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- Open Access
Self-assembled nanoparticles based on modified cationic dipeptides and DNA: novel systems for gene delivery
Journal of Nanobiotechnology volume 11, Article number: 18 (2013)
Gene therapy is most effective when delivery is both efficient and safe. However, it has often proven difficult to find a balance between efficiency and safety in case of viral or polymeric vectors for gene therapy. Peptide based delivery systems may be attractive alternatives but their relative instability to proteolysis is a major concern in realizing their potential application in biomedical sciences. In this work we report gene delivery potential of nanoparticles (Nps) synthesized from cationic dipeptides containing a non-protein amino acid α, β-dehydrophenylalanine (∆Phe) residue.
Dipeptides were synthesized using solution phase peptide synthesis method. Nps were formed using self-assembly. Nps were characterized using light scattering, electron microscopy. Transfection efficiency was tested in hepatocellular carcinoma (HuH 7) cells.
The cationic dipeptides condensed plasmid DNA into discrete vesicular nanostructures. Dipeptide Nps are non-cytotoxic, protected the condensed DNAs from enzymatic degradation and ferried them successfully inside different types of cells. GFP encoding plasmid DNA loaded dipeptide Nps showed positive transfection and gene expression in HuH 7 cells.
The cationic dipeptide Nps can successfully deliver DNA without exerting any cytotoxic effect. Owing to their simple dipeptide origin, ease of synthesis, enhanced enzymatic stability as well unmatched biocompatibility, these could be successfully developed as vehicles for effective gene therapy.
Gene therapy continues to hold great promise in the field of medicine. Replacing defective genes with functional genes offers many exciting possibilities for treating dreaded diseases such as cancers, autoimmune disorders and neurodegenerative diseases . Efficient gene delivery, fundamental to the success of gene therapy remains a complex process with many possible rate-limiting steps. The most serious intracellular barriers are lysosomal degradation, nucleolytic degradation in the cytosol and inefficient delivery to the nucleus [2–4], whereas, extracellular barriers include nucleolytic degradation in the serum, recognition by the reticulo-endothelial system and nonspecific delivery . Gene delivery in humans requires carriers that will transfer DNA into the nuclei of target cells. These carriers must be efficient in transfection, safe for human use, protect DNA from degradation before arriving at the target cell and possibly hold targeting qualities for the specific delivery of gene to required cells or tissue.
Most early applications of gene therapy were concentrated around viral vectors for efficient gene delivery. However, many of these vectors raised undesirable immune responses, hindering their therapeutic effect . Synthetic gene delivery methods surpassed viral vectors in many ways, such as low cytotoxicity, reduced ability to provoke an immune response, ease of manufacturing and scale up and high adaptability. Over the years many chemical synthetic approaches using polymers with ability to accommodate larger DNA molecules compared to viral vectors, have been developed. Nanoparticles (Nps) loaded with plasmid DNA may serve as efficient sustained release gene delivery systems due to their rapid escape from the degradative endo-lysosomal compartment to the cytoplasmic compartment . Successful oligonucleotide delivery to HeLa cells using anionic liposomes , lipid-derived Nps that carry oligonucleotides either in their core or via covalent attachment have been reported to have significant efficacy in vivo and in vitro. Many research groups have used quantum dots , magnetic  and gold Nps [12, 13] as well as carbon nanotubes  and enabled successful siRNA/antisense delivery [10, 14]. Much attention was given to the biodegradable and biocompatible poly PLGA polymers . Polycationic polymer based Nps as non-viral gene delivery vectors, have also been developed and polyethylenimines is currently the most popular polymer used to deliver genes into various cell types, including neurons. Polyethylenimines is able to condense genes into small Nps and protect the DNA from degradation by nucleases. Polymeric complexes such as PLGA– polyethylenimines Nps have been demonstrated as new delivery systems to carry genes to the lung epithelium . Cationic bovine serum albumin conjugated with poly(ethyleneglycol)–poly(lactide) nanoparticle, was also developed as a promising brain drug delivery carrier with low toxicity .
We have been working with very short peptides that self-assemble to form different kind of Nps. Here we report DNA condensation and cell delivery potential of cationic dipeptide Nps (CdNps) such as Arg-∆Phe and Lys-∆Phe. The cationic dipeptides were non-cytotoxic and could condense different plasmid DNAs into discrete Nps enhancing their cellular uptake and delivery, and may represent a novel platform that can be further developed for DNA delivery into cells of different kinds.
Materials used in this study are: THF, NMM, DMF, piperidine, DIPCDI, IBCF, TIPS, TFA, HFIP, phenol, DL-threo-phenylserine, MTT (Sigma-Aldrich, Munich, Germany) Boc-Arg(Mtr)-OH, Boc-Lys(Boc)-OH (Novabiochem, Merck, Darmstadt, Germany) sodium acetate, ethyl acetate, acetonitrile (Spectrochem Pvt Ltd, Mumbai, India), anhydrous sodium sulfate, citric acid (Merck, Munich, Germany), human ovarian cancer (HeLa), hepatocellular carcinoma (HuH-7) and human dermal fibroblast (L929) cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). RPMI-1640, DMEM, NHS-modified Alexa-488 (Invitrogen, Life Technologies Corp. NY, USA). Plasmids used in the study were generous gifts from Dr Reddy’s laboratory, ICGEB, India. Except for method 2.8 in which plasmids of different lengths [in base pairs (bp)] were used and the transfection experiment where EGFP plasmid was used, all other studies were carried out with pgem plasmid DNA.
Assembly of dipeptides
Assembly of dipeptides into Nps in water was done from their HFIP stock solutions at a concentration of 2 mg/ml. Briefly, 2 mg of cationic peptides were first dissolved in 20 μl of HFIP and then diluted with 1 ml of filtered MilliQ water to form self-assembled Nps.
Dynamic Light Scattering (DLS) studies
Light scattering studies were performed in Photocor complex (Photocor, Moscow, Russia) using multiple tau digital correlator. For DLS measurement samples were prepared in dust free environment. Experiments were carried out at an angle of 90° using 632 nm laser at room temperature. Data represent mean of three different sets.
Loading DNA in preformed peptide Nps
Arg-∆Phe and Lys-∆Phe, Nps (2 mg/ml) were prepared as described above and were titrated with increasing concentrations of plasmid DNA (25, 37.5, 50, 62.5, 75, 100 μg) and analyzed using DLS to determine the optimum DNA concentration needed to form compact nanostructures.
Determination of entrapment efficiency of DNA in Arg-∆Phe and Lys-∆Phe Nps
Plasmid DNA was first labeled with Pt-conjugated Alexa-488 dye using Ulysis DNA-labeling kit (Sigma-Aldrich, Munich, Germany) and then added to Nps (100 μg of plasmid DNA : 1 mg/ml of peptide Nps). Samples were incubated for 30–60 min, filtered through a filter plate with 10 kDa membrane cut-off (Millipore, Corp., Billerica, MA, USA) by centrifuging at 2800 rpm for 20 min at 25°C. Filtrates were analysed spectrophotometerically to measure the amount of free DNA and quantified for percentage entrapment of the nucleotides in Nps as described above by using the following formula:
Formation of DNA-peptide Nps by Arg-∆Phe and Lys-∆Phe
For making DNA-peptide Nps, 100 μg of plasmid DNA was titrated with increasing amounts of peptides (100, 500, 1000, 2000 μg) and analyzed using DLS.
Effect of plasmid DNA size on their interaction with Arg-∆Phe and Lys-∆Phe
Peptides (2 mg/ml) were incubated with 100 μg of plasmids with varying sizes for 30 min. The DNA-peptide Nps so obtained were analyzed using DLS and electron microscopy.
Transmission Electron Microscopy (TEM)
DNA-peptide Nps were adsorbed on a 300 mesh copper grid with carbon coated formvar support and stained with 1% uranyl acetate and viewed under transmission electron microscope operating at an accelerated voltage of 120 kV (Tecnai 12 BioTWIN, FEI Netherlands). Photomicrographs were digitally recorded using a Megaview II (SIS, Germany) digital camera. Image analysis was carried out using Analysis II (Megaview, SIS, Germany) and ImageJ (http://rsb.info.nih.gov/ij/) software packages.
Localization of DNA in DNA-peptide Nps
In order to determine the exact location of plasmid DNA in DNA-peptide Nps. plasmid DNA was first complexed with platinum using ULYSIS DNA labeling kit and then incubated with Arg-∆Phe (as better Nps were obtained with Arg-∆Phe) for 30 min. Nps so formed were analyzed under TEM.
Cellular uptake of DNA-peptide Nps
For estimating the cellular uptake of DNA-peptide Nps, plasmid DNA and peptide Nps were labeled with Alexa-488 using ULYSIS DNA labeling kit (Sigma-Aldrich, Munich, Germany) and NHS-modified Alexa-588 dyes (Invitrogen, Life Technologies Corp. NY, USA) respectively. DNA-peptide Nps were prepared as described above. HeLa cells were first cultured in tissue culture dishes in RPMI-1640 media, trypsinized and seeded in 6 well plates (1 x 105 cells/well). After 12 hrs of culture, cells were incubated with plasmid DNA, peptide Nps, DNA-peptide Nps for 18 hrs, washed with serum and phenol red free media and imaged under fluorescence microscope.
Nuclear localization of DNA-peptide Nps
HeLa cells were seeded on cover slips placed in 6-well plates, allowed to grow for 12 hrs for cell adherence and spreading and subsequently incubated with DNA peptide Nps carrying Alexa-488 labeled DNA for 12 hrs. After this, media was removed, cells were treated with DAPI for nucleus staining, washed and visualized under fluorescent and bright-field microscopes.
Agarose gel electrophoresis of DNA peptide Nps
