Escape Ability Research Articles

Enhanced transfection efficiency and targeted delivery of self-assembling h-R3-dendriplexes in EGFR-overexpressing tumor cells.

The efficient gene transfection, cellular uptake and targeted delivery in vivo are key issues for non-viral gene delivery vectors in cancer therapy. To solve these issues, we designed a new targeted gene delivery system based on epidermal growth factor receptor (EGFR) targeting strategy. An anti-EGFR monoclonal antibody h-R3 was introduced to dendriplexes of PAMAM and DNA via electrostatic interactions to form self-assembled h-R3-PAMAM-DNA complexes (h-R3-dendriplexes). Dendriplexes h-R3-dendriplexes represented excellent DNA encapsulation ability and formed unique nanostructures. Compared to dendriplexes, h-R3-dendriplexes presented lower cytotoxicity, higher gene transfection efficiency, excellent endosome escape ability and high nuclear accumulation in the EGFR-overexpressing HepG2 cells. Both ex vivo fluorescence imaging and confocal results of frozen section revealed that h-R3-dendriplexes showed higher targeted delivery and much better gene expression in the tumors than dendriplexes at the same N/P ratio, and h-R3-dendriplexes had accumulation primarily in the tumor and kidney. Moreover, h-R3-dendriplexes for p53 delivery indicated efficient cell growth inhibition and potentiated paclitaxel-induced cell death. These results indicate that the h-R3-dendriplexes represent a great potential to be used as efficient targeted gene delivery carriers in EGFR-overexpressing tumor cells.

Low dose photodynamic-therapy induce immune escape of tumor cells in a HIF-1α dependent manner through PI3K/Akt pathway.

As a regulatory approved antitumor therapy, photodynamic therapy (PDT) shows poor effect in clinical application. The current study aimed at investigating the mechanism through which low-dose PDT affects the immune escape ability of Lewis lung carcinoma (LLC) cells. Our data show that low-dose PDT could increase HIF-1α expression through the activation of the PI3K/Akt pathway. Our results also show that low-dose PDT treatment can increase tumor growth rate in C57BL/6 mice, reducing the survival of tumor-bearing mice. Furthermore, low-dose PDT could increase regulatory T cells (Tregs) number, decrease the cytotoxic T lymphocytes (CTLs) number, and induce CTLs apoptosis in co-culture system in vitro. The immunosuppression caused by low dose PDT could be partially abolished by knocking down the hypoxia-inducible factor 1α (HIF-1α) and blocking the phosphatidylinositol 3-kinase (PI3K). Our results suggest that low dose PDT may enhance immune escape of LLC cells in a HIF-1α dependent manner through PI3K/Akt pathway. The present study thus offers a new insight on elucidating the mechanism whereby low dose PDT induces tumor progression and therapy-resistance, providing a novel approach for cancer immunotherapy.

A family of cationic polyamides for in vitro and in vivo gene transfection

The purpose of this study is to develop biodegradable cationic polyamides for non-viral gene delivery and elucidate their structural effects on gene transfection activity. To this end, a group of novel cationic polyamides were synthesized by polycondensation reaction between different di-p-nitrophenyl esters and tertiary amine-containing primary diamines. These linear polyamides have flexible alkylene group (ethylene or propylene), protonable amino group and bioreducible disulfide linkage in the polyamide main chain. The alkylene group and disulfide linkage in these polyamides have a distinct effect on their gene delivery properties including buffering capacity, gene binding ability and intracellular gene release profile. Those cationic polyamides containing disulfide linkage and 1,4-bis(3-aminopropyl)piperazine (BAP) residue exhibited high buffering capacity (endosomal escape ability), high gene binding ability, and intracellular gene release ability, thus inducing fast gene nucleus translocation and robust gene transfection in vitro against different cell lines and rat bone marrow mesenchymal stem cells. Moreover, the transfection efficiencies in vitro were comparable or higher than those of 25kDa branched polyethylenimine and Lipofectamine 2000 transfection agent as positive controls. These cationic polyamides and their polyplexes were of low cytotoxicity when an optimal transfection efficacy was achieved. In vivo transfection tests showed that bioreducible BAP-based polyamides were applicable for intravenous gene delivery in a mouse model, leading to higher level of transgene expression in the liver as compared to 22kDa linear polyethylenimine as a positive control. These cationic polyamides provide a useful platform to elucidate the relationship between chemical functionalities and gene transfection activity.

