Functional analysis of AeSCP-2 using gene expression knockdown in the yellow fever mosquito, Aedes aegypti

The effect of gene expression knockdown was used to study the function of the sterol carrier protein-2 (AeSCP-2) in the yellow fever mosquito, Aedes aegypti. Injection of small double stranded AeSCP-2 RNAs into mosquito larvae resulted in the knockdown of gene products. The lack of AeSCP-2 in larvae coincided with a reduction in accumulated cholesterol in pupae, supporting the hypothesis that AeSCP-2 may be involved in cholesterol uptake in mosquito larvae. Knockdown of AeSCP-2 caused a high mortality rate in developing adult and reduced egg viability. Results from this study indicate that AeSCP-2 is important for adult development and for the viability of the eggs.     

Tissue‐specific expression and silencing phenotypes of mitochondrial phosphate carrier paralogues in several insect species

The mitochondrial phosphate carrier gene (PiC) encodes a membrane protein that mediates the supply of inorganic phosphate from the cytosol into the mitochondrial matrix. This substrate‐specific transport system plays an important role in efficient ATP synthesis. Mammals appear to have only one PiC with two alternative splicing variants whose functional differences remain unclear. The present study is the first to characterize the multiple genes that encode PiC in insects. Bombyx mori was found to have two PiC paralogues, one ubiquitous and one testis‐specific, the latter seeming to be present only in Lepidoptera. Drosophila melanogaster was found to harbour two PiC paralogues, whereas Liriomyza chinensis, another dipteran, has three PiC paralogues. Two PiCs were found to be present in Plautia stali, and silencing either of these genes affected the normal development of P. stali nymphs, although their expression patterns differed amongst tissues. Schistocerca gregaria and Locusta migratoria have two PiC each, with different expression patterns. Tribolium castaneum was found to have only one PiC, which appears to play an essential role in larval development. Thus, although the inorganic phosphate transport system appears to be conserved across eukaryotes, PiC has become specialized in the different tissues of different insect species.     

Amblyomma americanum tick saliva insulin‐like growth factor binding protein‐related protein 1 binds insulin but not insulin‐like growth factors

Silencing Amblyomma americanum insulin‐like growth factor binding protein‐related protein 1 (AamIGFBP‐rP1) mRNA prevented ticks from feeding to repletion. In this study, we used recombinant (r)AamIGFBP‐rP1 in a series of assays to obtain further insight into the role(s) of this protein in tick feeding regulation. Our results suggest that AamIGFBP‐1 is an antigenic protein that is apparently exclusively expressed in salivary glands. We found that both males and females secrete AamIGFBP‐rP1 into the host during feeding and confirmed that female ticks secrete this protein from within 24–48 h after attachment. Our data suggest that native AamIGFBP‐rP1 is a functional insulin binding protein in that both yeast‐ and insect cell‐expressed rAamIGFBP‐rP1 bound insulin, but not insulin‐like growth factors. When subjected to anti‐blood clotting and platelet aggregation assays, rAamIGFBP‐rP1 did not have any effect. Unlike human IGFBP‐rP1, which is controlled by trypsinization, rAamIGFBP‐rP1 is resistant to digestion, suggesting that the tick protein may not be under mammalian host control at the tick feeding site. The majority of tick‐borne pathogens are transmitted 48 h after the tick has attached. Thus, the demonstrated antigenicity and secretion into the host within 24–48 h of the tick starting to feed makes AamIGFBP‐rP1 an attractive target for antitick vaccine development.     

Preliminary evaluation of novel 68Ga‐DOTAVAP‐PEG‐P2 peptide targeting vascular adhesion protein‐1

Introduction: Expression of vascular adhesion protein‐1 (VAP‐1) is induced at the sites of inflammation where extravasation of leukocytes from blood to the peripheral tissue occurs. VAP‐1 is a potential target for anti‐inflammatory therapy and for in vivo imaging of inflammation. Purpose of this study was to preliminarily evaluate a novel VAP‐1‐targeting peptide as a potential PET imaging agent. Methods: Cyclic 17‐amino‐acid peptide selected from phage display libraries was 1,4,7,10‐tetraazacyclododecane‐N,N′,N′′,N′′′‐tetraacetic acid (DOTA) conjugated via 8‐amino‐3,6‐diooxaoctanoyl linker (polyethylene glycol, PEG derivative) and labelled with ⁶⁸Ga (⁶⁸Ga‐DOTAVAP‐PEG‐P2). In vitro stability of ⁶⁸Ga‐DOTAVAP‐PEG‐P2 was determined in saline, rat plasma and human plasma by radio‐HLPC. Lipophilicity was measured by calculating octanol‐water partition coefficient (logP). Whole‐body distribution kinetics and stability after intravenous injection in healthy rats was studied in vivo by PET imaging, ex vivo by measuring radioactivity of excised tissues, and by radio‐HPLC. Results: In vitro the ⁶⁸Ga‐DOTAVAP‐PEG‐P2 remained stable >4 h in saline and rat plasma, but degraded slowly in human plasma after 2 h of incubation. The logP value of ⁶⁸Ga‐DOTAVAP‐PEG‐P2 was ?1·3. In rats, ⁶⁸Ga‐radioactivity cleared rapidly from blood circulation and excreted quickly in urine. At 120 min after injection the fraction of intact ⁶⁸Ga‐DOTAVAP‐PEG‐P2 were 77 ± 6·0% and 99 ± 1·0% in rat plasma and urine, respectively. Conclusions: These basic and essential in vitro and in vivo studies of the new VAP‐1 targeting peptide revealed promising properties for an imaging agent. Further investigations to clarify in vivo VAP‐1 targeting are warranted.     