Complex formation between Nps and plasmid DNA was analyzed by 1% agarose gel electrophoresis. Free plasmid DNA (4 μg) and DNA peptide Nps complexes with peptide/DNA ratio of 20/1 (4 μg DNA/80 μg of peptide in 40 μl nuclease free water) were run on a 1% (w/v) agarose gel with TBE buffer (89 mmol/l Tris, 89 mmol/l boric acid, and 2 mmol/l EDTA pH 8.0) at 75 V for 90 minutes. DNA was visualized by staining gels with ethidium bromide (0.5 g/l) and images were acquired using an UV trans-illuminator (Vilber, Lourmat, France). Stability of DNA peptide Nps towards enzymatic degradation was determined by incubating plasmid DNA (4 μg) and DNA peptide Nps complexes with peptide/DNA ratio of 20/1 (4 μg DNA/80 μg of peptide in 40 μl nuclease free water) with 5 μl of DNase I solution (10 μg/ml in DNase/Mg2+ digestion buffer, which consisted of 50 mM Tris–HCl, pH 7.6, and 10 mM MgCl2) each at 37°C for 30 min, and degradation of plasmid DNA was analyzed by 1% agarose gel electrophoresis.
Cytotoxicity of DNA-peptide Nps
HeLa and L929 cells were cultured in RPMI-1640 and DMEM supplemented with 10% FBS, and maintained on TCTP plates at 37°C in a humified atmosphere containing 5% CO2, till 70-80% confluency. Cells were then harvested by trypsinization, washed and resuspended in RPMI-1640/DMEM medium (supplemented with 10% FBS). Live cells were counted by trypan blue dye exclusion test using a hemocytometer, diluted in completed media, seeded in 96 well plates (2 x 104 cells/well). After 12 hrs, cells were incubated with 20 μl each of dipeptide and DNA-dipeptide Nps (2 mg/ml) for a period of 24 hrs. As a control, cells were incubated with 20 μl of PBS. MTT assay was used to determine the cell viability of treated cells. The percentage viability was calculated using the equation given below:
Percentage viability of cells = [(Absorbance of MTT formazan produced by cells grown in presence of nps /Absorbance of MTT formazan produced by cells grown on TCTP control plate)] × 100.
In vitro transfection efficiency of DNA peptide Nps
HuH-7 cells were cultured in DMEM supplemented with 10% FBS and 1% pencillin-streptomycin and maintained on TCTP plates at 37°C in a humified atmosphere containing 5% CO2, till 70-80% confluency. 24 h prior to transfection cells were seeded in 6 well plates (2 x 105). DNA-Nps were prepared by incubating 4 μg of EGFP encoding plasmid DNA with 80 μg each of dipeptides Lys-∆Phe and Arg-∆Phe in 40 μl of nuclease free water. Nps so formed were visualized under TEM to determine particle size and morphology as described above. On the day of transfection, the culture medium in each well was replaced with 1 ml of complete medium containing 1 μg of DNA loaded in DNA-peptide Nps at (1: 200 DNA to peptide ratio) and 1 μg of DNA in lipofectamine. The cells were then incubated for 48 hrs, washed to remove excess Nps and DNA and analysed using fluorescence microscopy for GFP expression.
The results are presented as the mean ± SD calculated over at least three data points.
Results and discussion
Cationic polymer based systems have been widely used to condense DNA inside their core to protect it from intra-cellular nucleases and achieve efficient delivery [16, 20]. Arg-∆Phe and Lys-∆Phe, due to the presence of positive charges on them, were used to condense plasmid DNA of various sizes into discrete Nps. Plasmid DNAs were loaded on to dipeptide Nps either by incubating them with preformed Nps or by the formation of DNA-nanoparticle complexes by slow mixing of the aqueous solution of cationic dipeptides and plasmids.
Light scattering studies demonstrated that at a concentration of 2 mg/ml (in HFIP-water), Arg-∆Phe self-assembled into Nps with mean solution hydrodynamic radius (Rh) of 150 nm and a polydispersity index of 0.1. Nps formed by Lys-∆Phe at this concentration exhibited broad size distribution with a polydispersity of 0.38 (Figure 1a and b). In HFIP-water, the dipeptide formed particles with two major populations having mean Rh of 200 nm and 400 nm. TEM showed that Lys-∆Phe, at a concentration of 2 mg/ml self-assembled to form structures of irregular boundaries with a mean diameter of 50 nm (Figure 1c). These particles were different from the vesicular structures (370 nm) formed by the dipeptide at a concentration of 10 mg/ml , pointing towards concentration dependent assembly. However, Arg-∆Phe at a concentration of 2 mg/ml showed better assembly than Lys-∆Phe and formed well defined spherical Nps with an average size of 40 nm (Figure 1d). This is perhaps due to the fact that in arginine, the guanidinium group remains positively charged in neutral, acidic and even in low basic conditions promoting enhanced charge based interactions and hence self-assembly.
Light scattering technique was used to study the interaction of plasmid DNA with dipeptide Nps as well as to determine the optimum concentration of DNA needed for making compact and well defined Nps. Lys-∆Phe on its own formed Nps of Rh of 400 nm. Upon addition of increasing concentration of DNA [(25–100 μg) to 2 mg/ml of Nps], the Nps showed a reduction in their particle size. At a concentration of 100 μg of DNA, particles were further compacted and showed an average Rh of ~140 nm (Figure 2a). However, further addition of DNA did not lead to any significant change in particle size. When Arg-∆Phe Nps (2 mg/ml) were incubated with increasing concentrations of DNA (25–100 μg), their Rh decreased from approximately 170 nm to 110 nm (Figure 2c). The observed reduction in particle size of dipeptide Nps upon DNA addition could be due to charge-charge interactions of positively charged peptide Nps with negatively charged DNAs (Figure 2). Many earlier observations have also shown that cationic Nps bearing positive charge interact with DNA leading to DNA compaction and reduction in particle size [21–23].
From the above study, the optimum peptide to DNA ratio needed to form suitably compacted DNA-peptide Nps was found to be 20:1 (w/w). Entrapment efficiency determined from centrifugation experiments of the fluorescence labeled DNA showed almost 95% entrapment in both Arg-∆Phe and Lys-∆Phe Nps at this peptide to DNA ratio.
Cationic polymers condense DNA to yield compact Nps with well defined sizes [16, 24]. Nanoparticle formation was also observed when the cationic dipeptides were added to plasmid DNA. While plasmid DNA alone in water (100 μg/ml) formed irregular structures (no regular structure was found under TEM) with a mean Rh of 450 nm, it condensed into smaller and monodispersed Nps with the addition of increasing concentration of the cationic peptides. Nps with an average Rh of 120 nm were formed by the addition of 2 mg of Lys-∆Phe (Figure 2b) to the plasmid DNA. Thus, in this case nanoparticle formation occurred by the condensation of plasmid DNA in presence of cationic dipeptides and size of the resultant Nps depended on the amount of peptide added. Similar observation was seen in case of BSA-plasmid DNA polyplexes, where an increase in BSA/plasmid DNA mass ratio led to decrease in overall particle size of the resultant polyplexes . Likewise, addition of increasing concentration Arg-∆Phe also led to DNA condensation and decrease in overall particle size (Figure 2d). It was also observed that Arg-∆Phe led to DNA condensation at lower concentration as compared to Lys-∆Phe (250 μg of Arg-∆Phe vs 2000 μg Lys-∆Phe for same amount of DNA). Similar observation has been shown earlier where better DNA condensation was obtained with ariginine homopeptides than lysine ones . This could be explained by the difference in the nature of electrostatic interactions and DNA binding capacity between lysine and arginine residues . Where there is only one amine group in the lysine side chain guanidinium side chain of arginine has three amine groups. This promotes zwitterion hydrogen bonding of arginine with the phosphate group as well as guanine base of DNA, providing high binding strength and assembly than lysine .
Nps with a mean Rh of 117 nm were formed when 250 μg of Arg-∆Phe was added to 100 μg of DNA in 1 ml of water. A further addition of peptides did not cause any considerable change in particle size. TEM images further supported the idea of DNA condensation by Arg-∆Phe and showed initial formation of large, densely stained (due to the presence of negatively charged DNA) aggregates, which then rearranged into well defined spherical structures with time. The Nps were highly homogenous in nature with mean diameter ranging from 20–30 nm (Figure 3) a size suitable for endocytotic cellular uptake .
Effect of DNA size on the formation of DNA-peptide Nps was determined by incubating cationic dipeptides with plasmids of various sizes. Lys-∆Phe formed particles with mean Rh of 70 nm with pl, 117 nm with pgem, 165 nm with dh, 171 with md, and 112 with sod (Additional file 2: Figure S1a). Similarly, Arg-∆Phe formed particles with Rh of 103 nm with pl, 134 nm with pgem, 280 nm with dh, 479 nm with md and 280 nm with sod (Additional file 2: Figure S1b). Thus it was observed that there was no correlation between the size of DNA-peptide Nps with plasmid length (in base pairs) (Additional file 2: Figure S1).
The morphology of Nps formed by combination of different plasmid DNAs with Lys-∆Phe and Arg-∆Phe was determined under electron microscopy. Lys-∆Phe condensed plasmid DNA pl into well defined vesicular nanostructures (Figure 4) with mean size of 30 nm. The peptide formed micellar structures with md and dh DNAs with average particle size of approximately 20 nm. With sod plasmid DNA, the peptide formed densely stained ellipsoid like structures with mean size of 50 nm. Whereas, combination of Lys-∆Phe with pgem DNA resulted in the formation of roughly vesicular nanostructures with mean diameter of 100 nm. Similarly, Arg-∆Phe formed regular and well defined vesiclular nanostructures with plasmid DNAs of pgem, md, pl and md having mean diameters of 40, 30 and 42 and 50 nm respectively. However, with sod plasmid DNA, Arg-∆Phe formed nanostructures with irregular boundaries with average size of ~60 nm. Thus, both peptides formed Nps with variable morphology with different plasmid DNAs. Earlier studies have also demonstrated that multivalent cations such as polyamines, positively charged polymers, and peptides promote condensation of DNA to Nps that appear as rods, toroids, or spheroids under electron microscope [26, 30, 31].