The interplay between aerobic metabolism and antipredator performance: vigilance is related to recovery rate after exercise.

When attacked by a predator, fish respond with a sudden fast-start motion away from the threat. Although this anaerobically-powered swimming necessitates a recovery phase which is fueled aerobically, little is known about links between escape performance and aerobic traits such as aerobic scope (AS) or recovery time after exhaustive exercise. Slower recovery ability or a reduced AS could make some individuals less likely to engage in a fast-start response or display reduced performance. Conversely, increased vigilance in some individuals could permit faster responses to an attack but also increase energy demand and prolong recovery after anaerobic exercise. We examined how AS and the ability to recover from anaerobic exercise relates to differences in fast-start escape performance in juvenile golden gray mullet at different acclimation temperatures. Individuals were acclimated to either 18, 22, or 26°C, then measured for standard and maximal metabolic rates and AS using intermittent flow respirometry. Anaerobic capacity and the time taken to recover after exercise were also assessed. Each fish was also filmed during a simulated attack to determine response latency, maximum speed and acceleration, and turning rate displayed during the escape response. Across temperatures, individuals with shorter response latencies during a simulated attack are those with the longest recovery time after exhaustive anaerobic exercise. Because a short response latency implies high preparedness to escape, these results highlight the trade-off between the increased vigilance and metabolic demand, which leads to longer recovery times in fast reactors. These results improve our understanding of the intrinsic physiological traits that generate inter-individual variability in escape ability, and emphasize that a full appreciation of trade-offs associated with predator avoidance and energy balance must include energetic costs associated with vigilance and recovery from anaerobic exercise.

Prey size selection and cannibalistic behaviour of juvenile barramundi Lates calcarifer.

This study assessed the cannibalistic behaviour of juvenile barramundi Lates calcarifer and examined the relationship between prey size selection and energy gain of cannibals. Prey handling time and capture success by cannibals were used to estimate the ratio of energy gain to energy cost in prey selection. Cannibals selected smaller prey despite its capability of ingesting larger prey individuals. In behavioural analysis, prey handling time significantly increased with prey size, but it was not significantly affected by cannibal size. Conversely, capture success significantly decreased with the increase of both prey and cannibal sizes. The profitability indices showed that the smaller prey provides the most energy return for cannibals of all size classes. These results indicate that L. calcarifer cannibals select smaller prey for more profitable return. The behavioural analysis, however, indicates that L. calcarifer cannibals attack prey of all size at a similar rate but ingest smaller prey more often, suggesting that prey size selection is passively orientated rather than at the predator's choice. The increase of prey escape ability and morphological constraint contribute to the reduction of intracohort cannibalism as fish grow larger. This study contributes to the understanding of intracohort cannibalism and development of strategies to reduce fish cannibalistic mortalities.

Zwitterionic poly(carboxybetaine)-based cationic liposomes for effective delivery of small interfering RNA therapeutics without accelerated blood clearance phenomenon.

For efficient delivery of small interfering RNA (siRNA) to the target diseased site in vivo, it is important to design suitable vehicles to control the blood circulation of siRNA. It has been shown that surface modification of cationic liposome/siRNA complexes (lipoplexes) with polyethylene glycol (PEG) could enhance the circulation time of lipoplexes. However, the first injection of PEGylated lipoplexes in vivo induces accelerated blood clearance and enhances hepatic accumulation of the following injected PEGylated lipoplexes, which is known as the accelerated blood clearance (ABC) phenomenon. Herein, we developed zwitterionic poly(carboxybetaine) (PCB) modified lipoplexes for the delivery of siRNA therapeutics, which could avoid protein adsorption and enhance the stability of lipoplexes as that for PEG. Quite different from the PEGylation, the PCBylated lipoplexes could avoid ABC phenomenon, which extended the blood circulation time and enhanced the tumor accumulation of lipoplexes in vivo. After accumulation in tumor site, the PCBylation could promote the cellular uptake and endosomal/lysosomal escape of lipoplexes due to its unique chemical structure and pH-sensitive ability. With excellent tumor accumulation, cellular uptake and endosomal/lysosomal escape abilities, the PCBylated lipoplexes significantly inhibited tumor growth and induced tumor cell apoptosis.

