Structural and functional correlates of synaptic transmission in the vertebrate neuromuscular junction

Because vertebrate neuromuscular junctions are readily accessible for experimental manipulation, they have provided a superb model in which to examine and test functional correlates of chemical synaptic transmission. In the neuromuscular synapse, acetylcholine receptors have been localized to the crests of the junctional folds and visualized by a variety of ultrastructural techniques. By using ultrarapid freezing techniques with a temporal resolution of less than 1 msec, quantal transmitter release has been correlated with synaptic vesicle exocytosis at discrete sites called “active zones.” Mechanisms for synaptic vesicle membrane retrieval and recycling have been identified by using immunological approaches and correlated with endocytosis via coated pits and coated vesicles. In this review, available ultrastructural, physiological, immunological, and biochemical data have been used to construct an ultrastructural model of neuromuscular synaptic transmission that correlates structure and function at the molecular level.     

Cytoskeletal architecture of neuromuscular junction: Localization of vinculin

Cytoskeletons underneath the postsynaptic membrane of neuromuscular junctions were studied by using a quick-freeze deep-etched method and immunoelectron microscopy of ultrathin frozen sections. In a quick-freeze deep-etched replica of fresh, unfixed muscles, 8.9 ± 1.5-nm particles were present on the true postsynaptic membrane surface. Underneath this receptor-rich postsynaptic membrane, networks of fine filaments were observed. These cytoskeletal networks were more clearly observed in extracted samples. In these samples, diameters of the filaments which formed networks were measured. In the platinum replica, three kinds of filament were recognized—12 nm, 9 nm, and 7 nm in diameter. The 12-nm filament seemed to correspond to the intermediate filament. The other two filaments formed meshworks between intermediate filaments and plasma membrane. In ultrathin frozen sections vinculin label was localized just beneath the plasma membrane. Thirty-six percent of the label was within 18 nm from the cytoplasmic side of the plasma membrane and 50% was within 30 nm. Taking the size of the vinculin molecule into account, it was concluded that vinculin is localized just beneath the plasma membrane and might play some role in anchoring filaments which formed meshworks underneath the plasma membrane.     

Rodent nerve-muscle cell culture system for studies of neuromuscular junction development: refinements and applications

Understanding of vertebrate neuromuscular junction (NMJ) development has been advanced by experimentation with cultures of dissociated embryonic nerve and skeletal muscle cells, particularly those derived from Xenopus and chick embryos. We previously developed a rodent (rat) nerve-muscle coculture system that is characterized by extensive induction of acetylcholine receptor (AChR) aggregation at sites of axonal contact with myotubes (Dutton et al., 1995). In this article, we report modifications of this culture system and examples of its application to the study of NMJ development: (1) We describe improved methods for the enrichment of myoblasts to give higher yields of myotubes with equal or greater purity. (2) We demonstrate lipophilic dye labeling of axons in cocultures by injection of dye into neuron aggregates and show the feasibility of studying the growth of living axons on myotubes during synapse formation. (3) We describe the preparation of a better-defined coculture system containing myotubes with purified rat motoneurons and characterize the system with respect to axon-induced AChR aggregation. (4) We demonstrate dependence of the pattern of axon-induced AChR aggregation on muscle cell species, by the use of chick-rat chimeric co-cultures. (5) We provide evidence for the role of alternatively-spliced agrin isoforms in synapse formation by using single cell RT-PCR with neurons collected from co-cultures after observation of axon-induced AChR aggregation. Microsc. Res. Tech. 49:26-37, 2000. Published 2000 Wiley-Liss, Inc.     

Application of the Mesolens for subcellular resolution imaging of intact larval and whole adult Drosophila

In a previous paper, we showed a new giant lens called the Mesolens and presented performance data and images from whole fixed and intact fluorescently‐stained 12.5‐day old mouse embryos. Here, we show that using the Mesolens we can image an entire Drosophila larva or adult fly in confocal epifluorescence and show subcellular detail in all tissues. By taking several hundreds of optical sections through the entire volume of the specimen, we show cells and nuclear details within the gut, brain, salivary glands and reproductive system that normally require dissection for study. Organs are imaged in situ in correct 3D arrangement. Imaginal discs are imaged in mature larvae and it proved possible to image pachytene chromosomes in cells within ovarian follicles in intact female flies. Methods for fixing, staining and clearing are given.     

