Microstructure of the silk fibroin-based hydrogel scaffolds derived from the orb-web spider Trichonephila clavata.

Due to the unique properties of the silk fibroin (SF) made from silkworm, SF-based hydrogels have recently received significant attention for various biomedical applications. However, research on the SF-based hydrogels isolated from spider silks has been rtricted due to the limited collection and preparation of naïve silk materials. Therefore, this study focused on the microstructural characteristics of hydrogel scaffolds derived from two types of woven silk glands: the major ampullate gland (MAG) and the tubuliform gland (TG), in the orb-web spider Trichonephila clavate. We compared these spider glands with those of the silk fibroin (SF) hydrogel scaffold extracted from the cocoon of the insect silkworm Bombyx mori. Our FESEM analysis revealed that the SF hydrogel has high porosity, translucency, and a loose upper structure, with attached SF fibers providing stability. The MAG hydrogel displayed even higher porosity, as well as elongated fibrous structures, and improved mechanical properties: while the TG hydrogel showed increased porosity, ridge-like or wall-like structures, and stable biocapacity formed by physical crosslinking. Due to their powerful and versatile microstructural characteristics, the MAG and TG hydrogels can become tailored substrates, very effective for tissue engineering and regenerative medicine applications.

Fine structural characteristics of the chorionic microspheres on the egg surface of the orb web spider Trichonephila clavata.

The eggs laid by the orb web spider Trichonephila clavata must overwinter in bitterly freezing and dry conditions before hatching, but there does not seem to be any protection like a compact silk case covering the entire eggmass. Instead, the surface of the eggmass is completely coated with a milky coating called chorionic microspheres (CM). Therefore, we investigated the fine structural characteristics of CM to demonstrate their ecological importance. Although the diameter of CM in outer eggmass exhibits a significant variation, the chorionic surface is coated with a single layer of CM, characterized by a consistent diameter of approximately 2.3 µm. The surface structure of aggregated CM shows short papillary projections demonstrating segmental adhesion of mucous components. CM is insoluble in water but partially soluble in anhydrous ethanol, and its spherical structure is completely decomposed by hexafluoroisopropanol (HFIP), a strong organic solvent. Since our fine structural observations clearly show that CM is not derived from vitellogenic or choriogenetic processes, the CM adhesive coatings during ovipositional process appears to be equivalent to cocoon silk for various protective functions in silken eggcase.

Fine structural aspects on the web glue production in the golden orb-web spider Trichonephila clavata.

The water-soluble glue substance of the capture threads in Trichonephila clavata is solely produced from two pairs of aggregate silk glands. During the web glue production, secretory vesicles were synthesized via the extensive rough endoplasmic reticulum of epithelial cells. Unlike the clearly described fibrous web production in spiders, the process of aqueous web glue production appears to involve either a condensing or a packaging step by the Golgi complex. In particular, the fine structure of secretory vesicles varies from cell to cell and may represent the secretory cycle. The electron-dense multivesicular bodies were clearly visible as discrete droplets, and the mature secretory product in the glandular epithelium appeared as a spherical vacuole grown by fusion with surrounding small vesicles. Our fine structural observation reveals that the secretion occurs when the release of secreted material involves the loss of part of the cytoplasm. The bleb along the luminal surface of the secretory cells and membrane-bound extracellular vesicles which pinched off from the cell suggests that the secretory product is released by the mechanism of apocrine secretion.

Capture silk scaffold production in the cribellar web spider.

Spider capture silk is a natural scaffolding material that outperforms most synthetic materials in terms of its combination of strength and elasticity. Among the various kinds of silk threads, cribellar thread is the most primitive prey-capturing type of spider web material. We analyzed the functional organization of the sieve-like cribellum spigots and specialized calamistral comb bristles for capture thread production by the titanoecid spider Nurscia albofasciata. The outer cribellar surface is covered with thousands of tiny spigots, and the cribellar plate produces non-sticky threads composed of thousands of fine nanofibers. N. albofasciata cribellar spigots are typically about 10 µm long, and each spigot appears as a long individual shaft with a pagoda-like tiered tip. The five distinct segments comprising each spigot is a defining characteristic of this spider. This segmented and flexible structure not only allows for spigots to bend individually and join with adjacent spigots, but it also enables spigots to draw the silk fibrils from their cribella with rows of calamistral leg bristles to form cribellar prey-capture threads.

Molt-related changes in the major ampullate silk gland of the barn spider Araneus cavaticus.

