Therapy of cancer by insect cells containing recombinant baculovirus encoding genes

Provided are compositions and methods of use for insect cells comprising baculovirus encoding non-surface expressed proteins and peptides. The claimed invention particularly relates to compositions comprising insect cells containing baculovirus that express cytokines. Such compositions may be administered by, for example, direct intratumoral injection into tumors in mammals, resulting in tumor reduction or recission. Another aspect of the claimed invention concerns methods of promoting resistance to the reoccurence of tumors in mammals who have undergone such tumor recission. In a specific aspect of the claimed invention, the mammals are human subjects presenting with various forms of cancer.Board of Regents, University of Texas Syste

A practical method for efficient and optimal production of Seleno‐methionine‐labeled recombinant protein complexes in the insect cells

The use of Seleno‐methionine (SeMet) incorporated protein crystals for single or multi‐wavelength anomalous diffraction (SAD or MAD) to facilitate phasing has become almost synonymous with modern X‐ray crystallography. The anomalous signals from SeMets can be used for phasing as well as sequence markers for subsequent model building. The production of large quantities of SeMet incorporated recombinant proteins is relatively straightforward when expressed in Escherichia coli. In contrast, production of SeMet substituted recombinant proteins expressed in the insect cells is not as robust due to the toxicity of SeMet in eukaryotic systems. Previous protocols for SeMet‐incorporation in the insect cells are laborious, and more suited for secreted proteins. In addition, these protocols have generally not addressed the SeMet toxicity issue, and typically result in low recovery of the labeled proteins. Here we report that SeMet toxicity can be circumvented by fully infecting insect cells with baculovirus. Quantitatively controlling infection levels using our Titer Estimation of Quality Control (TEQC) method allow for the incorporation of substantial amounts of SeMet, resulting in an efficient and optimal production of labeled recombinant protein complexes. With the method described here, we were able to consistently reach incorporation levels of about 75% and protein yield of 60–90% compared with native protein expression

Controlling sap-sucking insect pests with recombinant endophytes expressing plant lectin

We developed a novel pest management strategy, which uses endophytes to express anti-pest plant lectins. Fungal endophyte of Chaetomium globosum YY-11 with anti-fungi activities was isolated from rape seedlings, and bacterial endophytes of SJ-10 (Enterobacter sp.) and WB (Bacillus subtilis) were isolated from rice seedlings. Pinellia ternate agglutinin gene was cloned into SJ-10 and WB for expression by a shuttle vector, and YY-11 was mediated by Agrobacterium tumefaciens. Positive transformants were evaluated using PCR and Western blot assay. Recombinant endophytes colonized most of crops, and resistance of rice seedlings, which were inoculated with the recombinant endophytic bacteria, to white backed planthoppers was dramatically enhanced by decreasing the survival and fecundity of white backed planthoppers. Rape inoculated with recombinant endophytic fungi significantly inhibited the growth and reproduction of aphids. Recombinant endophytes expressing PTA may endow hosts with resistance against sap-sucking pests

The genomes of two key bumblebee species with primitive eusocial organization

Background: The shift from solitary to social behavior is one of the major evolutionary transitions. Primitively eusocial bumblebees are uniquely placed to illuminate the evolution of highly eusocial insect societies. Bumblebees are also invaluable natural and agricultural pollinators, and there is widespread concern over recent population declines in some species. High-quality genomic data will inform key aspects of bumblebee biology, including susceptibility to implicated population viability threats. Results: We report the high quality draft genome sequences of Bombus terrestris and Bombus impatiens, two ecologically dominant bumblebees and widely utilized study species. Comparing these new genomes to those of the highly eusocial honeybee Apis mellifera and other Hymenoptera, we identify deeply conserved similarities, as well as novelties key to the biology of these organisms. Some honeybee genome features thought to underpin advanced eusociality are also present in bumblebees, indicating an earlier evolution in the bee lineage. Xenobiotic detoxification and immune genes are similarly depauperate in bumblebees and honeybees, and multiple categories of genes linked to social organization, including development and behavior, show high conservation. Key differences identified include a bias in bumblebee chemoreception towards gustation from olfaction, and striking differences in microRNAs, potentially responsible for gene regulation underlying social and other traits. Conclusions: These two bumblebee genomes provide a foundation for post-genomic research on these key pollinators and insect societies. Overall, gene repertoires suggest that the route to advanced eusociality in bees was mediated by many small changes in many genes and processes, and not by notable expansion or depauperation