Identification and characterization of the pyrokinin/pheromone biosynthesis activating neuropeptide family of G protein‐coupled receptors from Ostrinia nubilalis

Insects have two closely related G protein‐coupled receptors belonging to the pyrokinin/pheromone biosynthesis activating neuropeptide (pyrokinin/PBAN) family, one with the ligand PBAN or pyrokinin‐2 and another with diapause hormone or pyrokinin‐1 as a ligand. A related receptor is activated by products of the capa gene, periviscerokinins. Here we characterized the PBAN receptor and the diapause hormone receptor from the European corn borer, Ostrinia nubilalis. We also identified a partial sequence for the periviscerokinin receptor. Quantitative PCR of mRNA for all three receptors indicated differential expression in various life stages and tissues. All three splice variants of the PBAN receptor were identified with all variants found in pheromone gland tissue. Immunohistochemistry of V5 tags of expressed receptors indicated that all three variants and the diapause hormone receptor were expressed at similar levels in Spodoptera frugiperda 9 (Sf9) cells. However, the A‐ and B‐variants were not active in our functional assay, which confirms studies from other moths. Functional expression of the C‐variant indicated that it is has a 44 nM half effective concentration for activation by PBAN. The diapause hormone receptor was activated by diapause hormone with a 150 nM half effective concentration.     

Bone morphogenetic protein‐2 release profile modulates bone formation in phosphorylated hydrogel

The optimal release profile of locally delivered bone morphogenetic protein‐2 (BMP‐2) for safe and effective clinical application is unknown. In this work, the effect of differential BMP‐2 release on bone formation was investigated using a novel biomaterial oligo[(polyethylene glycol) fumarate] bis[2‐(methacryloyloxy) ethyl] phosphate hydrogel (OPF‐BP) containing poly(lactic‐co‐glycolic acid) microspheres. Three composite implants with the same biomaterial chemistry and structure but different BMP‐loading methods were created: BMP‐2 encapsulated in microspheres (OPF‐BP‐Msp), BMP‐2 encapsulated in microspheres and adsorbed on the phosphorylated hydrogel (OPF‐BP‐Cmb), and BMP‐2 adsorbed on the phosphorylated hydrogel (OPF‐BP‐Ads). These composites were compared with the clinically used BMP‐2 carrier, Infuse® absorbable collagen sponge (ACS). Differential release profiles of bioactive BMP‐2 were achieved by these composites. In a rat subcutaneous implantation model, OPF‐BP‐Ads and ACS generated a large BMP‐2 burst release (>75%), whereas a more sustained release was seen for OPF‐BP‐Msp and OPF‐BP‐Cmb (~25% and 50% burst, respectively). OPF‐BP‐Ads generated significantly more bone than did all other composites, and the bone formation was 12‐fold higher than that of the clinically used ACS. Overall, this study clearly shows that BMP‐2 burst release generates more subcutaneous bone than do sustained release in OPF‐BP‐microsphere composites. Furthermore, composites should not only function as a delivery vehicle but also provide a proper framework to achieve appropriate bone formation.     