Proteins in vacuo: denaturing and folding mechanisms studied with computer-simulated molecular dynamics

Mounting evidence from experiments suggests that the native fold in solution is metastable in dehydrated proteins. Results from a number of experiments that use mass spectrometry indicate also that folding-unfolding transitions take place in protein ions even in the absence of water. These observations on anhydrous proteins call for a re-evaluation of our understanding of the folding transition. In this context, computer-assisted simulations are an important complementary tool. Here, we provide an overview of recent progress on the simulation of proteins in vacuo. In particular, we discuss the response of proteins and protein ions to perturbations that trigger unfolding and re-folding transitions. By comparing the general patterns emerging from theory and experiment, we propose a series of new measurements that could help to validate, and improve, current simulation models.     

SARS-CoV-2 induced myocarditis: Current knowledge about its molecular and pathophysiological mechanisms

The existence of an inflammatory process in the heart muscle, related to a progressive worsening of myocardialfunction, different etiopathogenetic mechanisms concur and often overlap, thus making the diagnosis and the therapeuticapproach complex. As the COVID-19 pandemic progresses, the effects of the disease on the organ systems and inparticular on the cardiovascular system are becoming more and more profound. Cardiac involvement is a well-knownevent with a high percentage of findings in the heart’s magnetic field, even in asymptomatic areas. There arenumerous uncertainties regarding their evolution, in the long and short term, due not only to a difficult to determinethe varied clinical expression and the rarely performed intramyocardial biopsy which additionally presents diagnosticproblems but also in part to different clinical prognosis. Today, the new SARS-CoV-2 virus that uses the angiotensinconverting enzyme 2 (ACE2) which is present at high levels in myocardial cells as its entrance it can create evensevere heart injury. The pathophysiology in all of these cases can involve multiple immune and non-immunemechanisms within organs and vessels and can be occur in the clinical phases. Possible mechanisms of direct andindirect myocardial infarction in patients with COVID-19 include additional lesion and oxygen-rich and generalizedinflammation response with myocardial immune hyperactivity (myocarditis). Therefore, these can occur through theexcessive release of cytokines, the presence of thrombocytopenia, endocrine damage, heart failure, arrhythmias andmore. Patients can show average signs of myocardial damage, and some develop spontaneous cardiac complications,such as heart failure, arrhythmias and, rarely, rare cardiogenic disorders. Pathophysiology in all of these may involvemultiple mechanisms within the cytokine cephalic membrane, endocrine damage and thrombogenicity. The diagnosisof this myocardial injuri is mainly based on the myocardial enzyme troponin. This viewpoint paper explains today’sknowledge on viral myocarditis, in particular that from SARS-CoV-2 infection, if there is a connection with otherpossible biomolecular pathogenetic factors that can influence its natural course. In fact, it is for this reason that thepathogenetic mechanisms are analyzed and described. At the same time, its possible interaction with other parametersthat are documented risk factors for cardiovascular disease was examined. Although these biomolecular findings weremainly related to necrotic parts of the myocardium, it is important to recognize that myocardial damage early for abetter approach and prognosis.     

柔性构件和柔性机构自由度分析

柔性机构由刚性构件、刚性铰链、柔性构件和柔性铰链4个元素组成,4个元素的不同组合可以得到11种不同类型的柔性机构.文中分析了其中5种常用的结构形式与特征.柔性构件的自由度及其变形规律在机构功能中起重要作用,它们是几何特性、材料特性和载荷特性的函数.在分析柔性构件自由度的基础上,给出了柔性机构的自由度计算公式,并用实例进行了验算.     

