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Botulinum neurotoxins (BoNT) cause the disease botulism, a flaccid paralysis of the muscle. They are also very effective, widely used medicines applied locally in sub-nanogram quantities. BoNTs are released together with several non-toxic, associated proteins as progenitor toxin complexes (PCT) by Clostridium botulinum to become highly potent oral poisons ingested via contaminated food. They block the neurotransmission in susceptible animals and humans already in nanogram quantities due to their specific ability to enter motoneurons and to cleave only selected neuronal proteins involved in neuroexocytosis. BoNTs have developed a sophisticated strategy to passage the gastrointestinal tract and to be absorbed in the intestine of the host to finally attack neurons. A non-toxic non-hemagglutinin (NTNHA) forms a binary complex with BoNT to protect it from gastrointestinal degradation. This binary M-PTC is one component of the bi-modular 14-subunit ∼760 kDa large progenitor toxin complex. The other component is the structurally and functionally independent dodecameric hemagglutinin (HA) complex which facilitates the absorption on the intestinal epithelium by glycan binding. Subsequent to its transcytosis the HA complex disrupts the tight junction of the intestinal barrier from the basolateral side by binding to E–cadherin. Now, the L-PTC can also enter the circulation by paracellular routes in much larger quantities. From here, the dissociated BoNTs reach the neuromuscular junction and accumulate via interaction with polysialo gangliosides, complex glycolipids, on motoneurons at the neuromuscular junction. Subsequently, additional specific binding to luminal segments of synaptic vesicles proteins like SV2 and synaptotagmin leads to their uptake. Finally, the neurotoxins shut down the synaptic vesicle cycle, which they had exploited before to enter their target cells, via specific cleavage of soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, which constitute the core components of the cellular membrane fusion machinery. [ABSTRACT FROM AUTHOR]

Abstract: We have investigated two new methods to establish an electrically conductive interconnection between two contacts on a semiconductor device inside a Scanning Electron Microscope (SEM). Both methods use a nanowire that is transported to the contacts. The first interconnection is made using a glue that can be hardened inside the SEM to fix the nanowires at the contacts. The other approach makes use of a microsoldering unit that is mounted on a micromanipulator and can be heated up to 200°C. A special solder is used to make a low ohmic connection between the contacts. [Copyright &y& Elsevier]

Abstract: The high potency of clostridial neurotoxins relies predominantly on their neurospecific binding and specific hydrolysis of SNARE proteins. Their multi-step mode of mechanism can be ascribed to their multi-domain three-dimensional structure. The C-terminal HCC-domain interacts subsequently with complex polysialo-gangliosides such as GT1b and a synaptic vesicle protein receptor via two neighbouring binding sites, resulting in highly specific uptake of the neurotoxins at synapses of cholinergic motoneurons. After its translocation the enzymatically active light chain specifically hydrolyses specific SNARE proteins, preventing SNARE complex assembly and thereby blocking exocytosis of neurotransmitter. [Copyright &y& Elsevier]

Abstract: For centuries, poisons and other biological material have been considered as weapons. However, it has been merely 100 years that the use of biological toxins as weapons has been explored scientifically. Trichothecenes, ricin and botulinum neurotoxins are natural organic toxins with diverse potencies. Their molecular structure, mechanisms of action, detection, clinical diagnosis and therapy are reviewed and their potential as biological weapon is discussed. It is not only the median lethal dose of each toxin that decides on its usability as a biological weapon, but also the availability, scale of production, purity of the isolated material and route of distribution. In general, without a state infrastructure, the use of biological weapons is restricted to assassinations or strictly localised terrorist attacks. [Copyright &y& Elsevier]

Botulinum neurotoxins (BoNTs) induce muscle paralysis by selectively entering cholinergic motoneurons and subsequent specific cleavage of core components of the vesicular fusion machinery. Complex gangliosides are requisite for efficient binding to neuronal cells, but protein receptors are critical for internalization. Recent work evidenced that synaptotagmins I and II can function as protein receptors for BoNT/B (Dong, M., Richards, D. A., Goodnough, M. C., Tepp, W. H., Johnson, E. A., and Chapman, E. R. (2003) J. Cell Biol. 162, 12931303). Here, we report the protein receptor for a second BoNT serotype. Like BoNT/B, BoNT/G employs synaptotagmins I and II to enter phrenic nerve cells. Using pull-down assays we show that only BoNT/G, but neither the five remaining BoNTs nor tetanus neurotoxin, interacts with synaptotagmins I and II. In contrast to BoNT/B, interactions with both isoforms are independent of the presence of gangliosides. Peptides derived from the luminal domain of synaptotagmin I and II are capable of blocking the neurotoxicity of BoNT/G in phrenic nerve preparations. Pull-down and neutralization assays further established the membrane-juxtaposed 10 luminal amino acids of synaptotagmins I and II as the critical segment for neurotoxin binding. In addition, we show that the carboxyl-terminal domain of the cell binding fragment of BoNT/B and BoNT/G mediates the interaction with their protein receptor. [ABSTRACT FROM AUTHOR]

