Ultrastructural study of the salivary glands of the sugarcane spittlebug Mahanarva fimbriolata (Stal, 1854) (Euhemiptera: Cercopidae)

Spittlebugs are insects that suck sap from plants and regurgitate saliva containing toxic enzymes into the leaves. As a consequence, the conductive channels are blocked resulting in dry leaves, thus giving a burned aspect to the plantation. This work performed ultrastructural analyses of the salivary glands of the sugarcane spittlebug Mahanarva fimbriolata, since these organs produce the enzymes that are injected into the plants, thus being responsible for the economic losses in the production of sugarcane. Three kinds of secretory cells are found in principal gland, forming the lobules I-IV. The main differences among these cells relate to size, morphology and electron density of the secretory vesicles. The accessory glands contain different secretory vesicles to those in the principal gland. Muscular cells are found around the entire gland. The different secretory vesicles found in both principal and accessory glands indicate that the gland produces different substances or that the secretion in the interior of cells passes through a maturation process.     

3D reconstruction and scanning electron microscopy of salivary glands of the millipede Rhinocricus padbergi (Verhoef, 1938) (Diplopoda: Spirobolida)

The three-dimensional technique applied to the salivary gland of the millipides Rhinocricus padbergi showed that it is a single structure, dorsolaterally located to the animal gut and composed of two distinct portion: an acinar and a tubular one. The last one opening onto the oral cavity. This work shows that the salivary gland in R. padbergi, although is a single body, it displays two excretory ducts (one in each side of the anterior end of the gut) which has the function of carrying out the secretion produced toward to the oral cavity, contrary to the anterior data registered by other authors who suggested that the salivary gland in diplopod animals would be a paired structures.     

Proteolytic Cleavage of HIV-1 GFP-Vpr Fusions at Novel Sites Within Virions and Living Cells: Concerns for Intracellular Trafficking Studies

Fluorescent labelling of the highly conserved HIV-1 accessory protein Vpr (Viral Protein R) with GFP or variants thereof has proved a valuable approach to track Vpr and/or HIV-1 subcellular localisation in vivo. Our analysis in transfected mammalian cells expressing GFP-Vpr fusion protein, as well as within virus derived there from, documents site-specific proteolytic cleavage of the GFP-Vpr fusion protein. Western analysis revealed that transfected mammalian cells harbour a C-terminally truncated variant of Vpr in addition to full-length GFP-Vpr. Further, virions derived from these GFP-Vpr expressing cells show protein in which the GFP-tag has been additionally cleaved from the Vpr protein. Endogenous HIV protease (PR) activity was shown to be responsible for the latter, as addition of Saquinavir™, a potent PR inhibitor abolished the cleavage. Since many previous studies have relied on imaging the GFP fluorescence of GFP-Vpr, it would appear that the results may not reflect intact GFP-Vpr.     

茶刺蛾颗粒体病毒色涵体的拉曼光谱研究

得到了茶刺蛾颗粒体病毒(Darna trima Granulosis Virus 缩写为 DtGV)包涵体的拉曼光谱。分析表明,DtGV 包涵体蛋白的二级结构主要为β折叠和无规卷曲,后者含有大量β回折结构;蛋白的所有酪氨酸残基均“暴露”于溶剂中;其 C—C—S—S—C—C 构型属于扭曲—扭曲—扭曲式。     

The significance of the Golgi complex in envelopment of bovine herpesvirus 1 (BHV-1) as revealed by cryobased electron microscopy

Nucleocapsids of herpesviruses originate in the nucleus of host cells and bud through the inner nuclear membrane acquiring tegument and envelope. The release of the enveloped virus particle from the perinuclear space is unknown. Cryobased electron microscopic imaging revealed enveloped virus particles within cisterns associated with the perinuclear space, a pre-Golgi compartment connecting Golgi cisterns to the perinuclear space, and enveloped virus particles in Golgi cisterns where they are packaged into transport vacuoles by membrane fission. To our knowledge, our images show for the first time the connectivity from the perinuclear space to Golgi cisterns. The data strongly indicate an intracisternal transport of enveloped virus particles from the budding site to the packaging site. Budding starts by condensation at the inner membrane. Condensation involving the viral envelope and peripheral tegument was persistent in virus particles within perinuclear space and associated cisterns. Virus particles within Golgi cisterns and transport vacuoles originating by Golgi membrane fission, however, lacked condensation. Instead, spikes were clearly evident. The phenomenon of condensation is considered likely to be responsible for preventing fusion of the viral envelope with cisternal membranes and/or for driving virions from the perinuclear space to Golgi cisterns. Glycoprotein K is discussed to likely play a role in the intracisternal transportation of virions. In addition to the pathway including intracisternal transport and packaging, there were clear indications for the well-known pathway involving wrapping of cytoplasmic nucleocapsids by Golgi membranes. The origin of the cytoplasmic nucleocapsids, however, remains obscure. Lack of evidence for release of nucleocapsids at the outer nuclear membrane suggests that the process is very rapid, or that nucleocapsids pass the nucleocytoplasmic barrier via an alternative route.     