A protocol for processing the delicate larval and prepupal salivary glands of Drosophila for scanning electron microscopy

Although scanning electron microscopy (SEM) has been broadly used for the examination of fixed whole insects or their hard exoskeleton‐derived structures, including model organisms such as Drosophila, the routine use of SEM to evaluate vulnerable soft internal organs and tissues was often hampered by their fragile nature and frequent surface contamination. Here, we describe a simple four‐step protocol that allows for the reliable and reproducible preparation of the larval and prepupal salivary glands (SGs) of Drosophila for SEM devoid of any surface contamination. The steps are to: first, proteolytically digest the adhering fat body; second, use detergent washes to remove contaminating coarse tissue fragments, including sticky remnants of the fat body; third, use nonionic emulsifying polysorbate emulsifiers to remove fine contaminants from the SGs surface; and fourth, use aminopolycarboxylate‐based chelating agents to detach sessile hemocytes. Short but repeated rinses in 100 μL of a saline‐based buffer between steps ensure efficient removal of remnants removed by each treatment. After these steps, the SGs are fixed in glutaraldehyde, postfixed in osmium tetroxide, dehydrated, critically point‐dried, mounted on aluminum stubs, sputter coated with gold–palladium alloy and examined in the SEM.     

Concrete plasticity: Macro- and microapproaches

The development and the present state of some key aspects of the theory of concrete plasticity are summarized. Emphasis is placed on the representation and interpretation of the inelastic deformation of concrete materials in the postelastic range. The first part of the paper discusses the development of plasticity-based models in the prepeak stress range. A general formulation of softening behavior from the plasticity theory combined with the fracturing (damage) theory is then described. The difficulties of characterizing concrete deformations in the postpeak stress range led to an extensive study of the relationship between macrospace features and microscopic events in recent years. This is briefly outlined, and some key aspects of the development on strain softening and strain localization are reviewed. Against the background of this information, attempts are then made to develop an elastic-plastic-damage applicative model. Special emphasis is placed here on the practicality of the unified model combining the classical theory of plasticity with the modern theory of continuum damage mechanics. Some thoughts on possible numerical algorithms and program development for this type of model are also discussed. The role of cement-paste-sand interfaces in providing a deeper understanding of the behavior of concrete materials at the microscale are explored. Directions for further research are indicated.     

Anatomical and molecular design of the Drosophila antenna as a flagellar auditory organ

The molecular basis of hearing is less well understood than many other senses. However, recent studies in Drosophila have provided some important steps towards a molecular understanding of hearing. In this report, we summarize these findings and their implications on the relationship between hearing and touch. In Drosophila, hearing is accomplished by Johnston's Organ, a chordotonal organ containing over 150 scolopidia within the second antennal segment. We will discuss anatomical features of the antenna and how they contribute to the function of this flagellar auditory receptor. The effects of several mutants, identified through mutagenesis screens or as homologues of vertebrate auditory genes, will be summarized. Based on evidence gathered from these studies, we propose a speculative model for how the chordotonal organ might function.     

Machined surfaces: Final texture and underlying structure

It is commonly assumed that the surface texture after manufacture can be specified by the finishing cut which is taken. This cut often uses a relatively inefficient process such as grinding and so there is some motivation to minimize costs by making it as shallow as possible. If this happens it becomes increasingly likely that at least one of several plausible mechanisms whereby features of the previous process texture are reproduced in the final surface will become active.In this paper the nature and importance of any interactions which may be occurring are considered; we concentrate on the combination of an essentially periodic primary process and a random finish process. A preliminary investigation involving mainly spectral analysis techniques on milled and ground surfaces has given some evidence that such interaction does occur.     

Cellular and molecular mechanisms leading to cortical reaction and polyspermy block in mammalian eggs

Following fusion of sperm and egg, the contents of cortical granules (CG), a kind of special organelle in the egg, release into the perivitelline space (cortical reaction), causing the zona pellucida to become refractory to sperm binding and penetration (zona reaction). Accumulating evidence demonstrates that mammalian cortical reaction is probably mediated by activation of the inositol phosphate (PIP(2)) cascade. The sperm-egg fusion, mediated by GTP-binding protein (G-protein), may elicit the generation of two second messengers, inositol 1,4,5 triphosphate (IP(3)) and diacylglycerol (DAG). The former induces Ca(2+) release from intracellular stores and the latter activates protein kinase C (PKC), leading to CG exocytosis. Calmodulin-dependent kinase II (CaMKII) may act as a switch in the transduction of the calcium signal. The CG exudates cause zona sperm receptor modification and zona hardening, and thus block polyspermic penetration. Oolemma modification after sperm-egg fusion and formation of CG envelope following cortical reaction also contribute to polyspermy block.     