A novel analysis routine is proposed that visualizes the opening of a transistor channel using Electron Beam Induced Current (EBIC) with a net zero-volt bias across a channel. nFET devices on a 7 nm technology node chip were examined in two different regions of the sample. With varying gate voltage, the device turn-on was clearly evident in the resulting EBIC image. Quantitative analysis of the resulting currents is demonstrated, and shows the channel opening up at voltages earlier than shown by I-V measurement. A mechanism for this difference is proposed. The analysis, also involving a 2-dimensional map of the space, provides opportunities for detecting regional variations in the electrical properties or performance at the high resolutions afforded with a scanning electron microscope (SEM). [ABSTRACT FROM AUTHOR]

Highlights: [•] A microsoldering unit for use inside an SEM or FIB is presented. [•] A bonding wire is successfully soldered to a bond pad inside an SEM. [•] A broken bonding wire is sucessfully repaired inside an SEM. [•] A nanowire is fixed to a liquidized solder inside an SEM. [Copyright &y& Elsevier]

In-situ transmission electron microscopy is rapidly emerging as the premier technique for characterising materials in a dynamic state on the atomic scale. The most important aspect of in-situ studies is specimen preparation. Specimens must be electron transparent and representative of the material in its operational state, amongst others. Here, a novel fabrication technique for the facile preparation of lamellae for in-situ transmission electron microscopy experimentation using focused ion beam milling is developed. This method involves the use of rotating microgrippers during the lift-out procedure, as opposed to the traditional micromanipulator needle and platinum weld. Using rotating grippers, and a unique adhesive substance, lamellae are mounted onto a MEMS device for in-situ TEM annealing experiments. We demonstrate how this technique can be used to avoid platinum deposition as well as minimising damage to the MEMS device during the thinning process. Our technique is both a cost effective and readily implementable alternative to the current generation of preparation methods for in-situ liquid, electrical, mechanical and thermal experimentation within the TEM as well as traditional cross-sectional lamella preparation. [ABSTRACT FROM AUTHOR]

Using a compact nanoprobing setup comprising eight probe tips attached to piezo-driven micromanipulators, various techniques for fault isolation are performed on 28 nm samples inside an SEM. The employed techniques include nanoprobing as well as EBAC. The recently implemented Current Imaging technique is used to quickly image large arrays of contacts providing a means of locating faults. In this case, Current Imaging provides insight into the sample's behaviour yielding qualitatively comparable results to the more cumbersome cAFM technique. While the results of the TEM investigations including EDX mappings were inconclusive, the Current Imaging technique clearly shows that the root cause is located below the SiGe layer. By combining these techniques inside a FIB/SEM microscope, it is possible to locate and characterize a failure as well as prepare a TEM lamella for further investigation without the necessity to switch to a different tool. [ABSTRACT FROM AUTHOR]

Abstract: An increase in cytosolic free calcium concentration [Ca2+] i initiates the exocytotic activity in various types of secretory cells. The guanosine 5′-O-[3-thio]triphosphate (GTPγS), a non-hydrolysable analogue of GTP (guanosine 5′-triphosphate), is an effective secretagogue for different cell types of different species, like mast cells, neutrophils or eosinophils. Consequently, the internal administration of GTPγS causes degranulation of mouse and rat mast cells. Regarding rat mast cells, it is proved that Ca2+ can cooperate with GTP or GTPγS in accelerating and increasing amplitude of the secretory response. All the previous studies with respect to capacitance recordings and mast cells were performed using mouse or rat mast cells, usually derived from peritoneum or the rat basophilic leukaemia cell line RBL. In this study, we applied the capacitance measurement technique to the human mast cell line-1 (HMC-1) cells, an immature cell line established from a patient with mast cell leukaemia. Patch-clamp dialysis experiments revealed that high intracellular free Ca2+ and GTPγS concentrations are both required for considerable capacitance increases in HMC-1 cells. During degranulation of HMC-1 cells, the total membrane capacitance (C m ) increase appeared continuously and, in some cases, as a discrete capacitance change, developing in a stepwise manner. Then, we tested the effect of latrunculin B upon HMC-1 cell capacitance increase as well as of some classic mast cell stimulators like PMA, A23187 and IL-1β in hexosaminidase release. Finally, we could conclude that the HMC-1 cell line represents a suitable model for the study of human mast cell degranulation. [Copyright &y& Elsevier]