A3G蛋白结构与功能NMR研究的最新进展

A3G（apolipoprotin B mRNA-editing catalytic polypeptide 3 protein G）是一种天然抗病毒活性的胞嘧啶脱氨基酶,具有广谱的抗病毒作用,属于人体固有的免疫蛋白分子. 在HIV病毒复制过程中,A3G蛋白能进入新生成的病毒颗粒内部,诱导病毒cDNA胞嘧啶脱氨基化变为尿嘧啶,阻断病毒复制,从而导致病毒活性丧失. 最近几年,许多实验室以NMR技术和X射线衍射技术为基础,开展了一系列A3G蛋白的结构生物学和脱氨基化反应动态过程的研究,取得了具有重要意义的研究进展,为后期的研究工作提供了很好的新思路. 通过对这些工作进行总结,以期为我国艾滋病、乙肝病毒的防治,及相关药物分子的设计提供新的借鉴.     

Morphology and histology of the digestive system of the vector leafhopper Psammotettix striatus (L.) (Hemiptera: Cicadellidae)

The vector leafhopper Psammotettix striatus (L.) (Hemiptera: Cicadellidae) is an important economic pest that is a serious threat to wheat in Northwest China, causing great losses to agricultural production by transmitting wheat blue dwarf (WBD) in a persistent circulative manner. Studies on morphology and ultrastructure of the digestive system were made using light, scanning and transmission electron microscopy. The gross morphology of the alimentary canal consists of esophagus (foregut), midgut, hindgut, and special filter chamber. The esophagus, a narrow and slender tube, runs through the whole thorax extending into the filter chamber and connecting with the anterior midgut. The midgut consists of three major regions, anterior, middle and posterior midgut. The anterior midgut is composed of exceptionally thick epithelial cells surrounding a large lumen. Numerous well-developed microvilli occur at the basal region of the epithelium. A large number of electron-lucent and lipid-like vesicles are observed under the microvilli. The posterior midgut is smaller than the anterior midgut in diameter. Numerous different concentric circular bodies are characteristic of the posterior midgut. The hindgut arises from the posterior midgut at the boundary of the filter chamber. It extends downward toward the anus where the hindgut enlarges to form the rectum. The rectum is formed by various cells typical of epithelium, whose nuclei are relatively smaller than those of the midgut. Relatively large muscle cells are present at the well-defined and thick basement membrane. There are two pairs of Malpighian tubules. Numerous mitochondria and lamellar rough endoplasmic reticulum in the cells of the anterior segment of the Malpighian tubules; the sub-anterior segment of the tubules resemble the distal segment which are wave-like, amount vesicles in the cells; numerous tightly packed large vesicles contain two types of brochosomes in the inflated segment of the Malpighian tubules. The salivary system of P. striatus contains one pair of salivary glands, which are made up of the principal and accessory gland. The principal glands are divided into anterior, mid- and posterior lobes. About nineteen acini are observed in the principal glands of P. striatus, and vary in structure and colour. Five cellular types are found in the principal glands by transmission electron microscopy, which are rich in secretory granules, modified in size, electron-dense and shape. Rough endoplasmic reticulum in the shape of vesicular and lamellar, mitochondria and Golgi complexes are observed. The accessory gland has only an oval or a rod-shaped acinus.     

茶刺蛾颗粒体病毒包涵体的表面增强拉曼散射光谱研究

得到了茶刺蛾颗粒体病毒(DtGY)包涵体在银胶溶液中的表面增强拉曼散射(SERS)谱,并与其在水溶液中的一般拉曼散射(RS)谱作了比较分析。DtGV颗粒体外膜蛋白通过COO~-和NH_2基团吸附到银表面上。Tyr和Phe残基侧链远离金属表面,而Trp残基侧链接近银表面。多肽链被蛋白质中氨基酸残基侧链所屏蔽而妨碍有关振动的有效增强。     