Cathodoluminescence and transmission electron microscopy of an abrupt lateral junction in algaas grown by molecular beam epitaxy

We present evidence for a novel type of junction, lateral to the growth direction, in AlGaAs grown by molecular beam epitaxy. Cathodoluminescence data show an abrupt change in the bandgap of AlGaAs grown in grooves on a GaAs substrate at the boundary between adjacent facets. Transmission electron microscopy of a cross section of the groove reveals an unexpected quasiperiodic compositional modulation on the (111) facets of the grooves. The transition in the bandgap occurs as a result of abrupt termination of the compositional modulation at the facet boundaries.     

Network pharmacology and molecular docking identify mechanisms of medicinal plant-derived 1,2,3,4,6-penta-O-galloyl-beta-D-glucose treating gastric cancer

Background: 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG) is a natural polyphenolic compound derived from multiple medicinal plants with favorable anticancer activity. Methods: In this study, the mechanisms of PGG against gastric cancer were explored through network pharmacology and molecular docking. First, the targets of PGG were searched in the Herbal Ingredients’ Targets (HIT), Similarity Ensemble Approach (SEA), and Super-PRED databases. The potential targets related to gastric cancer were predicted from the Human Gene Database (GeneCards) and DisGeNET databases. The intersecting targets of PGG and gastric cancer were obtained by Venn diagram and then subjected to protein-protein interaction analysis to screen hub targets. Functional and pathway enrichment of hub targets were analyzed through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway databases. The differential expression and survival analysis of hub targets in gastric cancer were performed based on The Cancer Genome Atlas database. Finally, the affinity of PGG with hub targets was visualized by molecular docking. Results: Three hub targets were screened, including mitogen-activated protein kinase 14 (MAPK14), BCL2 like 1 (BCL2L1), and vascular endothelial growth factor A (VEGFA). MAPK14 had a higher expression, while BCL2L1 and VEGFA had lower expression in gastric cancer than in normal conditions. Enrichment analysis indicated enrichment of these hub targets in MAPK, neurotrophin, programmed death-ligand 1 (PD-L1) checkpoint, phosphatidylinositol 3-kinases/protein kinase B (PI3K-Akt), Ras, and hypoxia-inducible factor-1 (HIF-1) signaling pathways. Conclusion: Therefore, network pharmacology and molecular docking analyses revealed that PGG exerts a therapeutic efficacy on gastric cancer by multiple targets (MAPK14, BCL2L1, and VEGFA) and pathways (MAPK, PD-L1 checkpoint, PI3K-Akt, Ras, and HIF-1 pathways).     

Deconstructing the molecular mechanisms of cell cycle control in a mouse adrenocortical cell line: roles of ACTH

This is a progress report of an attempt to deconstruct the signaling network underlying cell cycle control in the mouse Y1 adrenocortical cell line, aiming to uncover ACTH growth regulatory pathways. Y1 adrenocortical tumor cells possess amplified and overexpressed c-Ki-ras proto-oncogene. Despite this oncogenic lesion, Y1 cells retain tight regulatory mechanisms of cell cycle control typified by the sequential events comprising the mitogenic response triggered by FGF2 in G0/G1-arrested Y1 cells: 1) activation of ERK1/2 and PI3K, by 5 minutes; 2) induction of c-Fos and c-Myc proteins by 2 hours; 3) induction of cyclin D1 protein by 5 hours; 4) phosphorylation of Rb protein between 6 and 8 hours; 5) onset of DNA synthesis by 8-9 hours. In this cell line, ACTH-receptor (ACTH-R) activates contradictory pathways of growth regulation. First, ACTH coordinately induces fos and jun gene families via activation of both ERK1/2 and cAMP/PKA pathways, resembling a mitogen. Second, ACTH-R triggers cAMP/PKA-mediated antimitogenic mechanisms comprised of Akt/PKB dephosphorylation/deactivation, c-Myc protein degradation, and p27(Kip1) protein induction. Induction of cyclin D1 depends on activation of both ERK1/2 and PI3K, but is not affected by ACTH action. As a consequence, ACTH antagonizes FGF2 mitogenic activity but ectopic expression of the c-Myc protein (via MycER fusion protein) is sufficient to abrogate this ACTH antagonistic effect over FGF2 mitogenic activity. Ectopic expression of both c-Myc and cyclin D1 is not sufficient to drive G0/G1-arrested Y1 cells into S phase, but when the sustained expression of these two proteins is complemented by ACTH treatment it promotes G1 phase progression and DNA synthesis initiation. In conclusion, ACTH-receptor lacks signaling potential sufficient to initiate a mitogenic response in Y1 adrenocortical cells and, therefore, cannot substitute for bona fide mitogens like FGF2.     

