@article{jeffers_shen_bissinger_khalil_gunnoe_roe_2014, title={Polymers for the stabilization and delivery of proteins topically and per os to the insect hemocoel through conjugation with aliphatic polyethylene glycol}, volume={115}, ISSN={["1095-9939"]}, DOI={10.1016/j.pestbp.2014.08.006}, abstractNote={Co-feeding of aliphatic polyethylene glycol (PEG), phospholipase A2, anionic and ionic detergents, and amphipathic glycoside with bovine serum albumin (BSA) as a model protein to fourth stadium tobacco budworms, Heliothis virescens, did not affect the levels of BSA in the hemolymph. Covalent conjugation of small proteins like the decapeptide trypsin modulating oostatic factor (TMOF) to polyethylene glycol was previously shown to protect the peptide from protease attack and enhance its accumulation in the insect hemocoel. Whether this polymer chemistry could do the same for larger proteins was examined. The chemistry for the synthesis of polydispersed aliphatic PEG350-insulin and monodispersed aliphatic PEG333-insulin are described herein. Insulin was used for this synthesis and not BSA to better control conjugation among the available free amine groups. When PEGylated insulin or free insulin were fed in artificial diet to fifth stadium budworms, greater concentrations of insulin using the PEGylated variants were found in the hemolymph than when free insulin was used (a 6.7 and 7.3-fold increase for the PEG350 and PEG333 conjugates, respectively). When insulin is topically applied to the dorsum of H. virescens, no insulin is found in the hemolymph. However, after topical application of the PEGylated insulins, PEG350-insulin and PEG333-insulin were detected in the hemolymph. After injections of insulin into the hemocoel of fourth stadium H. virescens, insulin is completely cleared from the hemolymph in 120 min. In comparison, PEG350-insulin and PEG333-insulin were present in the hemolymph for 300 and 240 min after injection, respectively, translating to a 3.3 and 2.7-fold increase in the length of time insulin remains in the hemolymph after injection.}, journal={PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY}, author={Jeffers, Laura A. and Shen, Hongyan and Bissinger, Brooke W. and Khalil, Sayed and Gunnoe, T. Brent and Roe, R. Michael}, year={2014}, month={Oct}, pages={58–66} }
 @article{jeffers_shen_khalil_bissinger_brandt_gunnoe_roe_2012, title={Enhanced activity of an insecticidal protein, trypsin modulating oostatic factor (TMOF), through conjugation with aliphatic polyethylene glycol}, volume={68}, ISSN={["1526-498X"]}, DOI={10.1002/ps.2219}, abstractNote={Abstract}, number={1}, journal={PEST MANAGEMENT SCIENCE}, author={Jeffers, Laura A. and Shen, Hongyan and Khalil, Sayed and Bissinger, Brooke W. and Brandt, Alan and Gunnoe, T. Brent and Roe, R. Michael}, year={2012}, month={Jan}, pages={49–59} }
 @article{khalil_donohue_thompson_jeffers_ananthapadmanaban_sonenshine_mitchell_roe_2011, title={Full-length sequence, regulation and developmental studies of a second vitellogenin gene from the American dog tick, Dermacentor variabilis}, volume={57}, ISSN={["1879-1611"]}, DOI={10.1016/j.jinsphys.2010.12.008}, abstractNote={Vitellogenin (Vg) is the precursor of vitellin (Vn) which is the major yolk protein in eggs. In a previous report, we isolated and characterized the first Vg message from the American dog tick Dermacentor variabilis. In the current study, we describe a second Vg gene from the same tick. The Vg2 cDNA is 5956 nucleotides with a 5775 nt open reading frame coding for 1925 amino acids. The conceptual amino acid translation contains a 16-residues putative signal peptide, N-terminal lipid binding domain and C-terminal von Willebrand factor type D domain present in all known Vgs. Moreover, the amino acid sequence shows a typical GLCG domain and several RXXR cleavage sites present in most isolated Vgs. Tryptic digest-mass fingerprinting of Vg and Vn recognized 11 fragments that exist in the amino acid translation of DvVg2 cDNA. Injection of virgin females with 20 hydroxyecdysone induced DvVg2 expression, vitellogenesis and oviposition. Using RT-PCR, DvVg2 expression was detected only in tick females after mating and feeding to repletion. Northern blot analysis showed that DvVg2 is expressed in fat body and gut cells of vitellogenic females but not in the ovary. DvVg2 expression was not detected in adult fed or unfed males. The characteristics that distinguish Vg from other similar tick storage proteins like the carrier protein, CP (another hemelipoglycoprotein) are discussed.