Negatively charged self-assembling DNA/poloxamine nanospheres for in vivo gene transfer

Over the past decade, numerous nonviral cationic vectors have been synthesized. They share a high density of positive charges and efficiency for gene transfer in vitro. However, their positively charged surface causes instability in body fluids and cytotoxicity, thereby limiting their efficacy in vivo. Therefore, there is a need for developing alternative molecular structures. We have examined tetrabranched amphiphilic block copolymers consisting of four polyethyleneoxide/polypropyleneoxide blocks centered on an ethylenediamine moiety. Cryo-electron microscopy, ethidium bromide fluorescence and light and X-ray scattering experiments performed on vector–DNA complexes showed that the dense core of the nanosphere consisted of condensed DNA interacting with poloxamine molecules through electrostatic, hydrogen bonding and hydrophobic interactions, with DNA molecules also being exposed at the surface. The supramolecular organization of block copolymer/DNA nanospheres induced the formation of negatively charged particles. These particles were stable in a solution that had a physiological ionic composition and were resistant to decomplexation by heparin. The new nanostructured material, the structure of which clearly contrasted with that of lipoplexes and polyplexes, efficiently transferred reporter and therapeutic genes in skeletal and heart muscle in vivo. Negatively charged supramolecular assemblies hold promise as therapeutic gene carriers for skeletal and heart muscle-related diseases and expression of therapeutic proteins for local or systemic uses.     

In Silico Analysis of Acinetobacter baumannii Phospholipase D as a Subunit Vaccine Candidate

The rate of human health care-associated infections caused by Acinetobacter baumannii has increased significantly in recent years for its remarkable resistance to desiccation and most antibiotics. Phospholipases, capable of destroying a phospholipid substrate, are heterologous group of enzymes which are believed to be the bacterial virulence determinants. There is a need for in silico studies to identify potential vaccine candidates. A. baumannii phospholipase D (PLD) role has been proved in increasing organism’s resistance to human serum, destruction of host epithelial cell and pathogenesis in murine model. In this in silico study high potentials of A. baumannii PLD in elicitation of humoral and cellular immunities were elucidated. Thermal stability, long half-life, non-similarity to human and gut flora proteome and non-allergenicity were in a list of A. baumannii PLD positive properties. Potential epitopic sequences were also identified that could be used as peptide vaccines against A. baumannii and various other human bacterial pathogens.     

Differentiation and Classification of Phytoplasmas Associated with Almond and GF‐677 Witches'‐Broom Diseases using rRNA and rp Genes

Stone fruits are affected by several diseases associated with plant pathogenic phytoplasmas. Previous studies have been shown that phytoplasma agents of almond and GF‐677 witches'‐broom (AlmWB and GWB, respectively) diseases belong to pigeon pea witches'‐broom (16SrIX) phytoplasma group. In this study, partial biological and molecular characterization was used to compare and classify phytoplasma agents of Khafr AlmWB (KAlmWB) and Estahban GWB (EGWB) diseases. Production of different symptoms in periwinkle indicated that agents of KAlmWB and EGWB are differentiable. Expected fragments were amplified from diseased almond and GF‐677 trees in direct PCR using phytoplasma universal primer pairs P1/P7 and rpF1/rpR1 and nested PCR using P1/P7 followed by R16F2n/ R16R2 primer pair. 16S‐rDNA Restriction fragment length polymorphism (RFLP) as well as phylogenetic analysis of rplV‐rpsC and 16S–23S rRNA spacer region sequences classified KAlmWB and EGWB phytoplasmas within 16SrIX‐C (rpIX‐C) and 16SrIX‐B (rpIX‐B) subgroups, respectively.     

Citric acid improves growth performance and phosphorus digestibility in Beluga (Huso huso) fed diets where soybean meal partly replaced fish meal

A 2 × 3 factorial experiment was conducted to evaluate the effect of partial substitution of dietary fish meal with soybean meal and citric acid (CA) supplementation on growth, food utilization, muscle composition and nutrient digestibility of Beluga, Huso huso. Three isonitrogenic and isoenergetic diets, as SBM₁ (soybean meal protein (SBP):fishmeal protein (FP) = 1:3), SBM₂ (SBP:FP = 2:3) and SBM₃ (SBP:FP = 1:1) containing two levels of CA (0 and 30 g kg⁻¹) were fed to triplicate groups of fish for 8 weeks. The results revealed that adding CA increased (P<0.05) the weight gain (WG), specific growth rate (SGR) and protein efficiency ratio (PER) and decreased (P<0.05) food conversion rate (FCR) whereas partial substitution of fishmeal with soybean meal decreased (P<0.05) growth performance. Partial replacement of fishmeal with SBM (P=0.021) as well as CA supplementation (P=0.019) and their interaction (P<0.001) affected hepatosomatic index (HSI). No differences (P>0.05) were observed for viscerosomatic index (VSI) among treatments (P>0.05). No differences (P>0.05) were detected in moisture, protein of muscle sample among treatments, but lipid content was reduced (P<0.05) while ash content increased (P=0.035) by CA. Soybean meal decreased (P<0.05) nutrient digestibility, whereas CA improved apparent protein and phosphorus (P) digestibility (P=0.011 and P<0.001 respectively). No interaction (P<0.05) between levels of SBM and CA was found on these parameters. Results of the present study indicate that Beluga has a limited ability to utilize SBM as a protein source in practical diets whereas CA can improve growth and nutrient utilization in Beluga.     