Aposematism increases acoustic diversification and speciation in poison frogs.

Multimodal signals facilitate communication with conspecifics during courtship, but they can also alert eavesdropper predators. Hence, signallers face two pressures: enticing partners to mate and avoiding detection by enemies. Undefended organisms with limited escape abilities are expected to minimize predator recognition over mate attraction by limiting or modifying their signalling. Alternatively, organisms with anti-predator mechanisms such as aposematism (i.e. unprofitability signalled by warning cues) might elaborate mating signals as a consequence of reduced predation. We hypothesize that calls diversified in association with aposematism. To test this, we assembled a large acoustic signal database for a diurnal lineage of aposematic and cryptic/non-defended taxa, the poison frogs. First, we showed that aposematic and non-aposematic species share similar extinction rates, and aposematic lineages diversify more and rarely revert to the non-aposematic phenotype. We then characterized mating calls based on morphological (spectral), behavioural/physiological (temporal) and environmental traits. Of these, only spectral and temporal features were associated with aposematism. We propose that with the evolution of anti-predator defences, reduced predation facilitated the diversification of vocal signals, which then became elaborated or showy via sexual selection.

Adaptive acceleration in growth and development of salamander hatchlings in cannibalistic situations

Summary In most animal species, the hatchling stage is a highly vulnerable life‐history stage. In many fish and amphibian species, hatchling abundance varies substantially among sites and years, with the result that selection strength in conspecific interactions such as cannibalism is also variable. The variability of selection leads species to evolve phenotypic plasticity, and adaptive trait changes in hatchlings that depend on the density of conspecifics can therefore be expected. However, plasticity strategies in response to cannibalistic interactions in this vulnerable life‐history stage have received little attention. Because cannibalism success is dependent on the size balance between predator capturing organ and prey body, and also on the prey‐capturing ability of the cannibal and the escape ability of its prey, we hypothesize that hatchlings will exhibit faster growth and development in response to conspecific interactions. We performed a series of experiments to test this hypothesis, using pre‐feeding larvae of a cannibalistic salamander (Hynobius retardatus). Many traits of the hatchlings reared with conspecifics were larger than the same traits in those reared alone, because the former hatchlings grew faster and were more advanced developmentally. The time to the beginning of feeding was shorter, and swimming speed as an indicator of escape performance was faster, for hatchlings reared with conspecifics. Hatchlings reared with conspecifics more successfully cannibalized small hatchlings and were also highly resistant to being cannibalized by large conspecifics, compared with hatchlings reared alone. These differences in cannibalism success and avoidance of cannibalization were well explained by differences in gape and head size, respectively, between the interacting hatchlings. Therefore, acceleration of growth and development of hatchlings in the proximate presence of conspecifics may confer significant fitness advantages on hatchlings in a high‐density cannibalistic situation. Developmental and growth plasticity may thus be a powerful adaptive mechanism that allows individuals to respond to the dynamic character of salamander populations in nature.