It was also observed that in most cases Nps formed by the combination of Arg-∆Phe with plasmid DNAs were more regular as compared to those formed by the Lys-∆Phe and plasmid DNAs (Figure 4). This correlates with our DLS results that showed better DNA condensation and assembly with Arg-∆Phe as compared to Lys-∆Phe.
In order to locate DNA in dipeptide Nps, plasmid DNA was labeled with Pt-complex; Pt being electron dense can aid in locating the labeled DNA inside DNA-peptide Nps in electron microscopy. Electron micrographs of Pt-DNA-dipeptide Nps showed densely stained Pt-DNA-complexes lying inside normal vesicular structures. High resolution micrographs demonstrated core-shell like structure with dark ring like DNA inside nanoparticle core (Figure 5).
Complexation of DNA with dipeptide Nps was further investigated using agarose gel electrophoresis [15, 24]. It was observed that DNA was retained around sample pore after being complexed with peptide Nps (Figure 6a). Nps bound to DNA remained positively charged, causing DNA binding to them to have retarded movement into the gel. Similar results and retention in DNA mobility were found in case of genes and DNAs when bound to Nps [15, 24].
A major hurdle in gene or siRNA delivery is the degradation of DNAs by intra and inter-cellular nucleases. Hence an ideal gene delivery vehicle should not only facilitate intracellular delivery of loaded DNAs but also protect them from degradation by inter or intra-cellular nucleases [32–34]. In order to test the stability of DNA-peptide Nps, Nps were digested with DNase I and analyzed using agarose gel electrophoresis. Free plasmid DNA was degraded into smaller fragments. However, DNA-peptide Nps digested by DNase I were retained around sample pore (Figure 6b). These results showed that DNA was largely protected from enzymatic degradation in the DNA-dipeptide Nps.
Efficacy of the DNA-dipeptide Nps to ferry DNA inside cells was determined in HeLa cells. It was observed that cells could easily take up the DNA-peptide Nps and appeared fluorescent in all four cases. Fluorescence intensity and hence cellular uptake was greater in case of Arg-∆Phe-DNA Nps as compared to Lys-∆Phe-DNA Nps (Figure 7). This may be attributed to better assembly and DNA condensing ability of Arg-∆Phe into discrete and compact nanostructures facilitating their cellular uptake. Similar phenomenon was observed in earlier reports that demonstrated that the cellular uptake of the DNA-peptide complexes and their intracellular state seem to be controlled by the peptide chemistry and homopeptides made of arginine demonstrated higher cellular uptake than their lysine counterparts [26, 35]. Cells were also incubated in presence of Alexa-488 labeled free DNA or void peptide Nps as controls. Results revealed that though HeLa cells could easily take-up void peptide Nps they showed very little uptake of free DNA (Figure 7). Thus, it was clearly seen that packaging DNA inside peptide Nps improved their cellular uptake ability and could be instrumental in non-viral mediated gene delivery. Moreover, the degree of nanoparticle uptake by cells correlated directly with their incubation time and was higher for an incubation period of 12 hrs as compared to that of 6 hrs (data not shown).
We next investigated if DNA-peptide Nps were localized in the nucleus of HeLa cells. Cells were incubated with fluorescent-labeled DNA-peptide Nps and imaged under microscope. Nuclei were labeled with DAPI staining. Fluorescent micrographs showed that DNA-peptide Nps were easily taken up by cells and the engulfed DNA was distributed all through the cytoplasm and the nucleus (Figure 8). These results suggest that small cationic peptide based Nps can condense DNA into discrete Nps and can carry them to cellular cytoplasm and nucleus protecting them from enzymatic degradation.
Toxicity is a major issue in synthetic polymer based gene delivery . Toxicity of the peptide and DNA-peptide Nps was determined using MTT assay. Results of MTT cell viability assay demonstrated that DNA-peptide Nps were biocompatible and showed no cytotoxicity to cell lines like HeLa and L929 (Figure 9). This is an important characteristic property of Arg-∆Phe and Lys-∆Phe Nps which make them suitable for gene therapeutic applications; several other cationic polymers have been found to be more toxic .
After establishing positive plasmid DNA delivery as well as biocompatibility of DNA-peptide Nps, we next evaluated the transfection efficiency of the Nps. For this the dipeptides were incubated with GFP encoding plasmid DNA (peptide to DNA ratio of 20:1) and analyzed using TEM. Images showed that Nps formed by the combination of Arg-∆Phe with EGFP plasmid DNA were vesicular in nature with mean diameter of 80 nm approximately, whereas those formed by the plasmid DNA and Lys-∆Phe were irregular with no proper shape and size (Figure 10a). These results corroborated with our earlier results carried out with other plasmid DNAs, where Arg-∆Phe demonstrated better DNA condensation and assembly than Lys-∆Phe. Since better Np formation was obtained with Arg-∆Phe, cellular transfection assay for determining EGFP expression was carried out with Arg-∆Phe. To test expression and transfection efficiency of DNA-dipeptide Nps, HuH7 cells were incubated with EGFP encoding plasmid DNA Arg-∆Phe and lipofectamine and analysed using fluorescence microscopy. Images showed positive green fluorescence in both cases depicting successful plasmid DNA delivery and expression by the dipeptide Nps (Figure 10b).
The work presented here showed that cationic dipeptides containing a chemically modified amino acid, ∆Phe, easily formed discrete nanoparticles with plasmid DNA of different lengths. Dipeptide nanoparticles protected plasmid DNA from enzymatic degradation and easily ferried them inside mammalian cells, both in nucleus and cytoplasm. GFP encoding plasmid DNA loaded Nps showed positive transfection and gene expression in HuH 7 cells. The dipeptides described here are easy to synthesize and also demonstrated no visible cytotoxic effect in tested mammalian cells. To conclude the study brings to light the potency of cationic dipeptide based systems as gene delivery vehicles and certainly adds new dimensions to non-viral based gene delivery.
Dynamic light scattering
Plasmid DNA of 5.2 kb [Topo 2.1 vector + mondehydroascorbate reductase gene]
Plasmid DNA of 6.1 kb [pET14b vector + dehydroascorbate reductase gene]
Plasmid DNA of 5.8 kb [pET14b + superoxide dismutase gene]
Plasmid DNA of 5.26 kb [pGreen + Rd2qA gene]
Plasmid DNA of 3.69 kb [PL12R34H]
Human cervical cancer
Human hepatocellular carcinoma
Bovine serum albumin
Fetal calf serum
tissue culture treated polystyrene
phosphate buffered saline
transmission electron microscopy
enhanced green fluorescent protein
Arginine-α, β-dehydrophenylalanine and Lys-∆Phe
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Tian HY, Deng C, Lin H, Sun J, Deng M, Chen X, Jing X: Biodegradable cationic PEG-PEI-PBLG hyperbranched block copolymer: synthesis and micelle characterization. Biomaterials. 2005, 26: 4209-4217. 10.1016/j.biomaterials.2004.11.002.
Vijayanathan V, Thomas T, Thomas TJ: DNA nanoparticles and development of DNA delivery vehicles for gene therapy. Biochemistry. 2002, 41: 14085-14094. 10.1021/bi0203987.
Percec V, Dulcey AE, Balagurusamy VS, Miura Y, Smidrkal J, Peterca M, Nummelin S, Edlund U, Hudson SD, Heiney PA: Self-assembly of amphiphilic dendritic dipeptides into helical pores. Nature. 2004, 430: 764-768. 10.1038/nature02770.
Mansouri S, Lavigne P, Corsi K, Benderdour M, Beaumont E, Fernandes JC: Chitosan-DNA nanoparticles as non-viral vectors in gene therapy: strategies to improve transfection efficacy. Eur J Pharm Biopharm. 2004, 57: 1-8. 10.1016/S0939-6411(03)00155-3.
Kim TH, Ihm JE, Choi YJ, Nah JW, Cho CS: Efficient gene delivery by urocanic acid-modified chitosan. J Control Release. 2003, 93: 389-402. 10.1016/j.jconrel.2003.08.017.
Je JY, Cho YS, Kim SK: Characterization of (amino ethyl) chitin/DNA nanoparticle for gene delivery. Biomacromolecules. 2006, 7: 3448-3451. 10.1021/bm060373d.
Naik RJ, Chandra P, Mann A, Ganguli M: Exogenous and cell surface glycosaminoglycans alter DNA delivery efficiency of arginine and lysine homopeptides in distinctly different ways. J Biol Chem. 2011, 286: 18982-18993. 10.1074/jbc.M111.227793.
Godbey WT, Mikos AG: Recent progress in gene delivery using non-viral transfer complexes. J Control Release. 2001, 72: 115-125. 10.1016/S0168-3659(01)00267-X.
Aramaki Y, Takano S, Tsuchiya S: Induction of apoptosis in macrophages by cationic liposomes. FEBS Lett. 1999, 460: 472-476. 10.1016/S0014-5793(99)01386-1.
This work is supported from a Nanoinitiative grant from Department of Science and Technology, India, Nanotechnology grant from Department of Biotechnology, India and core funding at ICGEB, New Delhi, India. JJP also thanks Unesco-loreal for women in science for fellowship.
Authors declare that they have no competing interests with any organization or body.
JJP conceived, designed and performed all experiments. AV carried out GFP transfection studies. VSC directed the research. JJP and VSC wrote the manuscript. All authors have read and approved the manuscript.
Electronic supplementary material
Additional file 1:Information S1. contains details about the synthesis of Arg-∆Phe and Lys-∆Phe dipeptides. (DOCX 13 KB)
Additional file 2: Figure S1: Change in overall size of DNA-peptide Nps when incubated with plasmid DNA of different lengths (in base pairs). (a) Change in size of Arg-∆Phe-DNA Nps. (b) Change in size of Lys-∆Phe-DNA Nps. [Blue bars show plasmid DNA size in base pairs (bp) and red bars show mean Rh of Nps determined through DLS (np size)]. (PDF 29 KB)
Authors’ original submitted files for images
Below are the links to the authors’ original submitted files for images.
About this article
Cite this article
Panda, J.J., Varshney, A. & Chauhan, V.S. Self-assembled nanoparticles based on modified cationic dipeptides and DNA: novel systems for gene delivery. J Nanobiotechnol 11, 18 (2013). https://doi.org/10.1186/1477-3155-11-18

