Strukturelle und immunologische Charakterisierung der Spinnenseide von Nephila clavipes

Ziel der Arbeit war es, die Spinnenseide des Sicherungsfadens der Goldenen Radnetzspinne Nephila clavipes zu charakterisieren und eventuelle Zusammenhänge zwischen Fadenbildung, strukturellem Aufbau und biochemischer Konsistenz herauszuarbeiten. Dabei war vor allem die noch weitgehend unverstandene Rolle der beiden bekannten Spidroine MASp1 und MASp2 sowie ihrer repetitiven und konservierten C-terminalen Domänen von Interesse. Hierzu wurden Antiseren gegen rekombinante Polypeptide mit den entsprechenden Sequenzen aus beiden Bereichen der beiden Gene hergestellt, affinitätsgereinigt und in den biochemischen und histochemischen Untersuchungen eingesetzt. Mithilfe der Antikörper konnte erstmalig gezeigt werden, dass die hochkonservierten C-Termini auch in den hochmolekularen Proteinfraktionen der ausgesponnenen Fäden wieder zu finden sind und nicht etwa vor der Fadenbildung abgespalten werden. Experimente zeigten, dass Sie zur Bildung von Disulfidbrücken befähigt sind. Ihre Bedeutung sowie ihre phylogenetische Entwicklung wird im Zusammenhang mit Ergebnissen aus Sequenzvergleichen diskutiert. Aus den biochemischen Untersuchungen des Spinnguts ergab sich, dass dies überraschend heterogen ist und mehrere hochmolekulare, lösliche Proteine im Bereich von 220-270 kDa aufweist. Durch Anfärbung mit Lektinen wurde gezeigt, dass die Spidroine aller Wahrscheinlichkeit nach O-glycosyliert sind. Als weitere Ursache für die Heterogenität wurden N-terminale Abbauprozesse diskutiert, die sich aus den mit den Antiseren erzielten Ergebnissen und Versuchen N-terminaler Sequenzierung ergaben. Die Ergebnisse der strukturellen und der immunologischen Studien wurden in einem Modell zum Aufbau der Tragfäden von Nephila clavipes zusammengefasst. Neben einem Kern besitzt die Seide eine aus drei Schichten bestehende Hülle. Während die beiden äußeren Schichten labil sind, zeigt die dritte innere Schicht eine hohe Resistenz gegen Laugen, Säuren und chaotrope Agenzien und verleiht dem Faden Stabilität. Der Kern lässt sich aufgrund der Verteilung der Spidroine in zwei Zonen unterteilen. Während MASp1homogen verteilt in beiden Zonen vorliegt, fehlt MASp2 in der äußeren Zone und liegt in der inneren Zone konzentriert in kleinen Inseln vor. Dieses Verteilungsmuster wird im Zusammenhang mit der Rolle von MASp2 bei der Fadenbildung und dem Auftreten von Fibrillen diskutiert

Conservation of a pH-sensitive structure in the C-terminal region of spider silk extends across the entire silk gene family

Spiders produce multiple silks with different physical properties that allow them to occupy a diverse range of ecological niches, including the underwater environment. Despite this functional diversity, past molecular analyses show a high degree of amino acid sequence similarity between C-terminal regions of silk genes that appear to be independent of the physical properties of the resulting silks; instead, this domain is crucial to the formation of silk fibres. Here we present an analysis of the C-terminal domain of all known types of spider silk and include silk sequences from the spider Argyroneta aquatica, which spins the majority of its silk underwater. Our work indicates that spiders have retained a highly conserved mechanism of silk assembly, despite the extraordinary diversification of species, silk types and applications of silk over 350 million years. Sequence analysis of the silk C-terminal domain across the entire gene family shows the conservation of two uncommon amino acids that are implicated in the formation of a salt bridge, a functional bond essential to protein assembly. This conservation extends to the novel sequences isolated from A. aquatica. This finding is relevant to research regarding the artificial synthesis of spider silk, suggesting that synthesis of all silk types will be possible using a single process

Temporal Decisions: Leveraging Temporal Correlation for Efficient Decisions in Early Exit Neural Networks

Deep Learning is becoming increasingly relevant in Embedded and Internet-of-things applications. However, deploying models on embedded devices poses a challenge due to their resource limitations. This can impact the model's inference accuracy and latency. One potential solution are Early Exit Neural Networks, which adjust model depth dynamically through additional classifiers attached between their hidden layers. However, the real-time termination decision mechanism is critical for the system's efficiency, latency, and sustained accuracy. This paper introduces Difference Detection and Temporal Patience as decision mechanisms for Early Exit Neural Networks. They leverage the temporal correlation present in sensor data streams to efficiently terminate the inference. We evaluate their effectiveness in health monitoring, image classification, and wake-word detection tasks. Our novel contributions were able to reduce the computational footprint compared to established decision mechanisms significantly while maintaining higher accuracy scores. We achieved a reduction of mean operations per inference by up to 80% while maintaining accuracy levels within 5% of the original model. These findings highlight the importance of considering temporal correlation in sensor data to improve the termination decision

Efficient Post-Training Augmentation for Adaptive Inference in Heterogeneous and Distributed IoT Environments