Complex Relationships amongst Parasite Load and Escape Behaviour in an Insular Lizard

Economic escape models predict escape decisions of prey which are approached by predators. Flight initiation distance (FID, predator–prey distance when prey begins to flee) and distance fled (DF) are major variables used to characterize escape responses. In optimal escape theory, FID increases as cost of not fleeing also increases. Moreover, FID decreases as cost of fleeing increases, due to lost opportunities to perform activities that may increase fitness. Finally, FID further increases as the prey's fitness increases. Some factors, including parasitism, may affect more than one of these predictors of FID. Initially, parasitized prey may have lower fitness as well as impaired locomotor ability, which would avoid predation and/or reduce their foraging ability, further decreasing the opportunity of fleeing. For example, if parasites decrease body condition, prey fitness is reduced and escape ability may be impaired. Hence, the overall influence of parasitism on FID is difficult to predict. We examined relationships between escape decisions and different traits: parasite load, body size and body condition in the Balearic lizard, Podarcis lilfordi. Lizards that showed higher haemogregarines load had longer FID and shorter DF. Although results did not confirm our initial predictions made on the basis of optimal escape theory, our findings suggest that parasites can alter several aspects of escape behaviour in a complex way.

Asymmetrical polymer vesicles with a "stealthy" outer corona and an endosomal-escape-accelerating inner corona for efficient intracellular anticancer drug delivery.

The efficient intracellular drug delivery is an important challenge due to the slow endocytosis and inefficient drug release of traditional delivery vehicles such as symmetrical polymer vesicles, which have the same coronas on both sides of the membrane. Presented in this paper is a noncytotoxic poly(ethylene oxide)-block-poly(caprolactone)-block-poly(acrylic acid) (PEO113-b-PCL132-b-PAA15) triblock copolymer vesicle with an asymmetrical structure. The biocompatible exterior PEO coronas are designed for stealthy drug delivery; The pH-responsive interior PAA chains are designed for rapid endosomal escape and enhanced drug loading efficiency. The hydrophobic PCL vesicle membrane is for biodegradation. Such asymmetrical polymer vesicle showed high doxorubicin (DOX) loading efficiency and good biodegradability under extracellular enzymatic conditions. Compared with three traditional symmetrical vesicles prepared from PEO113-b-PCL110, PEO43-b-PCL98-b-PAA25, and PAA21-b-PCL75 copolymers, the DOX-loaded asymmetrical PEO113-b-PCL132-b-PAA15 polymer vesicles exhibited rapid endocytosis rate and much faster endosomal escape ability, demonstrating promising potential applications in nanomedicine.

High gene delivery efficiency of alkylated low-molecular-weight polyethylenimine through gemini surfactant-like effect.

To our knowledge, the mechanism underlying the high transfection efficiency of alkylated low-molecular-weight polyethylenimine (PEI) is not yet well understood. In this work, we grafted branched PEI (molecular weight of 1,800 Da; bPEI1800) with lauryl chains (C12), and found that bPEI1800-C12 was structurally similar to gemini surfactant and could similarly assemble into micelle-like particles. Stability, cellular uptake, and lysosome escape ability of bPEI1800-C12/DNA polyplexes were all greatly enhanced after C12 grafting. bPEI1800-C12/DNA polyplexes exhibited significantly higher transfection efficiency than Lipofectamine™ 2000 in the presence of serum. Bioluminescence imaging showed that systemic injection of bPEI1800-C12/DNA polyplexes resulted in intensive luciferase expression in vivo and bioluminescence signals that could be detected even in the head. Altogether, the high transfection efficacy of bPEI1800-C12 was because bPEI1800-C12, being an analog of gemini surfactant, facilitated lysosome escape and induced the coil–globule transition of DNA to assemble into a highly organized micelle-like structure that showed high stability.

In vitro and in vivo therapeutic siRNA delivery induced by a tryptophan-rich endosomolytic peptide.

At the forefront of medicine, gene therapy provides an effective way to treat a range of diseases by regulating defective genes at the root of the disease. Short interfering RNAs (siRNAs) hold great promise as therapeutic agents in this domain; however, intracellular delivery remains a major obstacle to clinical applications of therapeutic siRNAs. Here we report a peptide designed to mediate siRNA delivery. This peptide, C6M1, is rationally designed to promote the endosomal escape ability of an existing peptide sequence. Formed C6M1-siRNA nanoscale complexes are able to deliver siRNA into cells and induce specific gene knockdown with low toxicity. The increased membrane disruption ability under acidic conditions of the peptide with tryptophan residue substitution may contribute to the enhanced gene silence efficacy. Intratumoral injection of the complexes results in a marked reduction of tumor growth through downregulation of antiapoptotic Bcl-2 protein in mice. In addition, the C6M1-siRNA complex was proven safe at transfection concentration by cytotoxicity assay. These results demonstrate that the C6M1-siRNA complex is a potent system for efficient gene delivery in vitro and in vivo.

