Enhanced gene expression in the brain following intravenous administration of lactoferrin-bearing polypropylenimine dendriplex

The possibility of using gene therapy for the treatment of brain diseases such as brain cancer, Alzheimer's and Parkinson's diseases, is currently hampered by the lack of gene delivery systems able to cross the blood–brain barrier and deliver DNA to the brain following intravenous administration. On the basis that lactoferrin can effectively reach the brain by using specific receptors for crossing the blood–brain barrier, we propose to investigate if a lactoferrin-bearing generation 3-diaminobutyric polypropylenimine (DAB) dendrimer would allow the transport of plasmid DNA to the brain after intravenous administration. In this work, we demonstrated that the conjugation of lactoferrin to the dendrimer led to an enhanced DNA uptake by 2.1-fold in bEnd.3 murine brain capillary endothelial cells compared to the unmodified dendriplex in vitro. In vivo, the intravenous administration of lactoferrin-bearing DAB dendriplex resulted in a significantly increased gene expression in the brain, by more than 6.4-fold compared to that of DAB dendriplex, while decreasing gene expression in the lung and the kidneys. Gene expression in the brain was significantly higher than in any other major organs of the body. Lactoferrin-bearing generation 3 polypropylenimine dendrimer is therefore a highly promising delivery system for systemic gene delivery to the brain

Two-color, two-photon imaging at long excitation wavelengths using a diamond Raman laser

We demonstrate that the second-Stokes output from a diamond Raman laser, pumped by a femtosecond Ti:Sapphire laser, can be used to efficiently excite red-emitting dyes by two-photon excitation at 1080 nm and beyond. We image red fluorescent protein (RFP) expressing HeLa cells, as well as dyes such as Texas Red and Mitotracker Red. We demonstrate the potential for simultaneous two-color, two-photon imaging with this laser by using the residual pump beam for excitation of a green-emitting dye. We demonstrate this for the combination of AlexaFluor 488 and AlexaFluor 568. Because the Raman laser extends the wavelength range of the Ti:Sapphire laser, resulting in a laser system tunable 680 nm-1200 nm it can be used for two-photon excitation of a large variety and combination of dyes

Label-free imaging of thick tissue at 1550nm using a femtosecond optical parametric generator

We have developed a simple wavelength tunable optical parametric generator (OPG), emitting broad band ultrashort pulses with peak wavelengths at 1530-1790 nm, for nonlinear label-free microscopy. The OPG consists of a periodically poled lithium niobate crystal, pumped at 1064 nm by a ultrafast Yb:fiber laser with high pulse energy. We demonstrate that this OPG can be used for label-free imaging, by third harmonic generation, of nuclei of brain cells and blood vessels in a >150 µm thick brain tissue section, with very little decay of intensity with imaging depth and no visible damage to the tissue at an incident average power of 15 mW

PEGylation of polypropylenimine dendrimers: effects on cytotoxicity, DNA condensation, gene delivery and expression in cancer cells

Diaminobutyric polypropylenimine (DAB) dendrimers have been shown to be highly efficient non-viral gene delivery systems for cancer therapy. However, their cytotoxicity currently limits their applications. To overcome this issue, PEGylation of DAB dendrimer, using various PEG molecular weights and dendrimer generations, has been attempted to decrease the cytotoxicity and enhance the DNA condensation, size and zeta potential, cellular uptake and transfection efficacy of these dendriplexes. Among all the PEGylated dendrimers synthesized, generation 3- and generation 4-DAB conjugated to low molecular weight PEG (2 kDa) at a dendrimer: DNA ratio of 20:1 and 10:1 resulted in an increase in gene expression on almost all tested cancer cells lines (by up to 3.2-fold compared to unmodified dendrimer in A431 cells). The highest level of β-galactosidase gene expression (10.07 × 10−3 ± 0.09 × 10−3 U/mL) was obtained following treatment of B16F10-Luc cells with G4-dendrimer PEGylated with PEG2K at a dendrimer: DNA ratio of 20:1. These delivery systems significantly decreased cytotoxicity on B16F10-Luc cells, by more than 3.4-fold compared to unmodified dendrimer. PEGylated generations 3- and 4-DAB dendrimers are therefore promising gene delivery systems for cancer therapy, combining low cytotoxicity and high transfection efficacy

Subterranean sympatry : an investigation into diet using stable isotope analysis

In the Western Cape three species of mole-rat occur in sympatry, however, little is known about differences in their dietary preferences. Dietary composition of the three species; the common mole-rat (Cryptomys hottentotus hottentotus), the Cape mole-rat (Georychus capensis) and the Cape dune mole-rat (Bathyergus suillus) were examined using stable isotope analysis. Blood, fur and claw samples were collected from 70 mole-rats, in addition to several potential food items, to assess food selection of the three species under natural conditions. Overall there was a significant difference in the isotopic composition (d13C and d15N) between all three species and significant differences in their diet composition. There were also significant differences between tissues in all three species suggesting temporal variation in diet. The small size and colonial lifestyle of C. h. hottentotus allows it to feed almost 100% on bulbs, while the solitary and larger species G. capensis and B. suillus fed to a greater extent on other resources such as grasses and clover. B. suillus, the largest of the species, had the most generalized diet. However, overall all species relied most heavily upon geophytes and consumed the same species suggesting competition for resources could exist. We also showed a high level of individual variation in diet choices. This was most pronounced in B. suillus and G. capensis and less so in C. h. hottentotus. We demonstrate that stable isotope analysis can successfully be applied to examine dietary patterns in subterranean mammals and provide insights into foraging patterns and dietary variation at both the inter and intra population level.G. Robb was funded by a University of Pretoria Post-doctoral fellowship. N. Bennett and fieldwork costs were funded through the Department of Science and Technology and National Research Foundation SARChI Chair of Mammal Behavioural Ecology and Physiology.http://www.plosone.orgab201