domain: medical


Our blue joint tape is made superior quality SPVC film coated with a rubber-based adhesive on one side. It has high elongation and excellent conformability for wire harness.
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Transform Scotland has responded to Transport Scotland’s ‘Consultation on the M8 and M9 Trunk Roads (Newbridge to Hermiston Gait) (Actively Managed Hard Shoulder and Speed Limit) Regulations’. The consultation remains open until 13 January.
Transform’s policy adviser Nigel Bagshaw commented:
“Any measures to improve bus reliability and journey times are to be welcomed and I look forward to these proposals being introduced unamended. However, to bring about the far-reaching changes needed to reduce transport-related climate emissions in Scotland, what is required is a huge switch in focus away from road-building towards public and active transport.”
Transform’s response in full reads:
In response to the above consultation, we would like to submit the following brief comments.
We support the provision of a full-time, permanent bus lane on the hard shoulder of the road and the extension of the speed limit on the M9 to M8 eastbound interchange link.
From the point of view of sustainable transport, it is well established that buses, like other forms of public transport, have the capacity to move people much more efficiently whilst producing only a fraction of the emissions per person in comparison with single-occupancy cars, and the proposed measures should, as intended, reduce journey times and increase journey time reliability for buses, thus making them a more attractive travel option.
The rationale behind the regulations made previously in 2012, which introduced an actively managed hard shoulder, continues to apply and the current proposals merely constitute an extension of those regulations.
We also support the exclusion of vehicles which cannot carry more than 23 seated passengers, for reasons of consistency with the existing regulations and for the purpose of prioritising more effective, high-capacity vehicles.
In summary, we support the proposals and look forward to their implementation with no changes which would reduce the effectiveness of the objective they pursue.


WHEELING, W.Va. – Today, at Independence Hall in Wheeling, State Senate Majority Whip Ryan Weld (R-Brooke) announced he will be seeking the Republican nomination for Attorney General. The announcement comes after current Attorney General, Patrick Morrisey, recently announced his candidacy for Governor.
“Over the next 12 months, my team and I are going to make it clear that my experiences as an officer in the Air Force, as a prosecuting attorney, and as a legislative leader all make me the most qualified candidate to be this state’s next Attorney General. We are running a campaign that is focused on getting the government out of people’s everyday lives, fighting the drug epidemic ravaging our communities, and protecting our citizens from fraud and abuse. We are putting the freedoms of West Virginians first in this campaign,” Weld said.
Weld’s platform for Attorney General will be based around three principles. First, he will continue to use the power of the office to fight back against federal overreach and excessive regulations. Second, he will hold accountable those responsible for the opioid and drug crisis that has ravaged our communities and devastated countless families across this state. And finally, he will protect West Virginia’s consumers from fraud and safeguard our seniors from abuse and neglect.
Weld’s commitment to public service began in 2005 when he commissioned into the Air Force Reserve. After spending several years on active duty with the U.S. Intelligence Community and a deployment to Afghanistan, he made the decision to come back home to West Virginia.
“When I came home in 2011, I realized that most of the folks I grew up with had been forced to leave in order to find opportunities for themselves and their families. I knew then that I had to do everything I could to turn West Virginia into a place with opportunities for both those who stayed, and those who wanted to come back. That is what motivates me as an elected official,” Weld said.
Noting recent accomplishments and momentum in the state, Weld said he plans to fight for West Virginia’s continued growth so more West Virginians can continue to call this state home.
“We are growing into a place that is full of new opportunities and growing optimism – and we need an attorney general who understands this change and is ready to fight for this new West Virginia,’ he said. “I firmly believe that my experience has uniquely prepared me to lead the office that will help protect the future that is now within West Virginia’s reach.”
He also recognized the important progress made by Attorney General Patrick Morrisey, who has served in the role for the past eleven years.
“D.C. is clearly out of touch with the needs and worries of everyday West Virginians. I vow to continue the tremendous work started by Patrick Morrisey against Washington’s overreach and ensure that the progress we have made in the past few years isn’t stopped by bureaucrats and politicians who have never been here,” Weld said.
About Ryan Weld:
State Sen. Ryan Weld is an accomplished leader with an impressive track record of service to his state and country.
As the Majority Whip in the West Virginia State Senate, he skillfully manages legislative priorities and effectively advances the state’s interests. In addition, Ryan’s extensive experience as chairman of the Military Committee and vice chairman of the Judiciary Committee reflects his exceptional ability to handle complex policy issues.
Ryan has served in the West Virginia Legislature since 2014, but Ryan’s commitment to public service began in 2005, when he commissioned into the Air Force Reserve, where he spent several years serving on active duty in the U.S. Intelligence Community.
In 2010 and 2011, he served as the Staff Intelligence Officer to the Provincial Reconstruction Team in the Zabul Province of southeastern Afghanistan. Prior to his time overseas, he served as the Executive Officer to the Director of Congressional Affairs of the Defense Intelligence Agency.
Ryan began his legal career as an assistant prosecutor at the Brooke County Prosecutor’s Office. He currently practices law with the firm of Spilman Thomas & Battle in their Wheeling office. He obtained his bachelor’s degree from Fairmont State University in 2003, and his law degree from Duquesne University in 2015.
Weld is a 1998 graduate of Brooke High School and a 2003 graduate of Fairmont State University. He commissioned into the Air Force Reserve in 2005, and spent several years serving on active duty in the U.S. Intelligence Community in Washington, D.C. Throughout 2010 and 2011, he served as the Staff Intelligence Officer to a joint Army-Air Force team in the Zabul Province of southeastern Afghanistan.
Following his deployment, Weld returned home to Wellsburg where he met his wife, Alex. They married in 2015 and continue to live in Wellsburg with their three dogs.
Weld obtained his law degree from the Thomas R. Kline School of Law of Duquesne University in 2015. Weld was elected to the House of Delegates in 2014 to represent the 2nd House District. In 2016, he was elected to the State Senate representing the 1st Senatorial District, and was reelected in 2020. In the Senate, Weld serves as the Majority Whip, chairman of the Military Committee, vice chairman of the Judiciary Committee, and member of several other Senate committees.


text:
Glyfada Seaside Residence FAQs
What are the hotel policies for check-in and check-out time?
Check-in begins at 13:00 and check-out can be done until 00:00.
What amenities/facilities are offered for guests at this hotel?
The Holiday homes offers Snorkelling, Tennis court, Cycling, Diving, Golf course (within 3 km), Bowling.
Does the Holiday homes - Glyfada Seaside Residence have on-site parking facility?
Yes, Parking, Free parking facility available on-site.

