Effect of temperature on development and reproduction of the carmine spider mite, Tetranychus cinnabarinus (Acari: Tetranychiae), fed on cassava leaves.

The effect of five constant temperatures (16, 20, 24, 28 and 32 °C) on the development, survival and reproduction of Tetranychus cinnabarinus (Boisduval) [= Tetranychus urticae Koch (red form)] fed on cassava leaves was examined in the laboratory at 85% relative humidity. Development time of various immature stages decreased with increasing temperature, with total egg-to-adult development time varying from 27.7 to 6.7 days. The lower thermal threshold for development was 10.8 °C and the thermal constant from egg to adult was 142.4 degree-days. Pre- and post-oviposition period and female longevity all decreased as temperature increased. The longest oviposition period was observed at 20 °C with 20.4 days. Under different temperatures, mated females laid, on average, 1.0, 2.9, 4.7, 4.7 and 4.9 eggs per day, respectively. The maximum fecundity (81.5 eggs per female) was at 28 °C and the intrinsic rate of increase (r m ) was highest (0.25) at 32 °C. The results of this study indicate that T. cinnabarinus population could increase rapidly when cassava leaves serve as a food source. At the appropriate temperature T. cinnabarinus could seriously threaten growth of cassava.

Impact of PEGylation on biodistribution and tumor accumulation of Lipid-Mu peptide-DNA.

For gene-based therapeutic approaches that require transfection of specific cell targets in vivo, it is important to design the delivery system to have optimized tissue distribution. Surface PEGylation of liposomes and polymer nanoparticles has been shown to lead to prolonged blood circulation and also preferential accumulation in tumor sites resulting from the enhanced permeability and retention (EPR) effect. The aim of this study was to investigate the effect of different surface PEGylation densities on resulted biodistribution profiles of Lipid-Mu peptide-DNA (LMD) nanoparticles. LMD particles containing the near infrared fluorescent lipid dye, Cy5.5-DSPE, were injected intravenously, and whole-body fluorescence images of the live animal were recorded. Analysis of these time series of images indicated that LMDs with different surface PEG(2000) densities had distinctively different biodistribution and tumor accumulation profiles. LMDs containing approximately 15-25% of surface PEG(2000) were shown to have the highest accumulation and longest residence time in tumor. LMD distribution and pharmacokinetic profiles in other organs were also observed to be different and were analyzed.

Efficient microbubble- and ultrasound-mediated plasmid DNA delivery into a specific rat liver lobe via a targeted injection and acoustic exposure using a novel ultrasound system.

To develop efficient gene delivery in larger animals, based on a previous mouse study, we explored the luciferase reporter gene transfer in rats by establishing a novel unfocused ultrasound system with simultaneous targeted injection of a plasmid and microbubble mixture into a specific liver lobe through a portal vein branch. Luciferase expression was significantly enhanced over 0-30 vol % of the Definity microbubbles, with a plateau between 0.5 and 30 vol %. The increase of gene delivery efficiency also depended on the acoustic peak negative pressure, achieving over 100-fold enhancement at 2.5 MPa compared with plasmid only controls. Transient, modest liver damage following treatment was assessed by transaminase assays and histology, both of which correlated with gene expression induced by acoustic cavitation. In addition, pulse-train ultrasound exposures (i.e., with relatively long quiescent periods between groups of pulses to allow tissue refill with microbubbles) produced gene expression levels comparable to the standard US exposure but reduced the extent of liver damage. These results indicated that unfocused high intensity therapeutic ultrasound exposure with microbubbles is highly promising for safe and efficient gene delivery into the liver of rats or larger animals.

Ultrasound-mediated gene delivery of factor VIII plasmids for hemophilia A gene therapy in mice.

Gene therapy offers great promises for a cure of hemophilia A resulting from factor VIII (FVIII) gene deficiency. We have developed and optimized a non-viral ultrasound-mediated gene delivery (UMGD) strategy. UMGD of reporter plasmids targeting mice livers achieved high levels of transgene expression predominantly in hepatocytes. Following UMGD of a plasmid encoding human FVIII driven by a hepatocyte-specific promoter/enhancer (pHP-hF8/N6) into the livers of hemophilia A mice, a partial phenotypic correction was achieved in treated mice. In order to achieve persistent and therapeutic FVIII gene expression, we adopted a plasmid (pHP-hF8-X10) encoding an FVIII variant with significantly increased FVIII secretion. By employing an optimized pulse-train ultrasound condition and immunomodulation, the treated hemophilia A mice achieved 25%-150% of FVIII gene expression on days 1-7 with very mild transient liver damage, as indicated by a small increase of transaminase levels that returned to normal within 3 days. Therapeutic levels of FVIII can be maintained persistently without the generation of inhibitors in mice. These results indicate that UMGD can significantly enhance the efficiency of plasmid DNA transfer into the liver. They also demonstrate the potential of this novel technology to safely and effectively treat hemophilia A.

Novel peptide ligand directs liposomes toward EGF-R high-expressing cancer cells in vitro and in vivo.