Characterisation of Bombyx mori odorant-binding proteins reveals that a general odorant-binding protein discriminates between sex pheromone components

In many insect species, odorant-binding proteins (OBPs) are thought to be responsible for the transport of pheromones and other semiochemicals across the sensillum lymph to the olfactory receptors (ORs) within the antennal sensilla. In the silkworm Bombyx mori, the OBPs are subdivided into three main subfamilies; pheromone-binding proteins (PBPs), general odorant-binding proteins (GOBPs) and antennal-binding proteins (ABPs). We used the MotifSearch algorithm to search for genes encoding putative OBPs in B. mori and found 13, many fewer than are found in the genomes of fruit flies and mosquitoes. The 13 genes include seven new ABP-like OBPs as well as the previously identified PBPs (three), GOBPs (two) and ABPx. Quantitative examination of transcript levels showed that BmorPBP1, BmorGOBP1, BmorGOBP2 and BmorABPx are expressed at very high levels in the antennae and so could be involved in olfaction. A new two-phase binding assay, along with other established assays, showed that BmorPBP1, BmorPBP2, BmorGOBP2 and BmorABPx all bind to the B. mori sex pheromone component (10E,12Z)-hexadecadien-1-ol (bombykol). BmorPBP1, BmorPBP2 and BmorABPx also bind the pheromone component (10E,12Z)-hexadecadienal (bombykal) equally well, whereas BmorGOBP2 can discriminate between bombykol and bombykal. X-ray structures show that when bombykol is bound to BmorGOBP2 it adopts a different conformation from that found when it binds to BmorPBP1. Binding to BmorGOBP2 involves hydrogen bonding to Arg110 rather than to Ser56 as found for BmorPBP1

The angiotensin-converting enzyme (ACE) gene family of Anopheles gambiae

Background Members of the M2 family of peptidases, related to mammalian angiotensin converting enzyme (ACE), play important roles in regulating a number of physiological processes. As more invertebrate genomes are sequenced, there is increasing evidence of a variety of M2 peptidase genes, even within a single species. The function of these ACE-like proteins is largely unknown. Sequencing of the A. gambiae genome has revealed a number of ACE-like genes but probable errors in the Ensembl annotation have left the number of ACE-like genes, and their structure, unclear. Results TBLASTN and sequence analysis of cDNAs revealed that the A. gambiae genome contains nine genes (AnoACE genes) which code for proteins with similarity to mammalian ACE. Eight of these genes code for putative single domain enzymes similar to other insect ACEs described so far. AnoACE9, however, has several features in common with mammalian somatic ACE such as a two domain structure and a hydrophobic C terminus. Four of the AnoACE genes (2, 3, 7 and 9) were shown to be expressed at a variety of developmental stages. Expression of AnoACE3, AnoACE7 and AnoACE9 is induced by a blood meal, with AnoACE7 showing the largest (approximately 10-fold) induction. Conclusion Genes coding for two-domain ACEs have arisen several times during the course of evolution suggesting a common selective advantage to having an ACE with two active-sites in tandem in a single protein. AnoACE7 belongs to a sub-group of insect ACEs which are likely to be membrane-bound and which have an unusual, conserved gene structure

Anopheline salivary protein genes and gene families: an evolutionary overview after the whole genome sequence of sixteen Anopheles species

