104 kDa irreC-rst protein, a member of the immunoglobulin superfamily, mediates homophilic adhesion in cell cultures. In larval optic chiasms, the protein is found on recently formed axon bundles, not on older ones. In developing visual neuropils, it is present in all columnar domains of specific layers. The number of irreC-rst-positive neuropil stratifications increases until the midpupal stage. Immunoreactivity fades thereafter. The functional importance of the restricted expression pattern is demonstrated by the severe projection errors of axons in the first and second optic chiasms in loss of function mutants and in transformants that express the irreC-rst protein globally. Epigenesis of the phenotypes can be explained partially on the bases of homophilic irreC-rst interactions.

Dynamic nuclear polarization (DNP) has recently emerged as a tool to enhance the sensitivity of solid-state NMR experiments. However, so far high enhancements (>100) are limited to relatively low magnetic fields, and DNP at fields higher than 9.4 T significantly drops in efficiency. Here we report solid-state Overhauser effect DNP enhancements of over 100 at 18.8 T. This is achieved through the unexpected discovery that enhancements increase rapidly with increasing magic angle spinning (MAS) rates. The measurements are made using 1,3-bisdiphenylene-2-phenylallyl dissolved in o-terphenyl at 40 kHz MAS. We introduce a source–sink diffusion model for polarization transfer which is capable of explaining the experimental observations. The advantage of this approach is demonstrated on mesoporous alumina with the acquisition of well-resolved DNP surface-enhanced 27Al cross-polarization spectra.

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Solid-state nuclear magnetic resonance under magic angle spinning (MAS) enhanced with dynamic nuclear polarization (DNP) is a powerful approach to characterize many important classes of materials, allowing access to previously inaccessible structural and dynamic parameters. Here, we present the first DNP MAS experiments using a 0.7 mm MAS probe, which allows us to reach spinning frequencies of 65 kHz, with microwave irradiation, at 100 K. At the highest magnetic field available for DNP today (21.1 T), we find that the polarizing agent HyTEK2 provides DNP enhancements as high as 200 at a spinning rate of 65 kHz at 100 K, and BDPA yields an enhancement of 106 under the same conditions. Fast spinning rates enable excellent DNP performance, but they also yield unprecedented 1 H resolution under DNP conditions. We report well-resolved 1 H-detected 1 H− 13 C and 1 H− 15 N correlation spectra of microcrystalline histidine•HCl•H 2 O.

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