domain: travel


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A brand new series of Celebrity SAS: Who Dares Wins is coming to Channel 4 next month with a huge line-up of celebrities set to take part. 14 famous faces will take on both physical and mental challenges as they are pushed to their limits in the Jordanian desert to raise money for cancer.
Love Island, Strictly Come Dancing and EastEnders stars are amongst those brave enough to attempt to pass the notoriously difficult SAS Selection test. Former 2019 Love Island winner Amber Rose Gill is part of the brand new line-up alongside former EastEnders actress Maisie Smith who played Tiffany Butcher and appeared as a contestant on Strictly in 2020.
Former footballer Ashley Cain will also take part in the military exercises for the all-important cause after losing his daughter, Azaylia Cain, at just eight-month-old after she was diagnosed with an aggressive form of leukaemia.
Speaking about his decision to do the show, Ashley paid tribute to his daughter Azaylia who died in April last year, saying: "Losing my daughter was the most difficult experience of my life.
"I think to combat trauma you need to step outside your comfort zone and see who you really are. There’s no better way to do that than on Celebrity SAS: Who Dares Wins, so when the opportunity came, I took it with both hands."
Also taking part are Brookside actress Jennifer Ellison, TV personality Calum Best, as well as Strictly Come Dancing professional dancer AJ Pritchard and his brother Curtis Pritchard who appeared on Love Island in 2019.
The Only Way Is Essex stars Pete Wicks and Ferne McCann are also taking part in the new series that will air on September 4 .
Ferne, who is newly engaged to her boyfriend Lorri Haines, said: "It just felt like the right time in my life to be challenged.
"I had no idea what to expect but I wanted to see how tough it really is. You’re stripped of everything, broken down to the point you have no choice but to build yourself back up again. It was the opportunity of a lifetime and I’m so glad I’ve done it."
Olympic sprinter Dwain Chambers, taekwondo Olympic gold medallist Jade Jones, javelin Olympic gold medallist Fatima Whitbread, boxer Shannon Courtenay and Paralympic high jumper Jonathan Broom-Edwards complete the line-up.
Former US Marine Rudy Reyes will take on his role as chief instructor once more, after stepping in for Ant Middleton.
Channel 4 reportedly severed ties with Ant over his 'personal conduct' but he said he had quit the programme as it was more like a "reality show" now.
The brand new series of Celebrity SAS: Who Dares Wins will air on Channel 4 on Sunday, September 4 at 9pm.

domain: news


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When doing any design work which is to be presented and then ultimately implemented in various ways, one of the most challenging prospects is how to exactly do this successfully. Sure, we've come up with ideas for the design, which typefaces suit, which colour scheme works - all fine. We have a solid proposal in our minds, collected some preliminary versions in various files but nothing that is ready to be reviewed.
Our sketches and scribbles which served us well during brainstorming and initial iterations should now become something tangible for the client to see, review and give feedback on. And if the design work involves a digital output, such as a website — how can we best present our design in a way that gives a clear impression of the concept and encourages good conversation?
Please do your own research to look into different people's workflow ~ there's plenty to explore, many to learn from and you'll end up finding what works for you. To illustrate the topics I'll be referencing some of my client work to give you actual examples.
styletil.es is open source and as far as I know, is a term coined by Samantha Warren. As opposed to a moodboard (which can be quite vague) or a static mockup (which can be too rigid) the style tile will collect the aspects of visual design relating them to their final purpose and use. It will feature the type choices and settings (for typical elements like sub/headings and paragraphs), the colour scheme as applied to layout and interface elements and additional UI related elements.
While it used to be norm to produce web pages as static mockups, this always had the obvious drawback of a static canvas, a set size and proportion. Therefore as designers we find ourselves creating pictures, snapshots of the design at a set size. This does not set expectations well ~ and is often the reason for the client's assumptions that this will look the same in all browsers.
Using a style tile however, this assumption is bylined as the presented material is a collection of different elements rather than aiming to demo a final design as it might look in the browser. An additional benefit of this approach is that it will keep the conversation focused on the design in principle, rather than specific little details of a given (and complete-looking) design.
How to work with style tiles
The best way to start is to simply think of this as a digital sketchbook, a place to collect the various ideas for type, colour, layout, UI etc. The file will be your drawing board where you can finalise the design you're going to propose. Once you're happy — you can revise a copy of this file and make it presentable, i.e. work on the presentation of these elements to best demonstrate the design concept.
Using the first style tile as basis, you can iterate the file following feedback and creating new versions will become a record of the design journey. Changes are evident and can clearly be reviewed, and referred back to if needed. Once the design is completed and signed off, your updated style tile can then be added to the final brand guide.
This is one of my client projects: a research project spanning 2 years. The design work started off with branding which was then to be carried through to an initial website for use during the active research phase. The next phase then expanded the branding and evolved the website to present research findings. (read case study)
This was the initial style tile after we'd confirmed the brand design and logo. Very minimal, it references typeface and colour alongside notes of purpose.
To work on the final version of the site, the original style tile was used as reference, adding new colours, making note of feedback and edits shown.
The principle of the style prototype is very much the same as the style tile - to present design aspects such as the typography, colour scheme and layout for review or final confirmation. It does so via a live webpage instead of a static version. This takes the style tile one level further via a real preview of the design concept in the browser - while still focusing on aspects of design instead of webpage layout.
Compared to a full-fledged page/site prototype, the style prototype does not offer the full design but remains focused on the foundation. It presents the cornerstones of the design alone. For example, it shows swatches for colour which are also applied to the page content elements as live demo of their use. Content of this should be exemplary of the final version or offer additional notes on the page in hand.
The style prototype offers a live preview and can easily be shared and viewed in any browser, any device and by anyone within the wider team. By previewing the design live, everyone will get a better sense of the final look and feel of the presented concept.
Going by my experiences after having used this approach for nearly a decade now - I can tell you that it works :) I generally use the style tile during the early stages of a brand design, for example, - or for print-related projects. Once the design is to be expanded to be used for digital presentation (i.e. screen-based) - the style prototype is the most effective approach to presenting design concepts.
This is the initial style prototype which was part of the first phase.
To illustrate the effectiveness of working with style tiles and prototypes, I'd like to tell you 2 little tales of their use. You'll see that this method is very flexible and great for demo of the design foundation.
paving the way
This is a wonderful joint project from the Challenging Behaviour Foundation and the Council for Disabled Children. It is an initiative to support children with extraordinary needs and their families and friends.
The initial work involved working with the brand colours of the two collaborating charities to create a fitting colour scheme and visual approach to the new website.
Work on the colour scheme was extensive to ensure clear legibility and meet the required standards. Considering content presentation as well as interactive elements, the original colours were fine-tuned to a nicely accessible contrast level.
While a lot of preliminary work could be done on the specific detail alone, only a live preview would be able to confirm the final colours and allow for user review/testing. The team took some time to get feedback from families, carers and support workers whose input was invaluable.
The style prototype for this project included the final title as well as the menu to demo colour treatments. Further iterations were done to optimise the colour scheme and design for further view modes, view the site to see it in action (read case study).
William is a Film Maker & Radio Broadcaster whose site was in dire need of an overhaul and design update. With a large archive of files his popular broadcast, he was keen to maintain a connection to the existing design and had quite firm ideas .
While I was quite certain that the proposed colours would not be as effective as he thought, it was the style prototype which offered William the best way to evaluate his requirements and suggestions.
We worked on iterations together, experimenting with the graphic aspects he requested and finalised the final design. The first prototype used the exact colours William proposed and was quite harsh and cold. The second prototype followed William's request for a dark theme with an ocre shade added for more warmth and hand-written font. A few iterations later, and the final prototype was produced, including more of the site's content. The end result did carry over many aspects of the old design and found a good medium between William's ideas for the feel of his site and changes for a clear update. Check out the end result on williamenglish.com.