Background: Mosquito saliva is a complex cocktail whose pharmacological properties play an essential role in blood feeding by counteracting host physiological response to tissue injury. Moreover, vector borne pathogens are transmitted to vertebrates and exposed to their immune system in the context of mosquito saliva which, in virtue of its immunomodulatory properties, can modify the local environment at the feeding site and eventually affect pathogen transmission. In addition, the host antibody response to salivary proteins may be used to assess human exposure to mosquito vectors. Even though the role of quite a few mosquito salivary proteins has been clarified in the last decade, we still completely ignore the physiological role of many of them as well as the extent of their involvement in the complex interactions taking place between the mosquito vectors, the pathogens they transmit and the vertebrate host. The recent release of the genomes of 16 Anopheles species offered the opportunity to get insights into function and evolution of salivary protein families in anopheline mosquitoes. Results: Orthologues of fifty three Anopheles gambiae salivary proteins were retrieved and annotated from 18 additional anopheline species belonging to the three subgenera Cellia, Anopheles, and Nyssorhynchus. Our analysis included 824 full-length salivary proteins from 24 different families and allowed the identification of 79 novel salivary genes and re-annotation of 379 wrong predictions. The comparative, structural and phylogenetic analyses yielded an unprecedented view of the anopheline salivary repertoires and of their evolution over 100 million years of anopheline radiation shedding light on mechanisms and evolutionary forces that contributed shaping the anopheline sialomes. Conclusions: We provide here a comprehensive description, classification and evolutionary overview of the main anopheline salivary protein families and identify two novel candidate markers of human exposure to malaria vectors worldwide. This anopheline sialome catalogue, which is easily accessible as hyperlinked spreadsheet, is expected to be useful to the vector biology community and to improve the capacity to gain a deeper understanding of mosquito salivary proteins facilitating their possible exploitation for epidemiological and/or pathogen-vector-host interaction studies

A deep insight into the sialome of male and female aedes aegypti mosquitoes

Only adult female mosquitoes feed on blood, while both genders take sugar meals. Accordingly, several compounds associated with blood feeding (i.e. vasodilators, anti-clotting, anti-platelets) are found only in female glands, while enzymes associated with sugar feeding or antimicrobials (such as lysozyme) are found in the glands of both sexes. We performed de novo assembly of reads from adult Aedes aegypti female and male salivary gland libraries (285 and 90 million reads, respectively). By mapping back the reads to the assembled contigs, plus mapping the reads from a publicly available Ae. aegypti library from adult whole bodies, we identified 360 transcripts (including splice variants and alleles) overexpressed tenfold or more in the glands when compared to whole bodies. Moreover, among these, 207 were overexpressed fivefold or more in female vs. male salivary glands, 85 were near equally expressed and 68 were overexpressed in male glands. We call in particular the attention to C-type lectins, angiopoietins, female-specific Antigen 5, the 9.7 kDa, 12–14 kDa, 23.5 kDa, 62/34 kDa, 4.2 kDa, proline-rich peptide, SG8, 8.7 kDa family and SGS fragments: these polypeptides are all of unknown function, but due to their overexpression in female salivary glands and putative secretory nature they are expected to affect host physiology. We have also found many transposons (some of which novel) and several endogenous viral transcripts (probably acquired by horizontal transfer) which are overexpressed in the salivary glands and may play some role in tissue-specific gene regulation or represent a mechanism of virus interference. This work contributes to a near definitive catalog of male and female salivary gland transcripts from Ae. aegypti, which will help to direct further studies aiming at the functional characterization of the many transcripts with unknown function and the understanding of their role in vector-host interaction and pathogen transmission

The Spermatophore in Glossina morsitans morsitans: Insights into Male Contributions to Reproduction.

Male Seminal Fluid Proteins (SFPs) transferred during copulation modulate female reproductive physiology and behavior, impacting sperm storage/use, ovulation, oviposition, and remating receptivity. These capabilities make them ideal targets for developing novel methods of insect disease vector control. Little is known about the nature of SFPs in the viviparous tsetse flies (Diptera: Glossinidae), vectors of Human and Animal African trypanosomiasis. In tsetse, male ejaculate is assembled into a capsule-like spermatophore structure visible post-copulation in the female uterus. We applied high-throughput approaches to uncover the composition of the spermatophore in Glossina morsitans morsitans. We found that both male accessory glands and testes contribute to its formation. The male accessory glands produce a small number of abundant novel proteins with yet unknown functions, in addition to enzyme inhibitors and peptidase regulators. The testes contribute sperm in addition to a diverse array of less abundant proteins associated with binding, oxidoreductase/transferase activities, cytoskeletal and lipid/carbohydrate transporter functions. Proteins encoded by female-biased genes are also found in the spermatophore. About half of the proteins display sequence conservation relative to other Diptera, and low similarity to SFPs from other studied species, possibly reflecting both their fast evolutionary pace and the divergent nature of tsetse's viviparous biology