Botulinum neurotoxins (BoNTs) are causative agents of the life-threatening disease botulism. They are naturally produced by species of the bacteria Clostridium botulinum as stable and non-covalent complexes, in which the BoNT molecule is assembled with several auxiliary non-toxic proteins. Some BoNT serotypes, represented by the well-studied BoNT serotype A (BoNT/A), are produced by Clostridium strains that carry the ha gene cluster, which encodes four neurotoxin-associated proteins (NTNHA, HA17, HA33, and HA70) that play an important role to deliver and protect BoNTs in the gastrointestinal tract during oral intoxication. In contrast, BoNT/E- and BoNT/F-producing strains carry a distinct gene cluster that encodes five proteins (NTNHA, P47, OrfX1, OrfX2, and OrfX3, termed the orfX cluster). The structures and functions of these proteins remain largely unknown. Here, we report the crystal structure of P47 resolved at 2.8 Å resolution. Surprisingly, P47 displays a structural topology that is similar to bactericidal/permeability-increasing (BPI) like proteins, which were previously identified only in eukaryotes. The similarity of a hydrophobic cleft of P47 with the phospholipid-binding groove of BPI suggests that P47 might be involved in lipid association to exert its function. Consistently, P47 associates and induces aggregation of asolectin-containing liposomes in a protein- and lipid-concentration dependent manner. These findings laid the foundation for future structural and functional studies of the potential roles of P47 and OrfX proteins in facilitating oral intoxication of BoNTs. [ABSTRACT FROM AUTHOR]

Biological toxins are a heterogeneous group of compounds that share commonalities both with biological and chemical agents. Based on their availability, toxicity, and the lack of medical countermeasures as well as their known history of military research, toxins such as ricin, botulinum neurotoxins, staphylococcal enterotoxins, and saxitoxin are classified as toxins of bioterrorism risk. At the same time, they are known to cause naturally occurring intoxication. Different technologies for toxin detection have been established, but hardly any universally agreed reference methods or reference materials are available. Regular proficiency tests have been lacking for most of the mentioned toxins. Therefore, objective comparison of method performance has not been possible. The recently completed EU-funded project EQuATox delineated the current status quo of toxin detection on the basis of a series of proficiency tests. This review provides an overview of the results obtained and highlights the need for future developments in the field. [ABSTRACT FROM AUTHOR]

Botulinum neurotoxins (BoNTs) cause the disease botulism manifested by flaccid paralysis that could be fatal to humans and animals. Oral ingestion of the toxin with contaminated food is one of the most common routes for botulism. BoNT assembles with several auxiliary proteins to survive in the gastrointestinal tract and is subsequently transported through the intestinal epithelium into the general circulation. Several hemagglutinin proteins form a multi-protein complex (HA complex) that recognizes host glycans on the intestinal epithelial cell surface to facilitate BoNT absorption. Blocking carbohydrate binding to the HA complex could significantly inhibit the oral toxicity of BoNT. Here, we identify lactulose, a galactose-containing non-digestible sugar commonly used to treat constipation, as a prototype inhibitor against oral BoNT/A intoxication. As revealed by a crystal structure, lactulose binds to the HA complex at the same site where the host galactose-containing carbohydrate receptors bind. In vitro assays using intestinal Caco-2 cells demonstrated that lactulose inhibits HA from compromising the integrity of the epithelial cell monolayers and blocks the internalization of HA. Furthermore, co-administration of lactulose significantly protected mice against BoNT/A oral intoxication in vivo . Taken together, these data encourage the development of carbohydrate receptor mimics as a therapeutic intervention to prevent BoNT oral intoxication. [ABSTRACT FROM AUTHOR]

Abstract: The modular four domain structure of clostridial neurotoxins supports the idea to reassemble individual domains from tetanus and botulinum neurotoxins to generate novel molecules with altered pharmacological properties. To treat disorders of the central nervous system drug transporter molecules based on catalytically inactive clostridial neurotoxins circumventing the passage of the blood–brain-barrier are desired. Such molecules can be produced based on the highly effective botulinum neurotoxin serotype A incorporating the retrograde axonal sorting property of tetanus neurotoxin which is supposed to be encoded within its C-terminal cell binding domain HC. The corresponding exchange of the tetanus neurotoxin HC-fragment in botulinum neurotoxin A yielded the novel hybrid molecule AATT which displayed decreased potency at the neuromuscular junction like tetanus neurotoxin but exerted equal activity in cortical neurons compared to botulinum neurotoxin A wild-type. Minimizing the tetanus neurotoxin cell binding domain to its N- or C-terminal half drastically reduced the potencies of AATA and AAAT in cortical neurons indicating that the structural motif mediating sorting of tetanus neurotoxin is predominantly encoded within the entire HC-fragment. However, the reciprocal exchange resulted in TTAA which showed a similar potency as tetanus neurotoxin at the neuromuscular junction indicating that the tetanus neurotoxin portion prevents a high potency as observed for botulinum neurotoxins. In conclusion, clostridial neurotoxin based inactivated drug transporter for targeting central neurons should contain the cell binding domain of tetanus neurotoxin to exert its tropism for the central nervous system. [Copyright &y& Elsevier]