domain: artistic


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R.S.V.P. (Retired & Senior Volunteer Program) is a National Service program designed to recruit skilled, experienced, active adults ages 55 and older for volunteer activities that work towards meeting the needs of their communities. The R.S.V.P. helps connect Seniors to rich volunteer opportunities based on their talents, skills, and experiences. Some of the various activies that R.S.V.P members do to help their community include:
- Build low income housing
- Provide food to the needy and isolated seniors
- Help families in need of clothing and goods
- Provide companionship to seniors
- Support veteran services
- Mentor and tutor the youth
- Provide support to local city government/ act as ambassadors to our region/ support CHP and local police departments
RSVP Impact Areas
RSVP Volunteer Requirements
Why should you volunteer?
Benefits to Volunteering
RSVP Overview Video
If you are 55 years of age or older and are interested in volunteering, feel free to fill out the R.S.V.P. application, email us, or call us!
RSVP Forms & Documents
Phone: (559) 237-3101
Meet Clifford and Madeline Gartin RSVP Volunteers
Madeline and Clifford Gartin have been happily married for 63 years. They have a long history of service to their community. This remarkable couple has over 5000 hours of documented volunteer service each, that’s over 10,000 hours of volunteer service together! This achievement was honored by HandsOn Central California who presented them with Presidential Lifetime Achievement Awards, and Mayor Lee Brand honored them with a proclamation declaring November 8th Gartin Day in the City of Fresno.
Madeline Gartin is currently volunteering at Fresno City Hall where she gives tours, directs residents to the departments they are looking for, and helps constituents find their city council representatives. In the past Madeline has volunteered with the Fresno CHP and as a docent with the Fresno Chaffee Zoo.
Clifford Gartin is an army veteran currently volunteering at the Fresno Yosemite International Airport and the Fresno Veterans Home. Clifford is part of a group of RSVP Volunteers that runs the information booth at the Airport. They greet visitors to our region and share information and history about the area, and help direct travelers to various areas and needs in the airport. Our airport ambassadors help our airport run more smoothly and put a friendly experienced face to what is available to do in our region. At the Fresno Veterans Home Clifford provides companionship and encouragement to resident veterans in the home. Clifford spent 20 years volunteering with the State of California General Machinist Joint Apprenticeship Division. Clifford was part of a group that help set the standards for machinist apprenticeships in California. Clifford volunteered as a Deputy Commissioner of Marriages for Fresno County where he performed 1273 marriage ceremonies. Clifford was named the RSVP Volunteer of the Year in 2015 at the Hands Across the Valley Awards.

domain: sociology


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2648 Margaret St
Mercer, WI 54547
Matt Dallman, director of conservation for The Nature Conservancy in Wisconsin, will talk about his organization and the conservation work they do in Wisconsin and world-wide in a free program at the Mercer Library on Friday, March 9 at 1:00pm. This program is part of the library’s celebration of the 2018 Aldo Leopold Week in Wisconsin. Light refreshments will be served. For more information, call the library at 715-476-2366.


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Alex Pike saluted Gosport Borough’s progress as they stretched their unbeaten run to eight matches.
Justin Bennett scored twice in the first half as the Privett Park outfit stunned second-placed Leamington at the New Windmill Ground last night.
Dan Wooden made sure of the Southern League premier division points late on as the game ended 3-1.
Pike was delighted with the win, especially as Leamington have been in fine form themselves.
He said: ‘To come away from home and beat a team who have lost just twice in 27 matches shows how far we have come.
‘It takes our unbeaten run to eight matches, too, which is really something considering our poor start to the season.’
The hosts began well but Lee Molyneaux and Brett Poate repelled the best strikers Tom Berwick and Lee Chilton had to offer.
And after soaking up the pressure for 15 minutes, Wooden forced a save from Leamington keeper Niall Cooper at his near post.
Borough then took the lead on 25 minutes when Poate and Wooden unlocked the Brakes defence to leave Bennett to blast home from 10 yards.
Berwick shot over as the hosts searched for a leveller.
And his wastfulness was punished as Bennett doubled Borough’s lead after latching onto a ball from Molyneaux and rounding the keeper.
Berwick did find the net on 34 minutes, though, halving the deficit as he caught Gosport napping.
But the visitors finished the half well, with Poate firing a free-kick narrowly over.
Nathan Ashmore saved well from Chilton early in the second half before Bennett twice went close to completing his hat-trick.
Ashmore then denied Alex Taylor before Wooden turned a cross home to seal the win.
LEAMINGTON: Cooper, Mace, Morley, Magunda, Hood, Daly, Dodd, Husband, Berwick, Chilton, Batchelor. Subs: Naylor, Perron, Taylor, Moore, Owen
GOSPORT: Ashmore, King, Poate, Dunford, Molyneaux, Pearce, Igoe, Smith, Wooden, Bennett, Wilde. Subs: Woodward, Martin, Ford, Brown, Wilson


A new film called “The Batman” is set to hit theaters on June 25, 2021 and it’s going to have both a new actor playing the protagonist and a new Batmobile. The actor, Robert Pattinson, was picked to play the role of Batman after Ben Affleck stepped down from playing Batman in 2019. If you haven’t seen Pattinson’s recent work, you should (e.g., The Lighthouse, The Childhood of a Leader, and Good Time).
For Pattinson’s Batmobile, the director Matt Reeves is going in a new and awesome direction. Reeves unveiled the new superhero car through his Twitter account and it’s phenomenal. Instead of a wild and over-the-top design, they went with an extremely strong-looking muscle car that has an exposed engine in the back. Unlike other Batmobiles, this one is powered by what looks like a V10 engine instead of a jet turbine.
We can’t wait to see this new film and see the Batmobile in action.
Image Source: Twitter/@mattreevesLA


text: Episode 25 of Fullmetal Alchemist is one of the most emotional yet. With Ed and Al seemingly giving up their search for the Philosophers Stone this episode concentrates quite a bit on Hughes' investigation into lab five leading to considerable danger for him. The episode also has some interesting flashbacks explaining some of his relationship with Mustang. Episode 26 is a lighter hearted episode than the last with Ed, Al and Winry taking time out in the town of Rush Valley, home of the best auto-mail in the world. While their Ed cheats a couple of conmen out of their winnings and Winry decides to teach Ed a lesson about good auto-mail. In episode 27 the brothers' hardnosed former teacher Izumi catches up with them and takes them back to her home village to explain themselves. This is a nice episode with some good back-story on the brothers. Episode 28 is another back-story episode. Having discovered the brothers' secrets involving human alchemy, Izumi decides they need to relearn a lesson they learned in their original training. Stranded on a seemingly deserted island the brothers' relive their previous experiences there.

domain: entertainment


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Botox, Dysport and Xeomin
Dr. Daria Hamrah is one of the most sought after facial cosmetic surgeons in Mclean and Northern Virginia for his artistic approach in administering Botox, Dysport and Xeomin to reduce lines and wrinkles and reverse the signs of age. In this blog posting, Dr. Hamrah offers and inside look into the aging process and reveals how Botox, Xeomin and Dysport actually work to erase lines and wrinkles.
Lines and Wrinkles Begin To Form at an Early Age
For most individuals wrinkles begin to form in the late teens and early-twenties. While genetics and lifestyle choices such as, cigarette smoking, sun exposure, caffeine, alcohol consumption and environmental pollution, play a pivotal role in how well we age, certain processes can’t be avoided.
Starting out as dynamic lines, wrinkles are initially visible when making certain facial expressions, such as a frown or smile. Squinting, stress and intense concentration also take their toll and with time the wrinkles caused by these repeated facial movements become static… meaning they are noticeable even when our face is perfectly relaxed and motionless.
While a few lines can add character to one’s features, multiple and deep lines serve to detract from the overall beauty and youthfulness of one’s appearance. Particularly vulnerable, and often the first to reveal the signs of age, are the forehead, the area between the eyebrows and the delicate skin around the eyes.
The lips are a bit more resilient, however with the passage of years vertical lines make their debut around the vermillion border. For smokers and sun worshippers these markers are more pronounced and can be readily evident in one’s thirties.
Botox, Dysport and Xeomin Put the Freeze on Lines and Wrinkles
Now a household name, Allergan’s Botox Cosmetic is a well-known treatment option, Medicis’s Dysport and Merz’s Xeomin are two others. Classified under a category of injectables referred to as neuromodulators, these FDA approved “denervating agents” temporarily block the muscles nerve receptors.
For a visual illustration of how neuromodulators work, one may think of gently “freezing” the muscles. This reduces both the quantity and intensity of the muscle’s ability to contract. No muscle contraction, no wrinkle. It’s that simple!
Typically, a small amount of the denervating solution is injected into the muscles directly above the area where lines and wrinkles appear. Within five to seven days of treatment these lines and wrinkles are thoroughly diminished, revealing a younger and happier looking you.
As a non-invasive procedure, Botox, Dysport or Xeomin is a quick and painless treatment that delivers outstanding results for expression related creases on the face and neck, with benefits that can last from three to six months (depending on individual variation on drug metabolism).
An Artistic Approach to Administering Botox, Dysport and Xeomin
Ecstatic with their results patients often want to know Dr. Hamrah’s secret. When the question arises, patients are surprised to find, there really is no secret.. JUST SKILL..
Botox, Dysport and Xeomin have been on the market for quite some time, and as the most popular non-invasive cosmetic procedure in the United States, many physicians and spas have jumped on the Botox band wagon, so to speak. This has given rise to a wave of practitioners with little training in facial anatomy, muscles and nerves to promote themselves as self-ordained experts in the field.
While it is true that treatment with a neuromodulator is relatively quick and painless: training, skill and expertise play a prominent role in the results that one achieves. Selecting the best therapy among the three options that are available today, and knowing when, where and precisely how much to administer is a specific talent acquired through years of dedication in the field of cosmetic surgery, facial structure and anatomy.
For the best results, patients should always choose a facial cosmetic surgeon or cosmetic dermatologist with specific training in anti-aging treatments and facial rejuvenation.


