Delivery of molecular cargoes in normal and cancer cell lines using non-viral delivery systems

Objective

In this study, transfection efficiency of human papillomavirus (HPV) E7 DNA and protein constructs into HEK-293T normal cell line, and A549 and TC-1 tumor cell lines was evaluated by four delivery systems including supercharge GFP, hPP10 cell penetrating peptide, TurboFect and Lipofectamine using fluorescence microscopy and flow cytometry.

Results

The results indicated that Lipofectamine 2000 and TurboFect produced more effective transfection for GFP and E7-GFP DNA constructs in HEK-293T cells compared to in A549 and TC-1 cells (p?<?0.05). In contrast, the supercharge GFP was efficient for E7 DNA and E7 protein delivery in both normal cell (~?83.94 and ~?77.01% for HEK-293T), and cancer cells (~?71.69 and ~?67.19% for TC-1, and ~?73.86 and ~?67.49% for A549), respectively. Indeed, in these cell lines, transfection efficiency by +36 GFP reached ~?60–80%. Moreover, the hPP10 produced the best transfection result for E7-GFP protein in HEK-293T cells (~?63.66%) compared to TurboFect (~?32.95%); however, the efficiency level of hPP10 was only ~?17.51 and ~?16.36% in TC-1 and A549 cells.

Conclusions

Our data suggested that the supercharge GFP is the most suitable transfection vehicle for DNA and protein delivery into TC-1 and A549 tumor cell lines compared to other carriers.

     

Osmoregulation in decapod crustaceans: physiological and genomic perspectives

Aquatic plants may face resource constraints or anthropogenic pollution, and effects might be heightened under multiple stress conditions. We investigated if arsenate effects on Myriophyllum spicatum L. would be stronger under CO₂ limitation and low phosphorus availability. In a factorial design, we exposed sediment-grown plants to either CO₂ (high carbon or HC) or bicarbonate (low carbon or LC) and four levels of arsenate. We observed strong effects of arsenate exposure on growth, biomass allocation (leaf, stem and root mass fractions), pigments and phenolic compounds. CO₂ availability strongly affected the content in phenolic compounds and a few other response variables, yet overall effects were less pronounced than expected. Strong interactive effects of CO₂ availability and arsenic concentration were only observed for carotenoids, the carotenoid/chlorophyll ratio and phenolic compounds in leaves. Only the carbon content declined with increasing arsenic concentration, otherwise leaf elemental content and stoichiometry were not affected by arsenic or CO₂ availability, suggesting that plants strived to maintain leaf functions. The observed effects on biomass allocation and plant quality, specifically dry matter content and phenolic compound content of M. spicatum not only show direct changes in plant performance but suggest also indirect effects on ecological interactions such as competition or herbivory.     

Changes in photosynthetic pigments and chlorophyll-<Emphasis Type="Italic">a</Emphasis> fluorescence attributes of sweet-forage and grain sorghum cultivars under salt stress

Water shortage leads to a low quality of water, especially saline water in most parts of agricultural regions. This experiment was designed to determine the effects of saline irrigation on sorghum as a moderately salt-tolerant crop. To study salinity effects on photosynthetic pigment attributes including the chlorophyll content and chlorophyll fluorescence, an experiment was performed in a climate-controlled greenhouse at two vegetative and reproductive stages. The experimental design was factorial based on a completely randomized design with five NaCl concentrations (control, 50, 100, 150, and 200 mM), two grain and sweet-forage sorghum cultivars (Kimia and Pegah, respectively) and four replications. According to the experimental data, there were no significant differences between two grain and sweet-forage cultivars. Except for 100 and 150 mM NaCl, salinity significantly decreased the chlorophyll index and pigment contents of the leaf, while it increased the chlorophyll-a fluorescence characteristics. Although salinity reduced photosynthetic pigments and the crop yield, either grain or sweet-forage cultivars could significantly control the effect of salinity between 100 and 150 mM NaCl at both developmental stages, showing the possibility of using saline water in sorghum cultivation up to 150 mM NaCl.     