Spiders molt periodically before reaching full maturity, but several spiders continue to molt after sexual maturity. This post-maturity molting (PMM) behavior has been observed in the barn spider Araneus cavaticus (Araneae: Araneidae) among the orb-web spiders. In this study, we investigated molt-related changes in the ampulla and tail regions of the major ampullate gland during the PMM sequences (intermolt, pre-molt, ecdysis, and post-molt). The results showed that all gland units consist of a monolayer of epithelial cells surrounding a large central lumen, and two types of secretory granules (Type-M and Type-S). During the molting period, most cells showed fine structural modification in their organelles, and conspicuous tissue swelling was detected at the glandular epithelium. Following the molting cycle, the amount of Type-M granules continues to increase in the cell with a corresponding swelling, but Type-S granules gradually disappeared during the process of ecdysis. This suggests that the molt-related changes in spider silk production originates from the periodic production of Type-S secretory granules in the ampulla region. As Type-M granules flow toward the funnel, it is coated with viscous liquid secretion of Type-S granules in order to produce dragline silk fibers. We provide fine structural evidence for Type-S granules of hexagonal crystalline substructures representing glycoprotein substances to maintain high level of water content.

Fine structure of the cardiac muscle cells in the orb-web spider Nephila clavata.

The fine structural characteristics of cardiac muscle cells and its myofibril organization in the orb web spider N. clavata were examined by transmission electron microscopy. Although myofibril striations are not remarkable as those of skeletal muscles, muscle fibers contain multiple myofibrils, abundant mitochondria, extensive sarcoplasmic reticulum and transverse tubules (T-tubules). Myofibrils are divided into distinct sarcomeres defined by Z-lines with average length of 2.0 µm, but the distinction between the A-band and the I-bands is not clear due to uniform striations over the length of the sarcomeres. Dyadic junction which consisted of a single T-tubule paired with a terminal cisterna of the sarcoplasmic reticulum is found mainly at the A-I level of sarcomere. Each cell is arranged to form multiple connections with neighboring cells through the intercalated discs. These specialized junctions include three types of intercellular junctions: gap junctions, fascia adherens and desmosomes for heart function. Our transmission electron microscopy (TEM) observations clearly show that spider's cardiac muscle contraction is controlled by neurogenic rather than myogenic mechanism since each cardiac muscle fiber is innervated by a branch of motor neuron through neuromuscular junctions.

Fine structure of the silk spinning system in the caddisworm, Hydatophylax nigrovittatus (Trichoptera: Limnephilidae).

Silk is produced by a variety of insects, but only silk made by terrestrial arthropods has been examined in detail. To fill the gap, this study was designed to understand the silk spinning system of aquatic insect. The larvae of caddis flies, Hydatophylax nigrovittatus produce silk through a pair of labial silk glands and use raw silk to protect themselves in the aquatic environment. The result of this study clearly shows that although silk fibers are made under aquatic conditions, the cellular silk production system is quite similar to that of terrestrial arthropods. Typically, silk production in caddisworm has been achieved by two independent processes in the silk glands. This includes the synthesis of silk fibroin in the posterior region, the production of adhesive glycoproteins in the anterior region, which are ultimately accumulated into functional silk dope and converted to a silk ribbon coated with gluey substances. At the cellular level, each substance of fibroin and glycoprotein is specifically synthesized at different locations, and then transported from the rough ER to the Golgi apparatus as transport vesicles, respectively. Thereafter, the secretory vesicles gradually increase in size by vesicular fusion, forming larger secretory granules containing specific proteins. It was found that these granules eventually migrate to the apical membrane and are exocytosed into the lumen by a mechanism of merocrine secretion.

Fine structure of the intercalated disc and cardiac junctions in the black widow spider Latrodectus mactans.

Arthropods have an open circulatory system with a simple tubular heart, so it has been estimated that the contractile pumping structure of the cardiac muscle will be less efficient than that of vertebrates. Nevertheless, certain arthropods are known to have far superior properties and characteristics than vertebrates, so we investigated the fine structural features of intercalated discs and cardiac junctions of cardiac muscle cells in the black widow spider Latrodectus mactans. Characteristically, the spider cardiac muscle has typical striated features and represents a functional syncytium that supports multiple connections to adjacent cells by intercalated discs. Histologically, the boundary lamina of each sarcolemma connects to the basement membrane to form an elastic sheath, and the extracellular matrix allows the cells to be anchored to other tissues. Since the intercalated disc is also part of sarcolemma, it contains gap junctions for depolarization and desmosomes that keep the fibers together during cardiac muscle contraction. Furthermore, fascia adherens and macula adherens (desmosomes) were also identified as cell junctions in both sarcolemma and intercalated discs. To enable the coordinated heartbeat of the cardiac muscle, the muscle fibers have neuronal innervations by multiple axons from the motor ganglion.