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‘The Incredible Mr. Limpet’ Remake Adds Jon Hamm, Sarah Silverman, Josh Gad and More
Director Richard Linklater recently impressed with 'Boyhood,' the incredible movie that took the filmmaker 12 years to make. But one of his next projects is decidedly much different: a remake of 'The Incredible Mr. Limpet,' with Zach Galifianakis filling the funny shoes of Don Knotts in the title role. The project is drawing boatloads of talent, including Jon Hamm, Sarah Silverman, Josh Gad, and many more.
The Wrap reports that a whole bunch of familiar faces are looking to join Galifianakis in Linklater's remake of the 1964 comedy classic 'The Incredible Mr. Limpet,' which blended animation and live-action and was based on the novel by Theodore Pratt. Jon Hamm is in talks to play the villainous role of a morally corrupt businessman, while Sarah Silverman and Danny McBride would play unspecified live-action characters. Kevin Hart, Josh Gad, and the duo of Jordan Peele and Keegan-Michael Key are looking to lend their voices as animated characters.
The original film told the tale of Henry Limpet, whose wish is granted when he's turned into a fish. Linklater's remake will move the story to present day, with Galifianakis' Limpet trying to help save a population of fish off the coast of a beach town.
Linklater has also written the script for the film, and will employ the same rotoscope technology he previously used on his films 'Waking Life' and 'A Scanner Darkly,' which should make for quite an interesting effect.


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Two pharmacists in Newfoundland facing hundreds of charges
Two pharmacists in Newfoundland have been charged with drug trafficking and defrauding the province’s prescription drug plan.
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ST. JOHN’S, N.L.—Two pharmacists in Newfoundland have been charged with drug trafficking and defrauding the province’s prescription drug plan.
They are to appear in court today for the start of a preliminary hearing.
The hearing will determine if there is enough evidence to go ahead with a trial.
John Gerald Whalen and Lloyd Matthew Bennett were pharmacists at East End Pharmacy in St. John’s.
Hundreds of charges were laid against them last year.
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There's something my friend Seven7 and I don't understand.
I've a Xvalve with ult on my rpg mag.
When I use it with a HP tank, it leaks by the barrel, when I use it with a SHP tank, it works perfectly.
The on/off orings are lubed and in good shape.
We checked and cleaned every part of the on/off.
I even tried to change the on/off
Have you ideas ?


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On Feb. 7, the provincial government announced legislation to make Truth and Reconciliation Day a statutory holiday, starting on Sept. 30, 2023.
The day was formally recognized as a federal statutory holiday in June of 2021, as a direct response to Truth and Reconciliation Commission Call to Action, 80. Though, this only allotted the statutory holiday to federally regulated workplaces.
Currently the federal government, Prince Edward Island, Northwest Territories, Nunavut, and Yukon are the jurisdictions that have declared Sept. 30 a statutory holiday.
The provincial legislation was in response to consultation with Indigenous groups throughout the province.
For Robert Dennis, chief counsellor for Huu-ay-aht, this legislation would “recognize the past wrongs” that have happened to First Nations people in Canada and work towards correcting those wrongs.
“This would be an opportunity for… Canadian society to work together to create a just society, [and] not a different level of treatment for different people,” said Dennis. “Things have to change, [they] can't remain the way they are, and a day like this would help us to make that change.”
Closing the economic gap for Indigenous people, a reduction in incarcerated Aboriginal individuals in the prison system, increasing graduates in the education system, and creating a level playing field in job opportunities are among some further changes that need to happen, said Dennis.
“Especially if people can change…their cultural attitudes [or] their cultural biases, they would realize that a segment of their society was definitely treated differently,” said Dennis. “This would just help them in educating them and realizing what needs to be changed, and then, more importantly, how it can be changed.”

domain: law


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Learning English Through Social Issues
st2012-2013 1 Semester
Food safety becomes a hot issue because of a series of food security accidents. It’s not a matter involving one person,but
everyone could be in danger of food poisoning. It has appealed public’s attention and should be regarded. Maybe it’s high time
to take severe measures to manage the food safety,especially for the government. We can make out how grim it is from following grid:
Year Accidents Harm
2003 Jinhua ham with DDVP Have bad effects to
2004 Fuyang inferior powdered Big-head baby
2005 Higher-than-standard Bad to thyroidea function
levels of iodine food in
Nestle milk powder
2006 Pork contaminated with Cause headache、nausea
clenbuterol and irregular heartbeat
2008 High levels of melamine in Kidney stone、renal failure
infant milk powder
2012 Firm yoguat Not clear yet
The features of food safety in china:
1. The range of food problem is expanding
In the past, it only referred to traditional staple and non-staple food like edible oil, meat. But now it reaches out into fruit,drinks,milk and so on. It takes on space formula and
2. It causes greater harm
Agricultural chemical and artificial fertilizer left over used to be the most common problem in food. Howevre, producers now put illegal food additives,which is an invisible harm to threaten people’s health.
3. Drugmaking is more and more diversified from external to internal,physical to chemical.
Cause of the issue:
1. The imperfection of legal system
The law system about food safety in china still has many disadvantages which gives the produce of unqualified food a room to exist. What’s more, china’s food standard lags behind
the international food standards. In addtion,there remains some important standards missing.
3. The consumers lack common sense in food safety
At present ,the majority of chinese belong to middle or lower class and have not that much money. When they’re faced witn
similar goods,the cheap ones will be their first choice and then
the quality or safety.People tend to have this consume attitude that besides me,there're so many people buying these food on special and it won't cause any problems according to the past
experience. Supported by these consumers, producers begin to expand their production and more dangerous food floods into the market. It can be a terrible cycling.
Suggestions to the issue:
1. We propose the government issuing the white paper of national food industry development planning,putting forward goals and policies from the microscopic angle. From the moment, the State Council has announced “The decision to
further strengthen the food safety” and most provinces have
offered their suggestions to launch the special remediation work aimed at the food safety, in the hope that nation would have a overall and far-reaching guild.
2. Improve the level of food industry;optimize the industrial structure;strengthen the whole-way quality management system of “stock-processing-circulation-marketing”.
3. Pay high attention to the food safety propaganda and supervision:Give full play to the role of the media’s supervision,
not only to continue exposing the illegal behavior of food safety, but also to promote the food safety knowledge and cultivate consumers the consciousness of protecting themselves. 4. Strengthen the supervision and punnishment power in the circulation field: Strictly implement that substandard food
should be withdrawed from the market system; consolidate the food safety logo and packaging management; rigidly control food additive producting and using.
5. Summary food safety accidents which expose system issues and moral failings.


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domain: game


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Guest - Spectre Premiere at grand opening of Cineworld Resorts World in Birmingham at Cineworld, Resorts World - Birmingham, United Kingdom - Monday 26th October 2015 (1 Picture)
Photo credit: James Watkins
Foo Fighters collect ''weird'' fan art. The 'Run' hitmakers are particularly fond of the pieces they are sent which are a ''little off'' and put them...
The film looks to be getting a reboot in a straight-to-TV Disney movie.


A buzz of excitement spreads through the halls of Spruce Avenue School as Jessy Nerval rushes by. “Is it hot lunch day?” someone asks. And like the pied piper, students follow him to the gym.
“We are all about stuff that’s just so good to eat, so good for you,” said Laurel Ferster, co-owner of The Calico Baking Company based in McCauley. Together with co-owner Zinovia Hardy, the partners believe their connection to the land makes their baked goods part of a bigger cycle.
They’ve had their share of challenges, like when the delivery driver dropped their oven off the back of the truck in 2014, significantly delaying when they opened their business, and putting extra financial pressure on them.
Continue reading Delicious and fresh from the oven Calico Baking Company offers locally-sourced baked goods


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Chanh muối is a salted, pickled lime in Vietnamese cuisine. Its name comes from the Vietnamese words chanh (meaning "lime" or "lemon") and muối (meaning "salt"). To make the chanh muối, many limes (often key limes) are packed tightly in salt in a glass container and placed in the sun until they are pickled. During the process, juices are drawn off the limes, which dissolves the salt and produces a pickling liquid which immerses the finished chanh muối.
Chanh muối are used to make a drink (with added sugar and water or carbonated water) that is called nước chanh muối or soda chanh muối, if made with carbonated water. Nước means water or "drink" in this context, when combined as nước chanh it means lemonade. The name of the drink is usually shortened to just chanh muối when the context is beverages and often appears on the menus of Vietnamese restaurants translated as "salty lemonade" or "salty limeade." To prepare the drink, a small piece of chanh muối (containing both rind and flesh) is cut, placed in a glass, and crushed slightly with a spoon or other utensil to release its juices, then the other ingredients are added.
Although the drink is typically served cold, with ice, as a refreshing summer drink, it may also be served hot, and is believed to be a remedy for the common cold. After finishing the drink, many Vietnamese people enjoy eating the piece of chanh muối left in the glass.
Outside Vietnam, lemons are sometimes used instead of limes to make chanh muối.
The first commercial brand was created by Dan Vo, sold in bottles and bags through Southeast Asia.
- Preserved lemon – condiment in South Asian and North African cuisine
- List of lemonade topics – Wikipedia list article
- List of pickled foods – List of links to Wikipedia articles on pickled foods
|This Vietnam-related article is a stub. You can help Wikipedia by expanding it.|

