Evaluation of a Novel Plasmid for Simultaneous Gene Electrotransfer-Mediated Silencing of CD105 and CD146 in Combination with Irradiation

Targeting tumor vasculature through specific endothelial cell markers represents a promising approach for cancer treatment. Here our aim was to construct an antibiotic resistance gene-free plasmid encoding shRNAs to simultaneously target two endothelial cell markers, CD105 and CD146, and to test its functionality and therapeutic potential in vitro when delivered by gene electrotransfer (GET) and combined with irradiation (IR). Functionality of the plasmid was evaluated by determining the silencing of the targeted genes using qRT-PCR. Antiproliferative and antiangiogenic effects were determined by the cytotoxicity assay tube formation assay and wound healing assay in murine endothelial cells 2H-11. The functionality of the plasmid construct was also evaluated in malignant melanoma tumor cell line B16F10. Additionally, potential activation of immune response was measured by induction of DNA sensor STING and proinflammatory cytokines by qRT-PCR in endothelial cells 2H-11. We demonstrated that the plasmid construction was successful and can efficiently silence the expression of the two targeted genes. As a consequence of silencing, reduced migration rate and angiogenic potential was confirmed in 2H-11 endothelial cells. Furthermore, induction of DNA sensor STING and proinflammatory cytokines were determined, which could add to the therapeutic effectiveness when used in vivo. To conclude, we successfully constructed a novel plasmid DNA with two shRNAs, which holds a great promise for further in vivo testing.     

Alterations in the Genomic Distribution of 5hmC in In Vivo Aged Human Skin Fibroblasts

5-Hydroxymethylcytosine (5hmC) is a functionally active epigenetic modification. We analyzed whether changes in DNA 5-hydroxymethylation are an element of age-related epigenetic drift. We tested primary fibroblast cultures originating from individuals aged 22–35 years and 74–94 years. Global quantities of methylation-related DNA modifications were estimated by the dot blot and colorimetric methods. Regions of the genome differentially hydroxymethylated with age (DHMRs) were identified by hMeDIP-seq and the MEDIPS and DiffBind algorithms. Global levels of DNA modifications were not associated with age. We identified numerous DHMRs that were enriched within introns and intergenic regions and most commonly associated with the H3K4me1 histone mark, promoter-flanking regions, and CCCTC-binding factor (CTCF) binding sites. However, only seven DHMRs were identified by both algorithms and all of their settings. Among them, hypo-hydroxymethylated DHMR in the intron of Rab Escort Protein 1 (CHM) coexisted with increased expression in old cells, while increased 5-hydroxymethylation in the bodies of Arginine and Serine Rich Protein 1 (RSRP1) and Mitochondrial Poly(A) Polymerase (MTPAP) did not change their expression. These age-related differences were not associated with changes in the expression of any of the ten-eleven translocation (TET) enzymes or their activity. In conclusion, the distribution of 5hmC in DNA of in vivo aged human fibroblasts underwent age-associated modifications. The identified DHMRs are, likely, marker changes.     

Beneficial Changes in Rat Vascular Endocannabinoid System in Primary Hypertension and under Treatment with Chronic Inhibition of Fatty Acid Amide Hydrolase by URB597

Our study aimed to examine the effects of hypertension and the chronic administration of the fatty acid amide hydrolase (FAAH) inhibitor URB597 on vascular function and the endocannabinoid system in spontaneously hypertensive rats (SHR). Functional studies were performed on small mesenteric G3 arteries (sMA) and aortas isolated from SHR and normotensive Wistar Kyoto rats (WKY) treated with URB597 (1 mg/kg; twice daily for 14 days). In the aortas and sMA of SHR, endocannabinoid levels and cannabinoid CB₁ receptor (CB₁R) expression were elevated. The CB₁R antagonist AM251 diminished the methanandamide-evoked relaxation only in the sMA of SHR and enhanced the vasoconstriction induced by phenylephrine and the thromboxane analog U46619 in sMA in SHR and WKY. In the sMA of SHR, URB597 elevated anandamide levels, improved the endothelium-dependent vasorelaxation to acetylcholine, and in the presence of AM251 reduced the vasoconstriction to phenylephrine and enhanced the vasodilatation to methanandamide, and tended to reduce hypertrophy. In the aortas, URB597 elevated endocannabinoid levels improved the endothelium-dependent vasorelaxation to acetylcholine and decreased CB₁R expression. Our study showed that hypertension and chronic administration of URB597 caused local, resistance artery-specific beneficial alterations in the vascular endocannabinoid system, which may bring further advantages for therapeutic application of pharmacological inhibition of FAAH.     

Microscopic Methods for Identification of Sulfate-Reducing Bacteria from Various Habitats

This paper is devoted to microscopic methods for the identification of sulfate-reducing bacteria (SRB). In this context, it describes various habitats, morphology and techniques used for the detection and identification of this very heterogeneous group of anaerobic microorganisms. SRB are present in almost every habitat on Earth, including freshwater and marine water, soils, sediments or animals. In the oil, water and gas industries, they can cause considerable economic losses due to their hydrogen sulfide production; in periodontal lesions and the colon of humans, they can cause health complications. Although the role of these bacteria in inflammatory bowel diseases is not entirely known yet, their presence is increased in patients and produced hydrogen sulfide has a cytotoxic effect. For these reasons, methods for the detection of these microorganisms were described. Apart from selected molecular techniques, including metagenomics, fluorescence microscopy was one of the applied methods. Especially fluorescence in situ hybridization (FISH) in various modifications was described. This method enables visual identification of SRB, determining their abundance and spatial distribution in environmental biofilms and gut samples.     