Early Exit Neural Networks (EENNs) present a solution to enhance the efficiency of neural network deployments. However, creating EENNs is challenging and requires specialized domain knowledge, due to the large amount of additional design choices. To address this issue, we propose an automated augmentation flow that focuses on converting an existing model into an EENN. It performs all required design decisions for the deployment to heterogeneous or distributed hardware targets: Our framework constructs the EENN architecture, maps its subgraphs to the hardware targets, and configures its decision mechanism. To the best of our knowledge, it is the first framework that is able to perform all of these steps. We evaluated our approach on a collection of Internet-of-Things and standard image classification use cases. For a speech command detection task, our solution was able to reduce the mean operations per inference by 59.67%. For an ECG classification task, it was able to terminate all samples early, reducing the mean inference energy by 74.9% and computations by 78.3%. On CIFAR-10, our solution was able to achieve up to a 58.75% reduction in computations. The search on a ResNet-152 base model for CIFAR-10 took less than nine hours on a laptop CPU. Our proposed approach enables the creation of EENN optimized for IoT environments and can reduce the inference cost of Deep Learning applications on embedded and fog platforms, while also significantly reducing the search cost - making it more accessible for scientists and engineers in industry and research. The low search cost improves the accessibility of EENNs, with the potential to improve the efficiency of neural networks in a wide range of practical applications

Biological responses to spider silk - antibiotic fusion protein

The development of a new generation of multifunctional biomaterials is a continual goal for the field of materials science. The in vivo functional behaviour of a new fusion protein that combines the mechanical properties of spider silk with the antimicrobial properties of hepcidin was addressed in this study. This new chimeric protein, termed 6mer + hepcidin, fuses spider dragline consensus sequences (6mer) and the antimicrobial peptide hepcidin, as we have recently described, with retention of bactericidal activity and low cytotoxicity. In the present study, mouse subcutaneous implants were studied to access the in vivo biological response to 6mer + hepcidin, which were compared with controls of silk alone (6mer), polylactic–glycolic acid (PLGA) films and empty defects. Along with visual observations, flow cytometry and histology analyses were used to determine the number and type of inflammatory cells at the implantation site. The results show a mild to low inflammatory reaction to the implanted materials and no apparent differences between the 6mer + hepcidin films and the other experimental controls, demonstrating that the new fusion protein has good in vivo biocompatibility, while maintaining antibiotic function.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/28603/2006, Chimera project (No. PTDC/EBB-EBI/109093/2008), Proteo-Light (No. PTDC/FIS/68517/2006), NIH (Grant No. P41 EB002520) Tissue Engineering Resource Center; and the NIH (Grant Nos EB003210 and DE017207).Tissue Engineering Resource Center - Bolsa No. P41 EB002520, bolsa Nos EB003210 and DE017207European - Projeto EXPERTISSUES (No. NMP3- CT-2004-500283

Mapping Nanostructural Variations in Silk by Secondary Electron Hyperspectral Imaging

Nanostructures underpin the excellent properties of silk. Although the bulk nanocomposition of silks has been well studied, direct evidence of the spatial variation of nanocrystalline (ordered) and amorphous (disordered) structures has remained elusive. Here we demonstrate that secondary electron hyperspectral imaging, can be exploited for direct imaging of hierarchical structures in carbon based materials which cannot be revealed by any other standard characterization methods. Applying this technique to silks from domesticated (Bombyx mori) and wild (Antheraea mylitta) silkworms, we report a variety of previously unseen features which highlight the local interplay between ordered and disordered structures. We conclude that our technique is able to differentiate composition on the nanoscale and enables in-depth studies into the relationship between morphology and performance of these complex biopolymer systems

Fiber optical dose rate measurement based on the luminescence of beryllium oxide

This work presents a fiber optical dose rate measurement system based on the radioluminescence and optically stimulated luminescence of beryllium oxide. The system consists of a small, radiation sensitive probe which is coupled to a light detection unit with a long and flexible light guide. Exposing the beryllium oxide probe to ionizing radiation results in the emission of light with an intensity which is proportional to the dose rate. Additionally, optically stimulated luminescence can be used to obtain dose and dose rate information during irradiation or retrospectively. The system is capable of real time dose rate measurements in fields of high dose rates and dose rate gradients and in complex, narrow geometries. This enables the application for radiation protection measurements as well as for quality control in radiotherapy. One inherent drawback of fiber optical dosimetry systems is the generation of Cherenkov radiation and luminescence in the light guide itself when it is exposed to ionizing radiation. This so called “stem” effect leads to an additional signal which introduces a deviation in the dose rate measurement and reduces the spatial resolution of the system, hence it has to be removed. The current system uses temporal discrimination of the effect for radioluminescence measurements in pulsed radiation fields and modulated optically stimulated luminescence for continuous irradiation conditions. This work gives an overview of the major results and discusses new-found obstacles of the applied methods of stem discrimination.</jats:p

Composition and Hierarchical Organisation of a Spider Silk

Albeit silks are fairly well understood on a molecular level, their hierarchical organisation and the full complexity of constituents in the spun fibre remain poorly defined. Here we link morphological defined structural elements in dragline silk of Nephila clavipes to their biochemical composition and physicochemical properties. Five layers of different make-ups could be distinguished. Of these only the two core layers contained the known silk proteins, but all can vitally contribute to the mechanical performance or properties of the silk fibre. Understanding the composite nature of silk and its supra-molecular organisation will open avenues in the production of high performance fibres based on artificially spun silk material