Fine structure of the aggregate silk nodules in the orb-web spider Nephila clavata.

Among the triad spinning units for capture thread producing system in the orb-web spiders, aqueous gluey substances are produced from two pairs of aggregate silk glands (ASG). Although biochemical analysis of glue substance is produced by way of their passage through the ASG, its structural modifications in the nodular area have been nearly neglected till now. This paper focused on identifying the fine structural characteristics of the aggregate nodules in the golden orb-web spider Nephila clavata using both of light and transmission electron microscopes. The ASG in N. clavata is composed of a multi-lobed secretory region and a thick excretory duct surrounded by large irregular nodules. Histological analysis of the nodules demonstrates that the nodule forming cells have extensive membrane-bound tubular system that is continuous with the surface membrane. In particular, the nodule forming cells contain numerous mitochondria and glycogen particles within their cytoplasms, and they are surrounded by the same sheath of thin connectives. As previously described, each gluey droplet is formed of a central glycoprotein mass surrounded by an aqueous covering components, the nodular organization in N. clavata indicates that the extensive membrane system is thought to have a function for gluey silk production in spider. The results of this study also strengthen the premise that spider glues are made of glycoproteins, and the aggregate nodule functions as a key component for the spider web glue production.

Roles of 5-HT on phase transition of neurite outgrowth in the identified serotoninergic neuron C1, Helisoma trivolvis.

Neurite outgrowth is a morphological marker of neuronal differentiation and neuroregeneration, and the process includes four essential phases, namely initiation, elongation, guidance and cessation. Intrinsic and extrinsic signaling molecules seem to involve morphological changes of neurite outgrowth via various cellular signaling cascades phase transition. Although mechanisms associated with neurite outgrowth have been studied extensively, little is known about how phase transition is regulated during neurite outgrowth. 5-HT has long been studied with regard to its relationship to neurite outgrowth in invertebrate and vertebrate culture systems, and many studies have suggested 5-HT inhibits neurite elongation and growth cone motility, in particular, at the growing parts of neurite such as growth cones and filopodia. However, the underlying mechanisms need to be investigated. In this study, we investigated roles of 5-HT on neurite outgrowth using single serotonergic neurons C1 isolated from Helisoma trivolvis. We observed that 5-HT delayed phase transitions from initiation to elongation of neurite outgrowth. This study for the first time demonstrated that 5-HT has a critical role in phase-controlling mechanisms of neurite outgrowth in neuronal cell cultures.

Scoring model to predict massive post-partum bleeding in pregnancies with placenta previa: A retrospective cohort study.

AIM: We aimed to identify factors associated with massive post-partum bleeding in pregnancies with placenta previa and to establish a scoring model to predict post-partum severe bleeding. METHODS: A retrospective cohort study was performed in 506 healthy singleton pregnancies with placenta previa from 2006 to 2016. Cases with intraoperative blood loss (≥2000 mL), packed red blood cells transfusion (≥4), uterine artery embolization, or hysterectomy were defined as massive bleeding. After performing multivariable analysis, using the adjusted odds ratios (aOR), we formulated a scoring model. RESULTS: Seventy-three women experienced massive post-partum bleeding (14.4%). After multivariable analysis, seven variables were associated with massive bleeding: maternal old age (≥35 years; aOR 1.79, 95% confidence interval [CI] 1.00-3.20, P = 0.049), antepartum bleeding (aOR 4.76, 95%CI 2.01-11.02, P < 0.001), non-cephalic presentation (aOR 3.41, 95%CI 1.40-8.30, P = 0.007), complete placenta previa (aOR 1.93, 95%CI 1.05-3.54, P = 0.034), anterior placenta (aOR 2.74, 95%CI 1.54-4.89, P = 0.001), multiple lacunae (≥4; aOR 2.77, 95%CI 1.54-4.99, P = 0.001), and uteroplacental hypervascularity (aOR 4.51, 95%CI 2.30-8.83, P < 0.001). We formulated a scoring model including maternal old age (<35: 0, ≥35: 1), antepartum bleeding (no: 0, yes: 2), fetal non-cephalic presentation (no: 0, yes: 2), placenta previa type (incomplete: 0, complete: 1), placenta location (posterior: 0, anterior: 1), uteroplacental hypervascularity (no: 0, yes: 2), and multiple lacunae (no: 0, yes: 1) to predict post-partum massive bleeding. According to our scoring model, a score of 5/10 had a sensitivity of 81% and a specificity of 77% for predicting massive post-partum bleeding. The area under the receiver-operator curve was 0.856 (P < 0.001). The negative predictive value was 95.9%. CONCLUSION: Our scoring model might provide useful information for prediction of massive post-partum bleeding in pregnancies with placenta previa.