Epidermal growth factor receptor (EGF-R) is an important target in anticancer therapy. Here we report how a novel EGF-R peptide ligand (D4: Leu-Ala-Arg-Leu-Leu-Thr) is identified using a computer-aided design approach from a virtual peptide library of putative EGF-R binding peptides by screening against the EGF-R X-ray crystal structure in silico and in vitro. The selected peptide is conjugated with a polyethylene glycol (PEG) lipid, and the lipid moiety of the peptide-PEG-lipid conjugate is inserted into liposome membranes by a postmodification process. D4 peptide-conjugated liposomes are found to bind to and enter cells by endocytosis specifically and efficiently in vitro in a process apparently mediated by EGF-R high-expressing cancer cells (H1299). In vivo, the D4 peptide-conjugated liposomes are found to accumulate in EGF-R-expressing xenograft tumor tissues up to 80 h after intravenous delivery, in marked contrast to controls. These results demonstrate how structure-based peptide design can be an efficient approach to identify highly novel binding ligands against important receptors. These data could have important consequences for the development of peptide-directed drug delivery systems with engineered specificities and prolonged times of action.

The Predicting Power of Cognitive Fluency for the Development of Utterance Fluency in Simultaneous Interpreting.

Although simultaneous interpreting (SI) is generally recognized as a highly demanding cognitive activity in nature, the role of cognitive processes in SI fluency is yet to be determined. While utterance fluency refers to the set of objectively determined oral features of utterances, cognitive fluency means the speaker's efficient mobilization and integration of underlying cognitive processes responsible for utterance production. An investigation into the relationship of the two dimensions of fluency helps to reveal the cognitive bases of interpreting. This study explores the predicting power of cognitive fluency in the utterance fluency development of L2 (English)-L1 (Chinese) SI output of trainee interpreters. Cognitive fluency was operationalized as measures of lexical access, linguistic attention control, and working memory capacity. Measures of utterance fluency were obtained through simulated SI tasks under conditions of low and high input rates. Twenty-eight trainees interpreted two speeches, one with a high input rate and the other with a low input rate, at the beginning and end of an SI training period of 13 weeks. A bilingual corpus of the participants' SI output was built, and indicators of SI utterance fluency were annotated systematically. Utterance fluency was indexed by the speech rate, mean length of run, phonation time ratio, mean number of silent pauses, and mean number of disfluencies. Results of analyses indicated that (1) the predicting power of cognitive fluency for SI utterance fluency development was only shown under high cognitive load over a training period of 13 weeks; (2) predictors for the development of SI utterance fluency tended to be the efficiency of cognitive processes involved in the target language production stage; and (3) the inclusion of measures of working memory capacity significantly increased the predicting power of cognitive fluency for SI utterance fluency development. This study for the first time provides evidence for the role of cognitive fluency in trainee interpreters' SI utterance fluency development, having implications for the theoretical framework of cognitive fluency and the information processing mechanism in interpreting process, as well as for interpreter aptitude tests and interpreting pedagogy.

Peptide ligand-mediated liposome distribution and targeting to EGFR expressing tumor in vivo.

Epidermal growth factor receptor (EGFR) is an important anti-cancer therapy target that is applicable to many cancer types. We had previously reported the screening and discovery of a novel peptide ligand against EGFR named GE11. It was shown to bind to EGFR competitively with EGF and mediate gene delivery to cancer cells with high-EGFR expression. In this study, we conjugated GE11 on to liposome surface and examined their binding and distribution to EGFR expressing cancer cells in vitro and in vivo using fluorescence imaging techniques. GE11 liposomes were found to bind specifically and efficiently to EGFR high-expressing cancer cells. In vivo in H1299 xenograft mouse model, GE11 liposomes also extravasated and accumulated into the tumor site preferentially, and demonstrated better targeting and drug delivery capacities.

Prolonging pulse duration in ultrasound-mediated gene delivery lowers acoustic pressure threshold for efficient gene transfer to cells and small animals.

While ultrasound-mediated gene delivery (UMGD) has been accomplished using high peak negative pressures (PNPs) of 2 MPa or above, emerging research showed that this may not be a requirement for microbubble (MB) cavitation. Thus, we investigated lower-pressure conditions close to the MB inertial cavitation threshold and focused towards further increasing gene transfer efficiency and reducing associated cell damage. We created a matrix of 21 conditions (n = 3/cond.) to test in HEK293T cells using pulse durations spanning 18 µs-36 ms and PNPs spanning 0.5-2.5 MPa. Longer pulse duration conditions yielded significant increase in transgene expression relative to sham with local maxima between 20 J and 100 J energy curves. A similar set of 17 conditions (n = 4/cond.) was tested in mice using pulse durations spanning 18 µs-22 ms and PNPs spanning 0.5-2.5 MPa. We observed local maxima located between 1 J and 10 J energy curves in treated mice. Of these, several low pressure conditions showed a decrease in ALT and AST levels while maintaining better or comparable expression to our positive control, indicating a clear benefit to allow for effective transfection with minimized tissue damage versus the high-intensity control. Our data indicates that it is possible to eliminate the requirement of high PNPs by prolonging pulse durations for effective UMGD in vitro and in vivo, circumventing the peak power density limitations imposed by piezo-materials used in US transducers. Overall, these results demonstrate the advancement of UMGD technology for achieving efficient gene transfer and potential scalability to larger animal models and human application.