A doxycycline inducible,adenoviral bone morphogenetic protein‐2 gene delivery system to bone

We report the novel use of a tuneable, non‐integrating viral gene delivery system to bone that can be combined with clinically approved biomaterials in an 'off‐the‐shelf' manner. Specifically, a doxycycline inducible Tet‐on adenoviral vector (AdTetBMP‐2) in combination with mesenchymal stromal cells (MSCs), fibrin and a biphasic calcium phosphate ceramic (MBCP®) was used to repair large bone defects in nude rats. Bone morphogenetic protein‐2 (BMP‐2) transgene expression could be effectively tuned by modification of the doxycycline concentration. The effect of adenoviral BMP‐2 gene delivery upon bone healing was investigated in vivo in 4 mm critically sized, internally fixated, femoral defects. MSCs were transduced either by direct application of AdTetBMP‐2 or by pre‐coating MBCP granules with the virus. Radiological assessment scores post‐mortem were significantly improved upon delivery of AdTetBMP‐2. In AdTetBMP‐2 groups, histological analysis revealed significantly more newly formed bone at the defect site compared with controls. Newly formed bone was vascularized and fully integrated with nascent tissue and implanted biomaterial. Improvement in healing outcome was achieved using both methods of vector delivery (direct application vs. pre‐coating MCBP). Adenoviral delivery of BMP‐2 enhanced bone regeneration achieved by the transplantation of MSCs, fibrin and MBCP in vivo. Importantly, our in vitro and in vivo data suggest that this can be achieved with relatively low (ng/ml) levels of the growth factor. Our model and novel gene delivery system may provide a powerful standardized tool for the optimization of growth factor delivery and release for the healing of large bone defects. Copyright © 2016 John Wiley & Sons, Ltd.     

Bowman‐Birk inhibitor affects pathways associated with energy metabolism in Drosophila melanogaster

Bowman‐Birk inhibitor (BBI) is toxic when fed to certain insects, including the fruit fly, Drosophila melanogaster. Dietary BBI has been demonstrated to slow growth and increase insect mortality by inhibiting the digestive enzymes trypsin and chymotrypsin, resulting in a reduced supply of amino acids. In mammals, BBI influences cellular energy metabolism. Therefore, we tested the hypothesis that dietary BBI affects energy‐associated pathways in the D. melanogaster midgut. Through microarray and metabolomic analyses, we show that dietary BBI affects energy utilization pathways in the midgut cells of D. melanogaster. In addition, ultrastructure studies indicate that microvilli are significantly shortened in BBI‐fed larvae. These data provide further insights into the complex cellular response of insects to dietary protease inhibitors.     

Heparin–dopamine functionalized graphene foam for sustained release of bone morphogenetic protein‐2

The recently developed three‐dimensional (3D) graphene foam (GrF) is intriguing for potential bone tissue engineering applications because it provides stem cells with a 3D porous substrate for osteogenic differentiation. However, the nature of graphene's structure lacks functional groups, thus making it difficult for further modification such as immobilization or conjugation of growth factors, which are normally required to promote tissue regeneration. To explore the potential of GrF functionalization and sustained release of therapeutic proteins, we fabricated a modified 3D GrF scaffold with bio‐inspired heparin–dopamine (Hepa–Dopa) molecules using a highly scalable chemical vapour deposition method. Our data indicated that Hepa–Dopa modification resulted in significantly higher bone morphogenetic protein‐2 (BMP2) binding ability and longer release capacity compared with the untreated scaffolds. Importantly, the heparin‐functionalized 3D GrF significantly improved the exogenous BMP2‐induced osteogenic differentiation. Therefore, our study, for the first time, indicated that the 3D GrF can be biomimetically functionalized with Hepa–Dopa and be used for sustained release of BMP2, thereby inducing osteogenic differentiation and suggesting promising potential as a new multifunctional carrier for therapeutic proteins and stem cells in bone tissue engineering.     

Characterization of recombinant human bone morphogenetic protein‐2 delivery from injectable hyaluronan‐based hydrogels by means of 125I‐radiolabelling

This study presents a thorough in vitro and in vivo characterization of the delivery of bone morphogenetic protein 2 (BMP‐2) from a hyaluronan‐based hydrogel system. The in vitro release of BMP‐2 from similar hydrogels has previously been studied by enzyme‐linked immunosorbent assay (ELISA), by which only a fraction of the loaded protein is detected. In the current study, ¹²⁵I radiolabelling was used instead to monitor BMP‐2 in vitro and in vivo. To minimize protein loss during handling, ¹²⁵I‐BMP‐2 adsorption to different tubes was studied at different times and temperatures. The data showed that Protein LoBind tubes exhibited the lowest protein affinity. Furthermore, a biphasic release profile of biologically active BMP‐2 was observed both in vitro and in vivo, with the initial fast phase during the first week, followed by a slower release during the remaining 3 weeks. The initial fast‐release phase corresponded to the early bone formation observed after 8 days in an ectopic model in rats. Bone volume and mineral content increased until day 14, after which a decrease in bone volume was observed, possibly due to resorption in response to decreased amounts of released BMP‐2. Overall, the results suggested that cautious protein handling and a reliable quantification technique are essential factors for successful design of a BMP‐2 delivery system. Copyright © 2012 John Wiley & Sons, Ltd.