不同性别C57BL/6小鼠肠道胆汁酸组成的差异性研究

随着人们对宿主肠道菌群的日益关注,关于胆汁酸与肠道菌群之间复杂关系的研究也越来越多.因肠道组织结构的复杂性和肠道菌群分布差异性,不同肠段胆汁酸的分布、表达及其生物学功能仍有待进一步研究.本文采用基于超高效液相色谱-三重四极杆-质谱联用（UPLC-QqQ-MS）的靶向代谢组分析结合基于核磁共振（NMR）波谱的非靶向代谢组分析方法,通过比较不同性别C57BL/6小鼠肠道各区段胆汁酸及粪样代谢组的变化水平,发现雌、雄小鼠不同肠段中胆汁酸的分布各不相同.另外,肠道下段各胆汁酸的性别差异性更为明显,以盲肠的性别差异性最为显著,这可能与盲肠的菌群微生态更为丰富有关.该研究为不同性别模型小鼠胆汁酸的代谢表型及其与肠道菌群的相互作用提供了基础研究数据.     

Structure of the sexually dimorphic gland of Cycloramphus fuliginosus (Amphibia, Anura, Cycloramphidae)

Cycloramphus fuliginosus males (Amphibia, Leptodactylidae) have discoid glandular elevations on the abdominal inferior body region which are considered a characteristic of genus. In this work, this sexually dimorphic gland of C. fuliginosus was investigated by low vacuum scanning electron microscopy, light microscopy, and transmission electron microscopy. In this cycloramphid, the covered integument exhibits the basic structure: the epidermis, a keratinized squamous stratified epithelium with flask cells, and the dermis. The dermis is subdivided into spongious and compact dermal layers. In the iliac gland region, an aggregate of small mucous glands and larger syncytial tubuloalveolar glands occur in the spongious dermis, and they do not go beyond the Eberth-Katschenko layer limit. The adenocytes of the mucous gland produce neutral glycoproteins, in contrast to the larger tubuloalveolar glands that elaborate a proteinaceous secretion. Myoepithelial cells surround the alveoli, and play an important role in the secretion extrusion. The final secretion, elaborated by the iliac gland, is a mixture of mucus and protein. Both secretions are important to the cycloramphid biology, and may act as pheromone and/or as chemical parental care.     

Alhydrogel® adjuvant, ultrasonic dispersion and protein binding: a TEM and analytical study

Aluminium-based vaccine adjuvants have been in use since the 1920s. Aluminium hydroxide (alum) that is the chemical basis of Alhydrogel, a widely used adjuvant, is a colloid that binds proteins to the particular surface for efficient presentation to the immune system during the vaccination process. Using conventional TEM and cryo-TEM we have shown that Alhydrogel can be finely dispersed by ultrasonication of the aqueous suspension. Clusters of ultrasonicated aluminium hydroxide micro-fibre crystals have been produced (～ 10-100 nm), that are significantly smaller than those present the untreated Alhydrogel (～ 2-12 μm). However, even prolonged ultrasonication did not produce a homogenous suspension of single aluminium hydroxide micro-fibres. The TEM images of unstained and negatively stained air-dried Alhydrogel are very similar to those obtained by cryo-electron microscopy. Visualization of protein on the surface of the finely dispersed Alhydrogel by TEM is facilitated by prior ultrasonication. Several examples are given, including some of medical relevance, using proteins of widely ranging molecular mass and oligomerization state. Even with the smaller mass proteins, their presence on the Alhydrogel surface can be readily defined by TEM. It has been found that low quantities of protein tend to cross-link and aggregate the small Alhydogel clusters, in a more pronounced manner than high protein concentrations. This indicates that complete saturation of the available Alhydrogel surface with protein may be achieved, with minimal cross-linkage, and future exploitation of this treatment of Alhydrogel is likely to be of immediate value for more efficient vaccine production.     

Functional Dynamics of Substrate Recognition in TEM Beta-Lactamase

The beta-lactamase enzyme provides effective resistance to beta-lactam antibiotics due to substrate recognition controlled by point mutations. Recently, extended-spectrum and inhibitor-resistant mutants have become a global health problem. Here, the functional dynamics that control substrate recognition in TEM beta-lactamase are investigated using all-atom molecular dynamics simulations. Comparisons are made between wild-type TEM-1 and TEM-2 and the extended-spectrum mutants TEM-10 and TEM-52, both in apo form and in complex with four different antibiotics (ampicillin, amoxicillin, cefotaxime and ceftazidime). Dynamic allostery is predicted based on a quasi-harmonic normal mode analysis using a perturbation scan. An allosteric mechanism known to inhibit enzymatic function in TEM beta-lactamase is identified, along with other allosteric binding targets. Mechanisms for substrate recognition are elucidated using multivariate comparative analysis of molecular dynamics trajectories to identify changes in dynamics resulting from point mutations and ligand binding, and the conserved dynamics, which are functionally important, are extracted as well. The results suggest that the H10-H11 loop (residues 214-221) is a secondary anchor for larger extended spectrum ligands, while the H9-H10 loop (residues 194-202) is distal from the active site and stabilizes the protein against structural changes. These secondary non-catalytically-active loops offer attractive targets for novel noncompetitive inhibitors of TEM beta-lactamase.     