The Effect of Raffaelea quercus-mongolicae Inoculations on the Formation of Non-conductive Sapwood of Quercus mongolica.

In Korea, mass mortality of Quercus mongolica trees has become obvious since 2004. Raffaelea quercus-mongolicae is believed to be a causal fungus contributing the mortality. To evaluate the pathogenicity of the fungus to the trees, the fungus was multiple- and single-inoculated to the seedlings and twigs of the mature trees, respectively. In both the inoculations, the fungus was reisolated from more than 50% of inoculated twigs and seedlings. In the single inoculations, proportions of the transverse area of non-conductive sapwood at inoculation points and vertical lengths of discoloration expanded from the points were significantly different between the inoculation treatment and the control. In the multiple inoculations, no mortality was confirmed among the seedlings examined. These results showed that R. quercus-mongolicae can colonize sapwood, contribute to sapwood discoloration and disrupt sap flows around inoculation sites of Q. mongolica, although the pathogenicity of the fungus was not proven.

The effect of static magnetic fields on the aggregation and cytotoxicity of magnetic nanoparticles.

Biomedical applications of magnetic nanoparticles (MNP), including superparamagnetic nanoparticles, have expanded dramatically in recent years. Systematic and standardized cytotoxicity assessment to ensure the biosafety and biocompatibility of those applications is compulsory. We investigated whether exposure to static magnetic field (SMF) from e.g. magnetic resonance imaging (MRI) could affect the cytotoxicity of superparamagnetic iron oxide (SPIO) nanoparticles using mouse hepatocytes and ferucarbotran, a liver-selective MRI contrast agent as a model system. We show that while the SPIO satisfied the conventional cytotoxicity assessment, clinical doses combined with SMF exposure exerts synergistic adverse effects such as reduced cell viability, apoptosis, and cell cycle aberrations on hepatocytes in vitro and in vivo. Concomitant treatments with the SPIO and SMF generated SPIO aggregates, which demonstrated enhanced cellular uptake, was sufficient to induce the cytotoxicity without further SMF, emphasizing that the SPIO aggregates were the predominant source of the cytotoxicity. Interestingly, the apoptotic effect was dependent on levels of reactive oxygen species (ROS) and SPIO uptake while the reduced cell viability was independent of these factors. Moreover, long-term monitoring showed a significant increase in multinuclear giant cells in the cells concomitantly treated with the SPIO and SMF compared with the control. The results demonstrate that the SPIO produces unidentified cytotoxicity on liver in the presence of SMF and the SPIO aggregates predominantly exert the effect. Since aggregation of MNP in biological milieu in the presence of strong SMF is inevitable, a fundamentally different approach to surface fabrication is essential to increase the biocompatibility of MNP.

Relation between the outer cover of the egg case of Argiope aurantia (Araneae: Araneidae) and the emergence of its spiderlings.

To emerge from the egg case, Argiope aurantia spiderlings must penetrate a tightly woven outer cover composed primarily of large-diameter cylindrical gland fibers and small-diameter fibers, likely of aciniform gland origin. They accomplish this using enzymatic digestion and mastication to form a communal hole in the outer cover. The involvement of proteolytic enzymes in this process was demonstrated by zymography of spiderling homogenates and washes made from the edges of holes. The specific source(s) of the proteases is unknown, but histological examination of spiderling sections indicates that the digestive tract, venom glands, and gnathocoxal glands are all functioning at the time of emergence from the egg case. Observations on edges of holes indicate that spiderlings are able to solubilize the small-diameter fibers completely, but cylindrical gland fibers only partially. In the outer cover, cylindrical fibers are composed of numerous fibrils embedded within a matrix. Spiderlings appear to be unable to solubilize the fibrils, but digestion of the matrix allows the spiderlings to push the fibrils aside to create the opening.