}, number={3}, journal={JOURNAL OF INSECT PHYSIOLOGY}, author={Khalil, Sayed M. S. and Donohue, Kevin V. and Thompson, Deborah M. and Jeffers, Laura A. and Ananthapadmanaban, Usha and Sonenshine, Daniel E. and Mitchell, Robert D. and Roe, R. Michael}, year={2011}, month={Mar}, pages={400–408} }
 @misc{jeffers_roe_2008, title={The movement of proteins across the insect and tick digestive system}, volume={54}, ISSN={["1879-1611"]}, DOI={10.1016/j.jinsphys.2007.10.009}, abstractNote={The movement of intact proteins across the digestive system was shown in a number of different blood-feeding and non-blood-feeding insects in the orders Blattaria, Coleoptera, Diptera, Hemiptera, Lepidoptera, Orthoptera, Neuroptera and Siphonaptera, as well as in two tick families Ixodidae and Argasidae. Protein movement was observed for both normal dietary and xenobiotic proteins, which suggest that the mechanism for transfer is not substrate specific. The number of studies on the mechanism of movement is limited. The research so far suggests that movement can occur by either a transcellular or an intercellular pathway in the ventriculus with most of the research describing the former. Transfer is by continuous diffusion with no evidence of pinocytosis or vesicular transport common in mammalian systems. Proteins can move across the digestive system without modification of their primary or multimeric structure and with retention of their functional characteristics. Accumulation in the hemolymph is the result of the protein degradation rate in the gut and hemolymph and transfer rate across the digestive system and can be highly variable depending on species. Research on the development of delivery systems to enhance protein movement across the insect digestive system is in its infancy. The approaches so far considered with some success include the use of lipophilic-polyethylene glycol (PEG) polymers, the development of fusion proteins with lectins, reduced gut protease activity and the development of amphiphilic peptidic analogs. Additional research on understanding the basic mechanisms of protein delivery across the insect digestive system, the importance of structure activity in this transfer and the development of technology to improve movement across the gut could be highly significant to the future of protein and nucleic acid-based insecticide development as well as traditional chemical insecticidal technologies.}, number={2}, journal={JOURNAL OF INSECT PHYSIOLOGY}, author={Jeffers, Laura A. and Roe, R. Michael}, year={2008}, month={Feb}, pages={319–332} }
 @article{deborah_khalil_jeffers_sonenshine_mitchell_osgood_roe_2007, title={Sequence and the developmental and tissue-specific regulation of the first complete vitellogenin messenger RNA from ticks responsible for heme sequestration}, volume={37}, ISSN={["0965-1748"]}, DOI={10.1016/j.ibmb.2007.01.004}, abstractNote={The first full-length mRNA for vitellogenin (Vg) from ticks was sequenced. This also represents the first complete sequence of Vg from the Chelicerata and of a heme binding Vg. The Vg cDNA from the American dog tick, Dermacentor variabilis was 5744nt in length (GenBank Accession number AY885250), which coded for a protein of 1843 aa with a calculated molecular weight of 208 kD. This protein had an 18 aa signal sequence, a single RXXR cleavage signal that would generate two subunits (49.5 and 157K in molecular weight) and lipoprotein N-terminal and carboxy von Willebrand factor type D domains. Tryptic digest MS analysis of vitellin protein confirmed the function of the cDNA as the