Ultrasound-Mediated Gene Therapy in Swine Livers Using Single-Element, Multi-lensed, High-Intensity Ultrasound Transducers.

We have achieved significant enhancement of gene delivery into livers of large animals using ultrasound (US)-targeted microbubble (MB) destruction methods. An infusion of pGL4 (encoding a luciferase reporter gene) plasmid DNA (pDNA) and MBs into a portal-vein segmental branch of a porcine liver was exposed to US for 4 min. Therapeutic US induced cavitation of MBs to temporarily permeabilize the vascular endothelium and cell membranes, allowing entry of pDNA. We obtained a 64-fold enhancement in luciferase expression in pig livers compared to control without US using an unfocused, dual-element transducer (H105, center frequency [fc] = 1.10 MHz) at 2.7 MPa peak negative pressure (PNP). However, input electrical energy was limited, and modified transducers were designed to have spherical (H185A, fc = 1.10 MHz) or cylindrical foci (H185B, fc = 1.10 MHz; H185D, fc = 1.05 MHz) to enhance PNP output. The revised transducers required less electrical input to achieve 2.7 MPa PNP compared to H105, thereby allowing PNP outputs of up to 6.2 MPa without surpassing the piezo-material limitations. Subsequently, luciferase expression significantly improved up to 9,000-fold compared to controls with minor liver damage. These advancements will allow us to modify our current protocols toward minimally invasive US gene therapy.

Development of therapeutic microbubbles for enhancing ultrasound-mediated gene delivery.

Ultrasound (US)-mediated gene delivery has emerged as a promising non-viral method for safe and selective gene delivery. When enhanced by the cavitation of microbubbles (MBs), US exposure can induce sonoporation that transiently increases cell membrane permeability for localized delivery of DNA. The present study explores the effect of generalizable MB customizations on MB facilitation of gene transfer compared to Definity®, a clinically available contrast agent. These modifications are 1) increased MB shell acyl chain length (RN18) for elevated stability and 2) addition of positive charge on MB (RC5K) for greater DNA associability. The MB types were compared in their ability to facilitate transfection of luciferase and GFP reporter plasmid DNA in vitro and in vivo under various conditions of US intensity, MB dosage, and pretreatment MB-DNA incubation. The results indicated that both RN18 and RC5K were more efficient than Definity®, and that the cationic RC5K can induce even greater transgene expression by increasing payload capacity with prior DNA incubation without compromising cell viability. These findings could be applied to enhance MB functions in a wide range of therapeutic US/MB gene and drug delivery approach. With further designs, MB customizations have the potential to advance this technology closer to clinical application.

A synthetic peptide mediated active targeting of cisplatin liposomes to Tie2 expressing cells.

Tie2 receptor is a receptor tyrosine kinase that plays important roles in vascular angiogenesis, and also highly expressed by a number of cancer cells. In this study, we reported an active targeting liposome system directed by a novel peptide ligand PH1 that can improve drug efficacies specifically to Tie2 expressing cells. The PH1 peptide (TMGFTAPRFPHY) was selected by phage display library screening combined with surface plasmon resonance binding assays. It was covalently conjugated to the distal end of DSPE-PEG(2000)-Maleimide lipid and loaded onto liposome membranes as the targeting ligand. These PH1-PEG-liposomes containing the anticancer drug cisplatin were showed to bind tightly to Tie2 positive cells, mediate active endocytosis of the drug containing liposomes, and result in much higher cell specific cytoxicities than mPEG coated liposomes. They can be used not only to target vascular endothelial cells for anti-angiogenesis effects, but also to improve drug delivery and release in Tie2 expressing cancer cells. Such liposome formulation may be developed into a very useful agent for metronomic chemotherapy.

A convenient and adjustable surface-modified complex containing poly-L-glutamic acid conjugates as a vector for gene delivery.

In order to quantify the amount of ligands or poly(ethylene glycol) (PEG) on each vector, here we developed a system in which poly-L-glutamic acid (PLG) was used as surface modification loading backbone, to which one PEG (MW 5000, 10000, 20000) or epidermal growth factor (EGF) was linked. The PLG conjugates can electro-statically adsorb upon DNA/ polycation complex with positive charge, and, the amount of EGF or PEG on the surface of complexes could be varied. We have made a series of complexes containing the various PLG conjugates and examined their physicochemical properties, and made a comparison of properties and transfection efficiency between these complexes. EGF- and PEG-modified complexes showed 10-25-folds higher cell transfection efficiency than unmodified complexes in medium with or without serum.