Crystal Structure of the Pre-fusion Nipah Virus Fusion Glycoprotein Reveals a Novel Hexamer-of-Trimers Assembly

Nipah virus (NiV) is a paramyxovirus that infects host cells through the coordinated efforts of two envelope glycoproteins. The G glycoprotein attaches to cell receptors, triggering the fusion (F) glycoprotein to execute membrane fusion. Here we report the first crystal structure of the pre-fusion form of the NiV-F glycoprotein ectodomain. Interestingly this structure also revealed a hexamer-of-trimers encircling a central axis. Electron tomography of Nipah virus-like particles supported the hexameric pre-fusion model, and biochemical analyses supported the hexamer-of-trimers F assembly in solution. Importantly, structure-assisted site-directed mutagenesis of the interfaces between F trimers highlighted the functional relevance of the hexameric assembly. Shown here, in both cell-cell fusion and virus-cell fusion systems, our results suggested that this hexamer-of-trimers assembly was important during fusion pore formation. We propose that this assembly would stabilize the pre-fusion F conformation prior to cell attachment and facilitate the coordinated transition to a post-fusion conformation of all six F trimers upon triggering of a single trimer. Together, our data reveal a novel and functional pre-fusion architecture of a paramyxoviral fusion glycoprotein.     

Essential Oil from Bark of Aniba parviflora (Meisn.) Mez (Lauraceae) Reduces HepG2 Cell Proliferation and Inhibits Tumor Development in a Xenograft Model

Aniba parviflora (Meisn .) Mez (Lauraceae) is an aromatic plant of the Amazon rainforest, which has a tremendous commercial value in the perfumery industry; it is popularly used as flavoring sachets and aromatic baths. In Brazilian folk medicine, A. parviflora is used to treat victims of snakebites. Herein, we analyzed the chemical composition of A. parviflora bark essential oil (EO) and its effect on the growth of human hepatocellular carcinoma HepG2 cells in vitro and in vivo. EO was obtained by hydrodistillation and characterized by GC-MS and GC-FID. The main constituents of EO were linalool (16.3±3.15), α-humulene (14.5±2.41 %), δ-cadinene (10.2±1.09 %), α-copaene (9.51±1.12 %) and germacrene B (7.58±2.15 %). Initially, EO's cytotoxic effect was evaluated against five cancer cell lines (HepG2, MCF-7, HCT116, HL-60 and B16-F10) and one non-cancerous one (MRC-5), using the Alamar blue method after 72 h of treatment. The calculated IC₅₀ values were 9.05, 22.04, >50, 15.36, 17.57, and 30.46 μg/mL, respectively. The best selectivity was for HepG2 cells with a selective index of 3.4. DNA Fragmentation and cell cycle distribution were quantified in HepG2 cells by flow cytometry after a treatment period of 24 and 48 h. The effect of EO on tumor development in vivo was evaluated in a xenograft model using C.B-17 SCID mice engrafted with HepG2 cells. In vivo tumor growth inhibition of HepG2 xenograft at the doses of 40 and 80 mg/kg were 12.1 and 62.4 %, respectively.     

Identification and culture of proliferative cells in abnormal Taenia solium larvae: Role in the development of racemose neurocysticercosis

Racemose neurocysticercosis is an aggressive disease caused by the aberrant expansion of the cyst form of Taenia solium within the subarachnoid spaces of the human brain and spinal cord resulting in a mass effect and chronic inflammation. Although expansion is likely caused by the proliferation and growth of the parasite bladder wall, there is little direct evidence of the mechanisms that underlie these processes. Since the development and growth of cysts in related cestodes involves totipotential germinative cells, we hypothesized that the expansive growth of the racemose larvae is organized and maintained by germinative cells. Here, we identified proliferative cells expressing the serine/threonine-protein kinase plk1 by in situ hybridization. Proliferative cells were present within the bladder wall of racemose form and absent from the homologous tissue surrounding the vesicular form. Cyst proliferation in the related model species Taenia crassiceps (ORF strain) occurs normally by budding from the cyst bladder wall and proliferative cells were concentrated within the growth buds. Cells isolated from bladder wall of racemose larvae were established in primary cell culture and insulin stimulated their proliferation in a dose-dependent manner. These findings indicate that the growth of racemose larvae is likely due to abnormal cell proliferation. The different distribution of proliferative cells in the racemose larvae and their sensitivity to insulin may reflect significant changes at the cellular and molecular levels involved in their tumor-like growth. Parasite cell cultures offer a powerful tool to characterize the nature and formation of the racemose form, understand the developmental biology of T. solium, and to identify new effective drugs for treatment.     