tick yolk protein. Apparently, vitellin in D. variabilis is oligomeric (possibly dimeric) and is comprised of a mixture of the uncleaved monomer and subunits that were predicted from the single RXXR cleavage signal. The highly conserved GL/ICG motif close to the C-terminus in insect Vg genes was different in the tick Vg message, i.e., GLCS. This variant was also present in a partial sequence of Vg from Boophilus microplus. Phylogenic analysis showed that the full length Vg cDNA from D. variabilis and the partial cDNA from B. microplus were distinct from insects and Crustacea. The Vg message was not found in whole body RNA from unfed or fed males or in unfed and partially fed (virgin) females as determined by Northern blotting. The message was found in replete (mated) pre-ovipositional females, increased to higher levels in ovipositing females and was absent after egg laying was complete. The endocrine regulation of the Vg mRNA is discussed. The tissue sources of the Vg message are both the gut and fat body. Tryptic digest MS fingerprinting suggests that a second Vg mRNA might be present in the American dog tick, which needs further study.}, number={4}, journal={INSECT BIOCHEMISTRY AND MOLECULAR BIOLOGY}, author={Deborah, M. Thompson and Khalil, Sayed M. S. and Jeffers, Laura A. and Sonenshine, Daniel E. and Mitchell, Robert D. and Osgood, Christopher J. and Roe, R. Michael}, year={2007}, month={Apr}, pages={363–374} }
 @article{thompson_khalil_jeffers_ananthapadmanaban_sonenshine_mitchell_osgood_apperson_roe_2005, title={In vivo role of 20-hydroxyecdysone in the regulation of the vitellogenin mRNA and egg development in the American dog tick, Dermacentor variabilis (Say)}, volume={51}, ISSN={["1879-1611"]}, DOI={10.1016/j.jinsphys.2005.05.011}, abstractNote={Injection of the hormone 20-hydroxyecdysone (20-E) into partially fed (virgin) female adults of the American dog tick, Dermacentor variabilis, while they are attached and feeding on the rabbit host, initiated the expression of the vitellogenin (Vg) gene, and Vg protein secretion and uptake by the ovary. The induction of egg production by 20-E in this bioassay was dose dependent in the range of 1–50 times the concentration normally found in a replete, vitellogenic female. Ticks examined 4 d after the 50× treatment were still attached to the host, had numerous enlarged vitellin-filled (brown) oocytes in their ovaries, but had not engorged to repletion. The ovaries reached weights similar to those found in untreated, replete (mated) females (pre-oviposition) while solvent-injected controls demonstrated no increase in oocyte size or increase in ovary weight. An increase in the levels of a putative Vg protein was observed in hemolymph samples collected 1, 2 and 3 d post-20-E injection but was not observed in the corresponding solvent controls as determined by native PAGE. Analysis of the ecdysteroid-induced protein by tryptic digestion-mass fingerprinting and BLASTP found that the putative Vg had the strongest match to GP80 (U49934), the partial sequence for the vitellogenin protein from Boophilus microplus. A partial Vg cDNA was cloned and sequenced from replete females of D. variabilis with a high similarity to GP80. Using this message as a probe, Northern blots conducted with RNA collected from partially fed, virgin females 1, 2 and 3 d post-20-E injection showed upregulation of the Vg mRNA on all 3 days. Controls injected with solvent only showed no Vg mRNA. Injections with juvenile hormone III did not stimulate Vg expression, oocyte growth or full engorgement. These studies indicate that ecdysteroids and not JH can initiate expression of the Vg gene, Vg protein synthesis and release into hemolymph, and Vg uptake into developing oocytes under bioassay conditions mimicking normal feeding on the host.}, number={10}, journal={JOURNAL OF INSECT PHYSIOLOGY}, author={Thompson, DM and Khalil, SMS and Jeffers, LA and Ananthapadmanaban, U and Sonenshine, DE and Mitchell, RD and Osgood, CJ and Apperson, CS and Roe, RM}, year={2005}, month={Oct}, pages={1105–1116} }