Widefield two-photon excitation without scanning : live cell microscopy with high time resolution and low photo-bleaching

We demonstrate fluorescence imaging by two-photon excitation without scanning in biological specimens as previously described by Hwang and co-workers, but with an increased field size and with framing rates of up to 100 Hz. During recordings of synaptically-driven Ca2+ events in primary rat hippocampal neurone cultures loaded with the fluorescent Ca2+ indicator Fluo-4 AM, we have observed greatly reduced photo-bleaching in comparison with single-photon excitation. This method, which requires no costly additions to the microscope, promises to be useful for work where high time-resolution is required

The influence of food quantity on carbon and nitrogen stable isotope values in southern African spiny mice (Acomys spinosissimus)

Stable isotope analysis is frequently applied as a tool to examine dietary patterns in animals. However, some of the underlying assumptions associated with using this approach are increasingly being questioned. We carried out a controlled diet experiment on the southern African spiny mouse (Acomys spinosissimus Peters, 1852) to test a number of aspects relating to these assumptions and also examine the hypothesis that stable isotopes, especially δ15N, can be used to provide evidence of nutritional stress. We compared the δ13C and δ15N values of livers and blood from animals that were fed ad libitum with animals undergoing a 10% reduction in food supply. Food-restricted animals showed no significant difference in δ15N; however, δ13C values of both liver and blood were depleted. Restricted animals also had a significantly lower C:N ratio. We examined the role of lipids and found following lipid extraction that both livers and lipids still showed the same separation in carbon values. Tissue–diet discrimination values were also calculated and found to be higher for both Δ13C and Δ15N compared with other mice species. Empirical values for discrimination rates were then compared with values calculated using an alternative method based on employing generic values and were found to be dissimilar, suggesting the use of generic values are not always appropriate. Our results highlight the need for greater understanding of the assumptions associated with using stable isotope analysis to examine diet and we suggest that studying a single species under captive conditions presents an ideal method to begin to test these hypotheses.Department of Science and Technology – National Research Foundation. South African Research Chair in mammal behavioural ecology and physiology. University of Pretoria.http://www.nrcresearchpress.com/journal/cjzhb2016Zoology and Entomolog

PEGylation of polypropylenimine dendrimers : effects on cytotoxicity, DNA condensation, gene delivery and expression in cancer cells

Diaminobutyric polypropylenimine (DAB) dendrimers have been shown to be highly efficient non-viral gene delivery systems for cancer therapy. However, their cytotoxicity currently limits their applications. To overcome this issue, PEGylation of DAB dendrimer, using various PEG molecular weights and dendrimer generations, has been attempted to decrease the cytotoxicity and enhance the DNA condensation, size and zeta potential, cellular uptake and transfection efficacy of these dendriplexes. Among all the PEGylated dendrimers synthesized, generation 3- and generation 4-DAB conjugated to low molecular weight PEG (2 kDa) at a dendrimer: DNA ratio of 20:1 and 10:1 resulted in an increase in gene expression on almost all tested cancer cells lines (by up to 3.2-fold compared to unmodified dendrimer in A431 cells). The highest level of β-galactosidase gene expression (10.07x10E-3 ± 0.09x10E-3 U/mL) was obtained following treatment of B16F10-Luc cells with G4-dendrimer PEGylated with PEG2K at a dendrimer: DNA ratio of 20:1. These delivery systems significantly decreased cytotoxicity on B16F10-Luc cells, by more than 3.4-fold compared to unmodified dendrimer. PEGylated generations 3- and 4-DAB dendrimers are therefore promising gene delivery systems for cancer therapy, combining low cytotoxicity and high transfection efficacy

Comparative isotopic natural history of two native passerines (Troglodytes cobbi and Cinclodes antarcticus) and the invasive rats (Rattus norvegicus) that extirpate them

While several studies have shown that invasive rats can have negative effects on island birds through predation (both direct predation and nest predation), other mechanisms for the effects of invasives on island biota have been given less attention. Here we explore another potential mechanism by which invasive rats can affect native island birds: the competitive use of common resources. We used stable isotope analyses to estimate the fraction of marine and terrestrial sources incorporated into the tissues of two species of passerines (Troglodytes cobbi, Troglodytidae; and Cinclodes antarcticus, Furnariidae) and Norway rats (Rattus norvegicus, Muridae) in the Falkland Islands. These two passerines are absent on islands where rats are present. We found significant incorporation of marine resources in the three species, with the highest incorporation in tissues of T. cobbi. This species appears to be one of the passerines most reliant on marine sources and the most marine member of the family Troglodytidae. We also used the results of these isotopic analyses to estimate the isotopic niche breadth of each of these species and the isotopic niche overlap among them. Rattus norvegicus had a large isotopic niche that overlapped broadly with those of the two passerine species. We propose that different ways of both depicting and estimating isotopic niche widths are complementary rather than alternative. Our results are consistent with the notion that invasive rats might have an impact on these two species of Falkland Island passerines by using common resources, but do not rule out the possibility that part of their effect is through direct predation.MT and CMR were partially funded by National Science Foundation Grants # 0841298 and DIOS-0848028, respectively. SP and KP were funded by Antarctic Research Trust, UK Overseas Territories Environment Programme, Royal Society for Protection of Birds, Joint Nature Conservation Committee, and the Falkland Islands Government.http://onlinelibrary.wiley.com/journal/10.1111/(ISSN)1442-99932017-10-31hb2017Zoology and Entomolog