Volutin granules of Eimeria parasites are acidic compartments and have physiological and structural characteristics similar to acidocalcisomes

The structural organization of parasites has been the subject of investigation by many groups and has lead to the identification of structures and metabolic pathways that may represent targets for anti-parasitic drugs. A specific group of organelles named acidocalcisomes has been identified in a number of organisms, including the apicomplexan parasites such as Toxoplasma and Plasmodium, where they have been shown to be involved in cation homeostasis, polyphosphate metabolism, and osmoregulation. Their structural counterparts in the apicomplexan parasite Eimeria have not been fully characterized. In this work, the ultrastructural and chemical properties of acidocalcisomes in Eimeria were characterized. Electron microscopy analysis of Eimeria parasites showed the dense organelles called volutin granules similar to acidocalcisomes. Immunolocalization of the vacuolar proton pyrophosphatase, considered as a marker for acidocalcisomes, showed labeling in vesicles of size and distribution similar to the dense organelles seen by electron microscopy. Spectrophotometric measurements of the kinetics of proton uptake showed a vacuolar proton pyrophosphatase activity. X-ray mapping revealed significant amounts of Na, Mg, P, K, Ca, and Zn in their matrix. The results suggest that volutin granules of Eimeria parasites are acidic, dense organelles, and possess structural and chemical properties analogous to those of other acidocalcisomes, suggesting a similar functional role in these parasites.     

Biochemical properties of an extracellular trehalase from <Emphasis Type="Italic">Malbranchea pulchella</Emphasis> var. Sulfurea

The thermophilic fungus Malbranchea pulchella var. sulfurea produced good amounts of extracellular trehalase activity when grown for long periods on starch, maltose or glucose as the main carbon source. Studies with young cultures suggested that the main role of the extracellular acid trehalase is utilizing trehalose as a carbon source. The specific activity of the purified enzyme in the presence of manganese (680 U/mg protein) was comparable to that of other thermophilic fungi enzymes, but many times higher than the values reported for trehalases from other microbial sources. The apparent molecular mass of the native enzyme was estimated to be 104 kDa by gel filtration and 52 kDa by SDS-PAGE, suggesting that the enzyme was composed by two subunits. The carbohydrate content of the purified enzyme was estimated to be 19 % and the pi was 3.5. The optimum pH and temperature were 5.0–5.5 and 55° C, respectively. The purified enzyme was stimulated by manganese and inhibited by calcium ions, and insensitive to ATP and ADP, and 1 mM silver ions. The apparent K_M values for trehalose hydrolysis by the purified enzyme in the absence and presence of manganese chloride were 2.70±0.29 and 2.58±0.13 mM, respectively. Manganese ions affected only the apparent V_max, increasing the catalytic efficiency value by 9.2-fold. The results reported herein indicate that Malbranchea pulchella produces a trehalase with mixed biochemical properties, different from the conventional acid and neutral enzymes and also from trehalases from other thermophilic fungi.     

Examining the interaction of light,nutrients and carbohydrates on seed germination and early seedling development of <Emphasis Type="Italic">Bletia purpurea</Emphasis> (Orchidaceae)

The effects of carbohydrate availability, carbohydrate source, nutrient availability and illumination on germination and early development of Bletia purpurea (Orchidaceae) seeds were investigated using asymbiotic seed germination. Of special interest was determining the minimum nutritional and light requirements for the completion of germination. Germination and development was limited when seeds were cultured in darkness without sucrose. Seeds were able to germinate under illuminated conditions even in the absence of sucrose and this effect was enhanced when mineral nutrients were incorporated into media. Sucrose, fructose, glucose and trehalose enhanced germination and seedling development while mannitol and sorbitol did not. These data suggest that carbohydrates, either as products of photosynthesis, from symbiotic fungi in situ or as exogenously supplied sugars in vitro, play an important role in regulating seed germination by fulfilling an energy requirement. This hypothesis has been often expressed but rarely satisfactorily tested. Mineral nutrients appear to be less important for germination than carbohydrates. The differential effect of sucrose, fructose, glucose and trehalose at two different concentrations on rhizoid production indicates carbohydrates may play a role in regulating rhizoid production.     

A novel role of periostin in postnatal tooth formation and mineralization

Periostin plays multiple functions during development. Our previous work showed a critical role of this disulfide-linked cell adhesion protein in maintenance of periodontium integrity in response to occlusal load. In this study, we attempted to address whether this mechanical response molecule played a direct role in postnatal tooth development. Our key findings are 1) periostin is expressed in preodontoblasts, and odontoblasts; and the periostin-null incisor displayed a massive increase in dentin formation after mastication; 2) periostin is also expressed in the ameloblast cells, and an enamel defect is identified in both the adult-null incisor and molar; 3) deletion of periostin leads to changes in expression profiles of many non-collagenous protein such as DSPP, DMP1, BSP, and OPN in incisor dentin; 4) the removal of a biting force leads to reduction of mineralization, which is partially prevented in periostin-null mice; and 6) both in vitro and in vivo data revealed a direct regulation of periostin by TGF-β1 in dentin formation. In conclusion, periostin plays a novel direct role in controlling postnatal tooth formation, which is required for the integrity of both enamel and dentin.