A Recombinant Probiotic, <Emphasis Type="Italic">Lactobacillus casei,</Emphasis> Expressing the <Emphasis Type="Italic">Clostridium perfringens</Emphasis> α-toxoid,as an Orally Vaccine Candidate Against Gas Gangrene and Necrotic Enteritis

The alpha-toxin is one of the virulence factors of Clostridium perfringens for gas gangrene in humans and animals or necrotic enteritis in poultry. The C-terminal domain of this toxin ( cpa _247-370 ) was synthesized and cloned into pT1NX vector to construct the pT1NX-alpha plasmid. This surface-expressing plasmid was electroporated into Lactobacillus casei ATCC 393, generating the recombinant L. casei strain expressing alpha-toxoid (LC-α strain). Expression of this modified alpha-toxoid was confirmed by SDS-PAGE, immunoblotting, and direct immunofluorescence microscopy. BALB/c mice, immunized orally by the recombinant LC-α strain, elicited mucosal and significantly humoral immune responses (p < 0.05) and developed a protection against 900 MLD/mL of the standard alpha-toxin. This study showed that this recombinant LC-α strain could be a promising vaccine candidate against gas gangrene and necrotic enteritis.     

In Solution Cation-Induced Secondary and Tertiary Structure Alterations of Human Calprotectin

Calprotectin (CP) is widely considered to have diverse roles including growth inhibitory and apoptosis induction in a number of tumor cell lines and antimicrobial activities. As CP has been proposed to bind metal ions with high affinity, we have studied its functional and primarily its structural behavior upon Zn²⁺ and Mn²⁺ chelation solely and along with Ca²⁺. We employed fluorescence spectroscopy and circular dichroism to determine the resulting modifications. Based upon our findings it is clear that treating CP with ions effectively weakened its natural growth inhibitory activity. Moreover, structural analysis of Zn²⁺ and Mn²⁺-treated CPs indicated remarkable alterations in the regular secondary structures in favor of irregular structures while Zn²⁺ and Mn²⁺ treatment of CP after incubation with Ca²⁺ displayed no remarkable shifts. Tertiary structure investigation using fluorescence spectroscopy showed that CP undergoes conformational changes upon Zn²⁺ and Mn²⁺ treatment whereby Trp residues of protein is slightly exposed to the hydrophilic environment, compactness of CP is compromised, whereas in Ca²⁺-treated CP, the tertiary structure integrity is intact upon Zn²⁺ and Mn²⁺ chelation. Interestingly, CP structural modifications upon Zn²⁺ and Mn²⁺ treatment was significantly comparable, probably due to similar radii and charges of ions. Taken all together, we have concluded that CP maintains its normal nature in Ca²⁺-loaded state when treated with Zn²⁺ and Mn²⁺ ions. It can be suggested that Ca²⁺ not only stabilize CP structure but also helps CP to keep its structure upon metal ions chelation which is involved in host organism defense system.     

Prolongation of insulin-induced activation of mitogen-activated protein kinases ERK 1/2 and phosphatidylinositol 3-kinase by vanadyl sulfate, a protein tyrosine phosphatase inhibitor

Vanadium salts such as vanadyl sulfate (VS), potent inhibitors of protein tyrosine phosphatases, have been shown to mimic, augment, and prolong insulin's action. However, the molecular mechanism of responses to these salts is not clear. In the present studies, we examined if VS-induced effects on insulin action are associated with enhancement or augmentation in the activation state of key components of the insulin signaling pathway. Treatment of insulin receptor-overexpressing cells with insulin or VS resulted in a time-dependent transient increase in phosphorylation and activation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2) that peaked at about 5 min, then declined rapidly to about baseline within 30 min. However, when the cells were treated with VS before stimulation with insulin, sustained ERK 1/2 phosphorylation and activation were observed well beyond 60 min. VS treatment also prolonged the insulin-stimulated activation of phosphatidylinositol 3-kinase (PI3-K), which was associated with sustained interaction between insulin receptor substrate-1 (IRS-1) and the p(85 alpha) subunit of phosphatidylinositol 3-kinase (PI3-K) in response to insulin. These data indicate that prolongation of insulin-stimulated ERK 1/2 and PI3-K activation by VS is due to a more stable complex formation of IRS-1 with the p(85 alpha) subunit which may, in turn, be responsible for its ability to enhance and extend the biological effects of insulin.     