Dynamics Analysis of a Wireless Rechargeable Sensor Network for Virus Mutation Spreading

Virus spreading problems in wireless rechargeable sensor networks (WSNs) are becoming a hot topic, and the problem has been studied and discussed in recent years. Many epidemic spreading models have been introduced for revealing how a virus spreads and how a virus is suppressed. However, most of them assumed the sensors are not rechargeable sensors. In addition, most of existing works do not consider virus mutation problems. This paper proposes a novel epidemic model, including susceptible, infected, variant, low-energy and dead states, which considers the rechargeable sensors and the virus mutation factor. The stability of the proposed model is first analyzed by adopting the characteristic equation and constructing Lyapunov functions methods. Then, an optimal control problem is formulated to control the virus spread and decrease the cost of the networks by applying Pontryagin’s maximum principle. Finally, all of the theoretical results are confirmed by numerical simulation.     

Salivary system in leaf-cutting ants (Atta sexdens rubropilosa Forel, 1908) castes: a confocal study

The salivary system in ants is not only limited to digestory functions, but also has important role in the communication. The glands which compose the salivary complex are: the post-pharyngeal, hypopharyngeal, mandibular, and thoracic salivary gland, showing peculiar features which may vary according to the castes the individuals belong to and according to the functions they develop. The present study compared the morphological differences among the glands of Atta sexdens rubropilosa workers, males and queens focused on the organization of microfilaments and microtubules in these ants salivary system. Although the post-pharyngeal gland appeared to be more developed in queens, there were no significant gland differences among the analyzed castes. In what regards to the secretory units of the hypopharyngeal and mandibular glands, the association of F-actin with the collector duct seemed to be strong, being surrounded by a microtubules arrangement. The use of a laser scanning confocal microscopy with immunofluorescence whole mounting preparations revealed itself an efficient instrument for the understanding of the internal morphology of insects.     

Microscopy techniques in flavivirus research

The Flavivirus genus is composed of many medically important viruses that cause high morbidity and mortality, which include Dengue and West Nile viruses. Various molecular and biochemical techniques have been developed in the endeavour to study flaviviruses. However, microscopy techniques still have irreplaceable roles in the identification of novel virus pathogens and characterization of morphological changes in virus-infected cells. Fluorescence microscopy contributes greatly in understanding the fundamental viral protein localizations and virus–host protein interactions during infection. Electron microscopy remains the gold standard for visualizing ultra-structural features of virus particles and infected cells. New imaging techniques and combinatory applications are continuously being developed to push the limit of resolution and extract more quantitative data. Currently, correlative live cell imaging and high resolution three-dimensional imaging have already been achieved through the tandem use of optical and electron microscopy in analyzing biological specimens. Microscopy techniques are also used to measure protein binding affinities and determine the mobility pattern of proteins in cells. This chapter will consolidate on the applications of various well-established microscopy techniques in flavivirus research, and discuss how recently developed microscopy techniques can potentially help advance our understanding in these membrane viruses.     

Ultrastructure of the seminal vesicle and sperm storage in Panorpidae (Insecta: Mecoptera)

The histology and ultrastructure of the seminal vesicle, male accessory gland and the epididymis in Panorpa and Sinopanorpa were observed using light microscopy and scanning and transmission electron microscopy. The seminal vesicle consists of a mono-layered elongated columnar epithelium, which contains abundant electron-dense granules and secretory vesicles, and a small central lumen. In the apical region of the epithelium of the seminal vesicle, the intense secretory activity seems to be effected by means of merocrine mechanisms. The epithelium of the accessory gland is rich in rough endoplasmic reticulum and Golgi complex, and secretes seminal fluid into the lumen via both apocrine and merocrine mechanisms. The seminal vesicle is similar to the accessory gland in the epithelium structure and their secretory activity, mainly serving a secretory function rather than storing sperms. Instead, the sperms are stored in the epididymis, whose epithelium secretes nutrients into the large lumen by merocrine mechanisms. The secretory activity and function of the seminal vesicle are briefly discussed.     