Suppression of VEGF by reversible-PEGylated histidylated polylysine in cancer therapy.

A reversible-PEGylated polylysine is designed and developed for efficient delivery of siRNA. In this unique structure, the ε-amino groups of disulfide linked poly(ethylene glycol) (PEG) and polylysine (mPEG-SS-PLL) are partially replaced by histidine groups, in order to develop the histidylated reversible-PEGylated polylysine (mPEG-SS-PLH), for enhanced endosome escape ability. The transfection efficacy of mPEG-SS-PLH is found to closely correlate with histidine substitution. Its maximum transfection efficiencies are determined, respectively, to be 75%, 42%, and 24%, against 293T, MCF-7, and PC-3 cells. These data indicate that the transfection efficiencies can equal or even outweigh PEI-25k in the corresponding cells (80%, 38.5%, and 20%). The in vivo circulation and biodistribution of the polyplexes are monitored by fluorescent imaging. The in vivo gene transfection is carried out by intravenous injection of pEGFP to BALB/c mice using the xenograft models. The in vivo experimental results show effective inhibition of tumor growth by mPEG-SS-PLH/siRNA-VEGF, indicating its high potential for clinical applications.

Co-delivery of doxorubicin and RNA using pH-sensitive poly (β-amino ester) nanoparticles for reversal of multidrug resistance of breast cancer

An appropriate co-delivery system for chemotherapeutic agents and nucleic acid drugs will provide a more efficacious approach for the treatment of breast cancer by reversing multidrug resistance (MDR). In this work, a new amphiphilic poly (β-amino ester), poly[(1,4-butanediol)-diacrylate-β-5-polyethylenimine]-block-poly[(1,4-butanediol)-diacrylate-β-5-hydroxy amylamine] (PDP-PDHA) was synthesized, and the doxorubicin (DOX) and survivin-targeting shRNA (shSur) co-loading nanoparticle (PDNs) were prepared. The pH-sensitive poly[(1,4-butanediol) diacrylate-β-5-hydroxy amylamine] (PDHA) endowed PDNs both pH-triggered drug release characteristics and enhanced endo/lysosomal escape ability, thus improving the cytotoxicity of DOX and the transfection efficiency. PDNs also increased the DOX accumulation, down-regulated 57.7% survivin expression, induced 80.8% cell apoptosis and changed the cell cycle in MCF-7/ADR cells. In the MCF-7/ADR tumor-bearing mice models, after administrated intravenously, PDNs raised the accumulation of DOX and shSur in the tumor tissue by 10.4 and 20.2 folds, respectively, resulting in obvious inhibition of the tumor growth with tumor inhibiting rate of 95.9%. The combination of DOX and RNA interference showed synergistic effect on overcoming MDR. Therefore, PDNs could be a promising co-delivery vector for effective therapy of drug resistant breast cancer.

Carotenoid coloration predicts escape performance in the House Finch (Haemorhous mexicanus)

Carotenoid coloration has been repeatedly shown to serve as a sexually selected signal of individual quality. Across different species, individuals showing brighter carotenoid-based signals have been found to have superior foraging abilities, to recover faster from diseases and, in general, to enjoy a better body condition. Experiments with birds have also shown that carotenoid supplementation can enhance flight performance, allowing birds to take off faster from the ground. Healthy, agile individuals should be better prepared to avoid predators, so it could be expected that individuals displaying brighter carotenoid-based coloration would show a higher escape ability from predator attacks. To test this prediction, we measured the escape ability of male House Finches (Haemorhous mexicanus) from a human with a net in a large aviary and related the escape ability of each individual to its breast coloration. Males with redder feathers showed a higher individual ability to escape than duller individuals. The superior flight performance of redder birds would be an important asset in escape from predators, as well as when foraging or maintaining a territory. In the specific case of the House Finch, the higher escape ability of redder individuals could be the reason for their higher overwinter survival rate.