Ergonomical valorization of working spaces in multipurpose ships

In this work it is shown how anthropological data are among the most needed factors in ergonomical valorization of crew working spaces. Ship's working or living environment involves many unique human factors, which should be specially considered in our case as limitation of crew space. In this work we have chosen ships of different years of construction to prove this tendency. As a micro study, the work posture analysis using the pulling force experiment is performed in order to determine lumbar moment, intra-abdominal pressure as a measure of evaluating and comparing different crew work positions. As a macro-study, the "crew work posture analysis" was carried out by the use of the data collected from real cases. The most probable work postures in different spaces of a ship are classified and after some corrections of the work place the profile and its grade were determined. The "statistical analysis for real ship's spaces" is also performed, as well as another macro study, in order to show some real designed ship spaces from the point of view of the allocated volume.     

The RNA binding protein Musashi1 regulates apoptosis,gene expression and stress granule formation in urothelial carcinoma cells

The RNA‐binding protein Musashi1 (MSI1) is a marker of progenitor cells in the nervous system functioning as a translational repressor. We detected MSI1 mRNA in several bladder carcinoma cell lines, but not in cultured normal uroepithelial cells, whereas the paralogous MSI2 gene was broadly expressed. Knockdown of MSI1 expression by siRNA induced apoptosis and a severe decline in cell numbers in 5637 bladder carcinoma cells. Microarray analysis of gene expression changes after MSI1 knockdown significantly up‐regulated 735 genes, but down‐regulated only 31. Up‐regulated mRNAs contained a highly significantly greater number and density of Musashi binding sites. Therefore, a much larger set of mRNAs may be regulated by Musashi1, which may affect not only their translation, but also their turnover. The study confirmed p21^CIP1 and Numb proteins as targets of Musashi1, suggesting additionally p27^KIP1 in cell‐cycle regulation and Jagged‐1 in Notch signalling. A significant number of up‐regulated genes encoded components of stress granules (SGs), an organelle involved in translational regulation and mRNA turnover, and impacting on apoptosis. Accordingly, heat shock induced SG formation was augmented by Musashi1 down‐regulation. Our data show that ectopic MSI1 expression may contribute to tumorigenesis in selected bladder cancers through multiple mechanisms and reveal a previously unrecognized function of Musashi1 in the regulation of SG formation.     

Using paracrine effects of Ad-MSCs on keratinocyte cultivation and fabrication of epidermal sheets for improving clinical applications

Recent advances in wound healing have made cell therapy a potential approach for the treatment of various types of skin defects such as trauma, burns, scars and diabetic leg ulcers. Cultured keratinocytes have been applied to burn patients since 1981. Patients with acute and chronic wounds can be treated with autologous/allograft cultured keratinocytes. There are various methods for cultivation of epidermal keratinocytes used in cell therapy. One of the important properties of an efficient cell therapy is the preservation of epidermal stem cells. Mesenchymal Stem Cells (MSCs) are major regulatory cells involved in the acceleration of wound healing via induction of cell proliferation, angiogenesis and stimulating the release of paracrine signaling molecules. Considering the beneficial effects of MSCs on wound healing, the main aim of the present study is investigating paracrine effects of Adipose-derived Mesenchymal Stem Cell (Ad-MSCs) on cultivation of keratinocytes with focusing on preservation of stem cells and their differentiation process. We further introduced a new approach for culturing isolated keratinocytes in vitro in order to generate epidermal keratinocyte sheets without using a feeder layer. To do so, Ad-MSC conditioned medium was applied as an alternative to commercial media for keratinocyte cultivation. In this study, the expression of several stem/progenitor cell (P63, K19 and K14) and differentition (K10, IVL and FLG) markers was examined using real time PCR on days 7, 14 and 21 of culture in keratinocytes in Ad-MSC conditioned medium. P63 and α6 integrin expression was also evaluated via flow cytometry. The results were compared with control group including keratinocytes cultured in EpiLife medium and our data indicated that this Ad-MSC conditioned medium is a good alternative for keratinocyte cultivation and producing epidermal sheets for therapeutic and clinical purposes. The reasons are the expression of stem cell and differentiation markers and overcoming the requirement for feeder layer which leads to a xenograft-free transplantation. Besides, this approach has low cost and is easier to perform. However, more in vitro and in vivo experiments as well as safety evaluation required before clinical applications.