Low temperature techniques applied for CTEM and STEM analysis of cellular components at a molecular level

One of the most important problems in tissue preparation for electron microscopic analysis at a molecular level involves the preservation of the tissue without introducing extensive denaturation of the proteins. Low temperature is a most efficient condition for the inhibition of protein denaturation and freeze-drying offers favourable conditions for transferring proteins to a dry state with minimal denaturation of the proteins. However, the embedding of the dried tissue in a plastic leads to extensive denaturation of the proteins when performed in the conventional way. This eliminates very efficiently the advantages of the method. The situation becomes even worse when subjecting the tissue to freeze-substitution. To eliminate as far as possible the denaturing effect of plastic embedding, freeze-drying can be combined with low temperature embedding in a plastic. Freeze-fracturing allows a most efficient use of low temperature to reduce conformation changes in proteins. The value of the freeze-fracturing technique depends entirely on a precise knowledge of the location of the fracture planes. Since this location is not known, it must be determined on the basis of a deduction. If this deduction is wrong, the method becomes misleading. Two methods which allow a certain testing of the correctness of the deduced location of the fracture planes are mentioned.     

Exploring the mechanisms of magnolol in the treatment of periodontitis by integrating network pharmacology and molecular docking

Background: Magnolol, a bioactive extract of the Chinese herb Magnolia officinalis has a protective effect against periodontitis. This study is aimed to explore the mechanisms involved in the functioning of magnolol against periodontitis and provide a basis for further research. Methods: Network pharmacology analysis was performed based on the identification of related targets from public databases. The Protein-protein interaction (PPI) network was constructed to visualize the significance between the targets of magnolol and periodontitis. Subsequently, Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to predict the functions and the signal regulatory pathways involved in the action of magnolol against periodontitis. The “function-target-pathway” networks were constructed to analyze the core targets and pathways of magnolol against periodontitis. Molecular docking was used to verify the interaction of magnolol and core targets. Results: A total of 58 active targets of magnolol and 644 periodontitis-related targets were collected from public databases. A total of 25 targets of magnolol against periodontitis were identified based on the Venn diagram. GO analysis showed that magnolol has a role in the response to oxidative stress, nicotine, and lipopolysaccharide. KEGG enrichment analysis indicated that the mechanism of magnolol against periodontitis was mainly related to the tumor necrosis factor (TNF), phosphoinositide 3-kinase (PI3K/Akt), and mitogen-activated protein kinase (MAPK) signaling pathways. Combined with PPI network and molecular docking results, the core targets of magnolol against periodontitis included AKT1, MAPK8, MAPK14, TNF, and TP53. Conclusion: To summarize, the anti-periodontitis mechanisms of magnolol are potentially through regulating the TNF, PI3K/Akt, and MAPK signaling pathways.     

硅光微电机械系统:光学元件与传感器

1 Introduction Using Micro-Electro-Mechanical System(MEMS)technology, many devices and systems can be miniaturized. Micro pressure sensors and micro accelerometers(air-bag sensor) are examples that are widely commercialized. In the case of optical system,some optical components need to be combined to implement the function desired for optical processing.