domain: food


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Since the release of iPhone 7 and iPhone 7 Plus it’s been five months. Five months ago, Apple changed its strategy, keeping the first design for the new flagship without the prefix “S”. Many analysts believed that the iPhone 7 will not be of interest to users because of the familiar design. Despite this “seven” at one point became the best selling smartphone in the world. Anyway, for the first time the existence of a right and a wrong iPhone.
When it comes to larger screens Apple, of course, was late. The fashion for large screens Samsung has introduced and presented the 2011 Galaxy Note. The iPhone maker did not change the size of displays to 2014. Apple representatives walked away from answers about the diagonal of smartphones and claimed that the company will not release of the enlarged iPhone until you find a way to do this without sacrificing the convenience of users.
Despite the fact that many people prefer devices that are easy to use with one hand, the advantages of a big screen is also clear to most. Perhaps the situation with ergonomics will be resolved in 2017 with the appearance of the display “edge to edge”, but until Apple offers phablets with 5.5-inch screen is large and clumsy.
The Apple range includes several models, but the “iPhone”, uncompromising and advanced, offering users the most advanced developments, one. And that’s the iPhone 7 Plus.
First iPhone 7 Plus equipped with a capacious battery, allowing him to work the entire day without recharging. iOS 10 offers many exciting features, but most of them are meaningless because of the limited autonomy of gadgets. If you have an iPhone 7, it’s likely you have to choose between battery life and voracious features. Of course, you can buy a case with a battery, but it will spoil the look of the device.
The iPhone 7 Plus not having to worry about battery life. No need to buy a case with battery or external battery pack, disable the “Hey Siri” and use the power saving mode.
Part of the display of the iPhone 7 Plus iPhone 7 is also superior. First and foremost is the size of the display panel. Say what you will, but the difference between 4.7 and 5.5 inches plays a big role. Secondly, and importantly, the 5.5-inch model has a higher resolution of 1920 x 1080 pixels and a higher pixel density at inches.
But the most important advantage the iPhone 7 Plus is the camera. Both versions got a 12-megapixel main module with optical image stabilization and optics from Sony. But the iPhone 7 Plus there is an extra lens. It allows you to remove a two-fold optical zoom, without losing quality, and use of portrait mode shooting. Making the two cameras and analyzing the data, the iPhone 7 Plus blurs the background behind your subject, similar to what we see on professional SLR cameras.
Summing up, it becomes clear what Apple wants to see in the hands of its users. This is partly due to the fact that the older model brings the company more profit. However, iPhone 7 Plus really gives users much more than the original iPhone 7. The only compromise is ergonomics, because to use a 5.5-inch smartphone is not very convenient, and one hand is not impossible.


Promoters go bargain hunting post sharp declines in stocks
Recently, five companies announced buybacks through stock exchanges.
Since January 1, promoters of nearly 150 mid- and small-cap companies have increased their stake by either buying from the open market or through buybacks and preferential issues.
For example, Tata Sons has bought 1.18 crore shares of Tata Motors valued at Rs 183 crore from the open market last week after the company reported its biggest-ever quarterly loss in December quarter, which led to a steep fall in its stock price. Shares of Tata Motors declined 56 per cent from their 52-week highs.
Similarly, Bajaj Auto has acquired shares worth Rs 600 crore in Bajaj Holding since January 1, while Bajaj Holding has purchased Bajaj Finance shares worth Rs 367 crore. Likewise, promoters of Future Retail have acquired shares worth Rs 107 crore of the company after the stock fell 37 per cent from its yearly high.
According to analysts, promoters raising stakes in their own companies is often considered a good sign, signalling better prospects for the business in the immediate future.
“Market volatility and relentless selling pressure across midcaps is offering an opportunity for the companies to use free cash on the books while in many cases even the promoters have shown willingness to use personal money to increase stake in their entities,” said Gaurav Dua, head of research, Sharekhan by BNP Paribas. “In both the cases, it is a positive indicator for the minority shareholders as it reflects the management’s confidence in the company and its growth outlook.”
Recently, five companies — Cyient, Persistent Systems, Lakshmi Machine Works and Selam Exploration — announced buybacks through stock exchanges.
IIFL promoters have bought three lakh shares of the company from the open market between February 5 and February 12. Promoters of other companies, such as like Zydus Wellness, Autoline Industries, Commercial Engineers and Ador Multiproducts, have subscribed the preferential shares of their companies.
“Such a trend of promoters increasing their holding when stock prices are low and a desire to enhance control reinforces investor confidence” said G Chokkalingam, CEO, Equinomics Research & Advisory.
Among prominent companies, Srikalahasthi Pipes, Sadbhav Engineering, Voltamp Transformers, Raymond, Thyrocare and Action Construction have acquired shares in their own companies through open-market purchases since January 1 as the equity market reported its sharpest quarterly fall.
The BSE Midcap index and Smallcap indices have declined 10 per cent and 11 per cent respectively so far this year.


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About Lisa Barth
Lisa Barth is a wire jewelry artist and metal clay instructor from Atlanta, Georgia. She teaches wire artistry and metal clay and absolutely loves sharing her passion for this art. Her work has been featured in Bead&Button, Australian Beading, BeadStyle, Metal Clay Artist Magazine, Bead&Button's Creative Beading Vol. 4. Her work has also been featured in a polymer clay book by Barbara McGuire called Creative Canes.
In 2007 she took an introductory class in Art Clay Silver at a bead show, and it blew the doors open to the unlimited possibilities to add custom metal work to her designs. She was instantly fascinated and hooked.
Lisa Barth Methods
Lisa begins her designs and draws her inspiration from the stone she is working with because she feels itís the best way to create harmony between the stone and the setting. She plans out her designs completely, making drawings and templates to guide her in the fabrication of her work. She most often bezel sets her stones and feels that bezel setting is what she is best at.
Lisa teaches classes on metal clay and photography in her home studio. Bezel set stones is her specialty and in her classes she shares her expertise in designing organically, achieving harmony between stone and setting.


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The purchase of the Russian S-400 air defence systems was not Turkey's choice but a necessity since Ankara's priority is security, Turkish Minister of National Defense, Hulusi Akar said.
"This was not a choice, but a necessity. This is a system that will not harm anyone if there is no attack or threat. Our priority is the safety of 84 million inhabitants of our country. This is a system that our allies did not provide, but we ourselves provided from Russia", RIA Novosti quotes Akar's words with reference to the Turkish news agency "Anadolu".
"Reasonable and logical solutions" can be found on the S-400 issue, and "Turkey is right in this regard", concluded Akar.
Let us remind you that Russia and Turkey signed a contract for the supply of four divisions of the S-400 air defense system amounting to $2,5 billion in 2017, the deliveries to the Turkish side were carried out in the summer-autumn of 2019.The contract included an option for one more regimental set, and Turkish President Recep Tayyip Erdoğan stated last summer that he had no doubt about purchasing the second S-400 regiment from Russia.

domain: politics


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TuSimple to Haul Union Pacific Goods Between Tucson, Phoenix
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Self-driving truck developer TuSimple says Union Pacific Railroad will be its first customer to move freight on the company’s fully automated trucking route between Tucson and Phoenix, after making a test run on a similar route in December.
The driverless freight runs for Union Pacific will begin this spring from the Port of Tucson intermodal transportation center on the southeast side to a terminal in Phoenix, said San Diego-based TuSimple.
TuSimple, which has been running test runs with human monitors in the trucks since 2017, said it plans to expand its “Driver Out” program to incorporate daytime runs and new routes with a goal of reaching reach commercial viability by the end of 2023.
December’s nighttime test run, which was coordinated with transportation authorities and monitored by law enforcement, was the first time an autonomous heavy truck has operated on open public roads without a human in the vehicle and without human intervention.
Union Pacific Railroad will become the first customer to move freight on TuSimple's fully-automated trucking route in Arizona. Building on our recent "driver out" accomplishment, we will continue to expand new routes and to incorporate daytime runs. https://t.co/DIlzPmvsk4 pic.twitter.com/xtRTJXKV9A — TuSimple (@TuSimpleAI) February 2, 2022
TuSimple said that since then, the company has had an additional six successful fully autonomous runs, covering more than 550 cumulative miles on open public roads in varying traffic situations.
“Our repeatable and scalable ‘Driver Out’ operations marks a significant inflection point in our company’s history,” said TuSimple President and CEO Cheng Lu. “We are proud of our on-time delivery of this historic milestone and are excited to shift our full focus to commercializing our groundbreaking technology on an accelerated timeline.”
A Union Pacific official said TuSimple’s autonomous driving technology will help to scale the technology on its network and reduce supply chain congestion.
“Partnering with TuSimple allows us to extend our operations beyond our rail hubs and serve our customers faster and more efficiently,” said Kenny Rocker, executive vice president of marketing and sales at Union Pacific.
TuSimple also has been using its autonomous trucks to haul paid loads for partners including DHL and UPS as part of its testing program.
TuSimple said it will continue to work with transportation and law enforcement agencies in Arizona and the other states where it operates to gain approval for routine autonomous trucking.
Last July, the company opened a new freight facility in Dallas-Fort Worth to extend the company’s autonomous operations eastward, and allow for autonomous operations in the Texas Triangle, which includes Dallas, Houston, San Antonio and Austin.
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domain: geography