New antiprotozoal agents: Synthesis and biological evaluation of different 4-(7-chloroquinolin-4-yl) piperazin-1-yl)pyrrolidin-2-yl)methanone derivatives

In an endeavor to develop efficacious antiprotozoal agents 4-(7-chloroquinolin-4-yl) piperazin-1-yl)pyrrolidin-2-yl)methanone derivatives (5–14) were synthesized, characterized and biologically evaluated for antiprotozoal activity. The compounds were screened in vitro against the HM1: IMSS strain of Entamoeba histolytica and NF54 chloroquine-sensitive strain of Plasmodium falciparum. Among the synthesized compounds six exhibited promising antiamoebic activity with IC₅₀ values (0.14–1.26 μM) lower than the standard drug metronidazole (IC₅₀ 1.80 μM). All nine compounds exhibited antimalarial activity (IC₅₀ range: 1.42–19.62 μM), while maintaining a favorable safety profile to host red blood cells. All the compounds were less effective as an antimalarial and more toxic (IC₅₀ range: 14.67–81.24 μM) than quinine (IC₅₀: 275.6 ± 16.46 μM) against the human kidney epithelial cells. None of the compounds exhibited any inhibitory effect on the viability of Anopheles arabiensis mosquito larvae.     

Delivery of HIV-1 Nef Protein in Mammalian Cells Using Cell Penetrating Peptides as a Candidate Therapeutic Vaccine

The HIV-1 Nef protein expressed early in viral life cycle has been known as a potent candidate for therapeutic vaccine development. Due to different cell barriers, various cell penetrating peptides (CPPs) such as Pep-1 and CADY-2 have been known to deliver biologically active proteins to cytoplasmic compartments via the plasma membrane. In current study, we firstly evaluated the efficiency of lentiviral vector (pCDH-CMV-MCS-EF1-cGFP-T2A-puro) and eukaryotic expression vector (pEGFP-N1) for expression of HIV-1 Nef protein in HEK-293T cells using TurboFect transfection reagent. Our results showed that both vectors can effectively express the Nef proteins within the target cell. The pEGFP-N1 was more effective than pCDH-GFP for protein expression. Furthermore, Nef protein was expressed in E. coli as GST-Nef fusion and transfected by the amphipathic CPPs including Pep-1 and CADY-2 into HEK-293T cells. The size and morphology of the GST-Nef/CPP complexes were evaluated by scanning electron microscopy, and Zetasizer. Our data indicated that the recombinant GST-Nef protein generated in BL21 strain migrated as a clear band of ~50 kDa in SDS-PAGE. The CPP/GST-Nef nanoparticles were formed with a diameter of below 200 nm and notably delivered into HEK-293T cells. Generally, the Nef protein was expressed in prokaryotic and eukaryotic expression systems using different vectors and efficiently transfected in mammalian cells using various delivery systems. The in vitro efficient delivery of HIV-1 Nef gene and also its protein supports the potential of Nef DNA constructs and CPPs as potent carriers of Nef protein for HIV vaccine design in Future.     

Inhibitors of Escherichia coli serine acetyltransferase block proliferation of Entamoeba histolytica trophozoites

The protozoan parasite Entamoeba histolytica is the etiologic agent of amebiasis, a major global public health problem, particularly in developing countries. There is an effective anti-amoebic drug available, however its long term use produces undesirable side effects. As E. histolytica is a micro-aerophilic organism, it is sensitive to high levels of oxygen and the enzymes that are involved in protecting against oxygen-stress are crucial for its survival. Therefore serine acetyltransferase, an enzyme involved in cysteine biosynthesis, was used as a target for identifying potential inhibitors. Virtual screening with Escherichia coli serine acetyltransferase was carried out against the National Cancer Institute chemical database utilizing molecular docking tools such as GOLD and FlexX. The initial analysis yielded 11 molecules of which three compounds were procured and tested for biological activity. The results showed that these compounds partially block activity of the E. coli enzyme and the growth of E. histolytica trophozoites but not mammalian cells.