No evidence of selection by predators on tadpole boldness

Animals typically exhibit adaptive behaviors that reduce their risk of predation. The term ‘boldness’ describes individual variation in the propensity to exhibit risk-reducing behavior and is the subject of much research attention. Predators should select against boldness, and this has been supported by empirical studies and behavioral ecology theory. We tested whether a standardized assay of three boldness-associated behaviors in wood frog (Lithobates sylvaticus) tadpoles predicted survival when faced with a predator. Tadpole behavior was assayed in an open field and then tadpoles were placed, in pairs, in an enclosure with a predator (newt or larval dragonfly). Survival did not depend on differences in measured boldness, and this result held when we accounted for interactions between different boldness behaviors and between behavior and size or predator identity. The absence of selection by predators against bolder tadpoles is counterintuitive and inconsistent with our understanding of the behavioral ecology of these animals. Two possible explanations are offered for this result. First, selection against boldness may be minimized by other phenotypic traits, such as escape ability. Alternatively, the potential lack of consistency between standardized boldness assays and natural encounters with predators may limit our capacity to study the evolution of boldness, cautioning against this approach. These results highlight the complexities of the relationships between behavioral traits and fitness and the challenges associated with their study.

Core-Shell type lipid/rPAA-Chol polymer hybrid nanoparticles for in vivo siRNA delivery

Our previous study had reported that cholesterol-grafted poly(amidoamine) (rPAA-Chol polymer) was able to self-assemble into cationic nanoparticles and act as a potential carrier for siRNA transfection. In this study, the core–shell type lipid/rPAA-Chol hybrid nanoparticles (PEG-LP/siRNA NPs and T7-LP/siRNA NPs) were developed for improving in vivo siRNA delivery by modifying the surface of rPAA-Chol/siRNA nanoplex core with a lipid shell, followed by post-insertion of polyethylene glycol phospholipid (DSPE-PEG) and/or peptide (HAIYPRH, named as T7) modified DSPE-PEG-T7. The integrative hybrid nanostructures of LP/siRNA NPs were evidenced by dynamic light scattering (DLS), confocal laser scanning microscope (CLSM), cryo-transmission electron microscope (Cryo-TEM) and surface plasmon resonance (SPR) assay. It was demonstrated that the T7 peptide modified LP/siRNA NPs (T7-LP/siRNA NPs) exhibited uniform and spherical structures with particle size of 99.39 ± 0.65 nm and surface potential of 42.53 ± 1.03 mV, and showed high cellular uptake efficiency and rapid endosomal/lysosomal escape ability in MCF-7 cells. Importantly, in vitro gene silencing experiment demonstrated that both of pegylated and targeted LP/siEGFR NPs exhibited significantly stronger downregulation of EGFR protein expression level in MCF-7 cells, compared to that of the physical mixture of siRNA lipoplexes and rPAA-Chol/siRNA nanoplexes. In vivo tumor therapy on nude mice bearing MCF-7 tumors further confirmed that the targeted T7-LP/siEGFR NPs exhibited the greatest inhibition on tumor growth via transferrin receptor-mediated targeting delivery, without any activation of immune responses and significant body weight loss following systemic administration. These findings indicated that the core-shell type T7-LP/siRNA nanoparticles would be promising siRNA delivery systems for in vivo tumor-targeted therapy.