Ultrastructural studies of the mandibular glands of the minima, media and soldier ants of Atta sexdens rubropilosa (Forel 1908) (Hymenoptera: Formicidae)

The mandibular glands of Hymenoptera are structures associated with the mandibles and constitute part of the salivary glands system. Histological studies in workers of Atta sexdens rubropilosa revealed that this gland contains two portions: a secretory and a storage portion or reservoir. Both portions are connected by means of canaliculi. The object of the present work was the study of the ultratructure of the mandibular glands of minima, media and soldier ant of A. s. rubropilosa by TEM techniques. The glands, in the three castes studied, possess a reservoir, constituted by a simple pavementous epithelium surrounded by the cuticular intima and the secretory portion is constituted by cells of rounded shape. The secretory cells, mainly of minima and soldier, were rich in smooth endoplasmic reticulum. The media worker and soldier presented a large number of mitochondria, of varying shape. Well-developed Golgi complexes were also present in the soldiers. The secretory cells in minima, media and soldier were provided with collecting intracellular canaliculi, which were linked to the reservoir through the extracellular portion. The cytoplasm of the canaliculi-forming cell was poor in organelles. In the individuals of the three castes of A. s. rubropilosa, the presence of lipid secretion granules suggested, beyond the other functions, also a possible pheromonal action. The different roles executed by the different insect castes are directly dependent on the glandular products and, consequently, on the secretory cellular characteristics.     

Male reproductive system structure and accessory glands ultrastructure of two species of Triatoma (Hemiptera,Reduviidae, Triatominae)

Morphological studies in male reproductive system of insect, and accessory glands functions, can contribute to the knowledge of reproductive aspects in Triatominae. The present study aims to describe the structure of male reproductive system under scanning electron microscopy, and the ultrastructure of accessory glands during the process of maturation, in T. brasiliensis and T. melanica of different ages.These system consist of two testis, two vas deferens, two seminal vesicles, four pair of accessory glands and one ejaculatory duct. The four mesodermal male accessory glands of T. melanica and T. brasiliensis, present secretion storage in the firsts days of adult life in starved males. During the maturation process of male accessory glands of T. brasiliensis and T. melanica, granules with different sizes, shapes and electron density were seen. In T. melanica small vacuoles are released into the gland lumen in one-day-old males, however, after three and five days few granules are found in the cell cytoplasm. The secretory granules in T. brasiliensis increase in size and amount, in the five days old adult. The secretory pathway in the male accessory glands of the two species seems to be merocrine and apocrine suggesting that different substances are being produced at different times. The cell microvilli in T. brasiliensis are longer and narrower, these can be absent in some regions of the cell surface of T. melanica. Maturation of male accessory glands in the triatomine species studied occurred without adult blood meal. Its function can led to new insights for the comprehension of reproductive aspects in Triatominae.     

Dehiscent organs used for defensive behavior of kamikaze termites of the genus Ruptitermes (Termitidae,Apicotermitinae) are not glands

During Isoptera evolution, the caste of soldiers disappeared in some Apicotermitinae termites as in the Neotropical Ruptitermes. Paired dorsolateral structures located between the metathorax and abdomen of foraging workers of Ruptitermes were previously denominated dehiscent glands, and are responsible for releasing an adhesive secretion that immobilizes enemies, causing their death. In this study, we investigated the morphology of dehiscent organs of workers of Ruptitermes reconditus, Ruptitermes xanthochiton, and Ruptitermes pitan and also second instar larvae of R. reconditus using light, laser scanning confocal, and transmission electron microscopy. Additionally, we performed a preliminary protein analysis using SDS-PAGE to further characterize the secretion of Ruptitermes dehiscent organs. Our results showed that the dehiscent organs do not exhibit the typical characteristics of the exocrine glandular cells class I, II or III of insects, suggesting that they constitute a new type of defensive organ. Thus, the denomination dehiscent gland was not used but dehiscent organ. Dehiscent organs in larvae are formed by fat body cells. In workers, dehiscent organs are composed by compact masses of cells that accumulate a defensive secretion and are poor in organelles related to the production of secretion. Since the dehiscent organs are not glands, we hypothesize that the dehiscent organs originate from larval fat body. The defensive secretion may have been produced at younger developmental stages of worker or the defensive compounds were absorbed from food and accumulated in the worker fat body. Histochemical techniques and SDS-PAGE revealed that the secretion of Ruptitermes dehiscent organs is constituted mainly by a protein of high molecular weight (200 kDa). In conclusion, the dehiscent organs are extremely different from the exocrine glands of termites and other insects described until now. In fact, they seem to be a specialized fat body that is peculiar and exclusive of Ruptitermes termites.