Polylysine-modified polyethylenimine inducing tumor apoptosis as an efficient gene carrier

Polyethylenimine (PEI) is receiving increasing attention as a gene carrier with high transfection efficiency. However, its high charge density and cytotoxic effects limit its application. Polylysine (PLL) is another polymeric gene carrier with good biodegradability and biocompatibility, although its lack of endosomal escape ability strongly impairs its transfection efficiency. In this study, PLL was introduced to PEI by ring-opening polymerization of ε-benzyloxycarbonyl-l-lysine N-carboxyanhydride, followed by deprotection of carbobenzyloxy groups. As-prepared PEI-PLL multiarm hyperbranched copolymers were characterized as gene carriers in vitro by measuring their particle size, zeta potential, cytotoxicity, transfection efficiency, and cell internalization. The optimum transfected efficiency of PEI-PLL was nearly seven times higher than that of PEI with a molecular weight of 25kDa. Furthermore, pKH3-rev-casp-3 plasmid DNA was used as a gene for anti-tumor treatment in a xenograft model using nude mice. Compared with 25kDa PEI, PEI-PLL exhibited better tumor inhibition effects in 23days. In addition, terminal deoxynucleotidyl transferase dUTP nick end labeling, immunohistochemistry, and western blot analysis were used to determine the anti-tumor mechanism of PEI-PLL. The results showed that tumor cell apoptosis led to tumor inhibition, which could be attributed to pKH3-rev-casp-3 inducing poly(ADP-ribose) polymerase-1 cleavage. PEI-PLL is a promising gene carrier candidate for further application in vivo.

Gene Silencing via RNAi and siRNA Quantification in Tumor Tissue Using MEND, a Liposomal siRNA Delivery System

Small interfering RNA (siRNA) would be predicted to function as a cancer drug, but an efficient siRNA delivery system is required for clinical development. To address this issue, we developed a liposomal siRNA carrier, a multifunctional envelope-type nanodevice (MEND). We previously reported that a MEND composed of a pH-sensitive cationic lipid, YSK05, showed significant knockdown in both in vitro and in tumor tissue by intratumoral injection. Here, we report on the development of an in vivo siRNA delivery system that is delivered by systemic injection and an analysis of the pharmacokinetics of an intravenously administered siRNA molecule in tumor tissue. Tumor delivery of siRNA was quantified by means of stem-loop primer quantitative reverse transcriptase PCR (qRT-PCR) method. PEGylation of the YSK-MEND results in the increase in the accumulation of siRNA in tumor tissue from 0.0079% ID/g tumor to 1.9% ID/g tumor. The Administration of the MEND (3 mg siRNA/kg body weight) showed about a 50% reduction in the target gene mRNA and protein. Moreover, we verified the induction of RNA interference by 5' RACE-PCR method. The collective results reported here indicate that an siRNA carrier was developed that can deliver siRNA to a target cell in tumor tissue through an improved siRNA bioavailability.

Extravagant ornaments of male threadfin rainbowfish (Iriatherina werneri) are not costly for swimming

Summary Exaggeration of male sexual ornaments should be costly, in terms of metabolic expenditure, resource allocation or even locomotor function. For example, many male ornaments are predicted to affect the aerodynamics, drag or biomechanics of movement and thus inhibit the speed or manoeuvrability of individuals; but empirical support for this is equivocal. We tested the locomotor and metabolic costs of exaggerated male ornaments in the threadfin rainbowfish (Iriatherina werneri), an Australasian native fish characterized by excessively long fin streamers. We predicted that males with greater relative ornamentation would have reduced escape abilities (i.e. burst swim speeds) as well as higher metabolic costs when resting or swimming. Furthermore, we evaluated the benefits of the signal by comparing the preference of females for males with differing amounts of ornamentation. As expected, we found that females spent more time observing (i.e. preferred) males with longer relative fins. We also experimentally reduced threadfin length and found that females continued to show preference for males with longer fins, rather than a preference for particular males. Male I. werneri with longer ornaments had higher resting metabolic rates, but we found no effect of ornament size on metabolic rates during swimming. Males with longer threadfins tended to swim faster, but our manipulation of fin length had no effect on burst swimming speed, indicating swimming abilities are not causally related to threadfin length. Overall, we found no evidence that the extravagant ornaments of male threadfin rainbowfish increase the metabolic or functional costs associated with swimming. Our results are surprising, given the high viscosity of water and the extreme length of I. werneri's ornaments. We suggest that future work should focus on the fitness costs of threadfin length, relative to reproductive output or survival under more natural conditions.