Dipeptides of S-Substituted Dehydrocysteine as Artzyme Building Blocks: Synthesis,Complexing Abilities and Antiproliferative Properties

Background: Dehydropeptides are analogs of peptides containing at least one conjugate double bond between α,β-carbon atoms. Its presence provides unique structural properties and reaction centre for chemical modification. In this study, the series of new class of dipeptides containing S-substituted dehydrocysteine with variety of heterocyclic moieties was prepared. The compounds were designed as the building blocks for the construction of artificial metalloenzymes (artzymes). Therefore, the complexing properties of representative compounds were also evaluated. Furthermore, the acknowledged biological activity of natural dehydropeptides was the reason to extend the study for antiproliferative action of against several cancer cell lines. Methods: The synthetic strategy involves glycyl and phenylalanyl-(Z)-β-bromodehydroalanine as a substrate in one pot addition/elimination reaction of thiols. After deprotection of N-terminal amino group the compounds with triazole ring were tested as complexones for copper(II) ions using potentiometric titration and spectroscopic techniques (UV-Vis, CD, EPR). Finally, the antiproliferative activity was evaluated by sulforhodamine B assay. Results and Conclusions: A simple and efficient procedure for preparation of dipeptides containing S-substituded dehydrocysteine was provided. The peptides containing triazole appeared to be strong complexones of copper(II) ions. Some of the peptides exhibited promising antiproliferative activities against number of cancer cell lines, including cell lines resistant to widely used anticancer agent.     

Vanadium promoted Ni(Mg,Al)O hydrotalcite-derived catalysts for CO2 methanation

A series of V-promoted hydrotalcite-derived nickel catalysts (1.0, 2.0, and 4.0 wt%) were tested in CO₂ methanation. Ni–I–V2.0 with 2.0 wt% vanadium loading showed the highest catalytic activity, achieving 74.7% of CO₂ conversion and 100% of CH₄ selectivity at 300 °C. XRD and XANES analyses showed that the smallest Ni⁰ particles in Ni–I–V2.0 were consistent with the improved textural features observed for this catalyst. Moreover, CO₂-TPD revealed the highest sum of weak and medium basic sites in Ni–I–V2.0 that can positively influence catalytic behavior. For the studied catalysts, a clear correlation was demonstrated between the catalytic activity and specific surface area, as well as the basic properties.     

Synthesis and characterization of Co5Cr2(P2O7)4 - New pyrophosphate as a colouring substance

A new pyrophosphate(V) of the formula Co₅Cr₂(P₂O₇)₄ was obtained in the system Co₂P₂O₇–Cr₄(P₂O₇)₃ as a result of solid-state reactions taking place between different reactants. The new compound crystallizes in the orthorhombic system and belongs to the family of pyrophosphates of the general formula M₅²⁺M₂³⁺(P₂O₇)₄ and is probably isostructural with Fe₅²⁺Fe₂³⁺(P₂O₇)₄. Powder diffraction pattern, infrared spectrum and SEM image of the new compound were presented. As a new potential inorganic pigment, Co₅Cr₂(P₂O₇)₄ was tested for its thermal stability, particle size distribution and colour properties, which were studied both for powder and after introduction into organic matrix and leadless ceramic glaze. The colour of Co₅Cr₂(P₂O₇)₄ powder was defined as deep grey with the colour coordinates L*/a*/b* = 60.63/-1.42/-3.41 and according to the hue angle (h° = 247.39°) it belongs to the blue region. Co₅Cr₂(P₂O₇)₄, with its relatively high thermal stability (t _m = 1230 ± 10 °C) and appropriate colour properties, is a good candidate to be used as inorganic pigment for colouring of acrylic paints. In the case of leadless glaze, the obtained compound acts as a dye.     

The influence of volume fraction of amorphous phase on corrosion resistance of Mg67Zn 29Ca 4 alloy

The aim of this study was to investigate the structure and corrosion resistance of amorphous, amorphous‐crystalline, and crystalline Mg₆₇Zn₂₉Ca₄ alloy for biodegradable applications. This paper presents a preparation method and results of the structural characterization and corrosion resistance analysis of the material. Samples were prepared in the form of 3 mm diameter rods. The structure of the alloy was examined with the use of X‐ray diffractometry and scanning electron microscopy. The thermal properties of the samples were examined with differential scanning calorimetry (DSC). Results of DSC analysis were used to determine heat treatment temperatures, allowing to obtain different fractures of crystalline phase in the material. Corrosion resistance of heat‐treated samples was investigated by immersion tests and electrochemical measurements performed in the simulated body fluid. The X‐ray diffraction results confirmed that the prepared Mg₆₇Zn₂₉Ca₄ alloy's structure is fully amorphous. After heat treatment, samples with different fractions of amorphous phase in the structure were obtained. Immersion tests of the samples showed that the structure significantly influenced corrosion resistance in examined materials. It should be pointed out, that certain amounts of crystalline phase in amorphous matrix can greatly improve the corrosion resistance of Mg₆₇Zn₂₉Ca₄ alloy.     

Linear correlation between pH value of stimulated beef and electrical current intensity

The aim of the present study was to determine mathematical relationships between pH changes in beef 24 h post-slaughter and changes in the intensity of electrical current flowing through bull and heifer carcasses during high-voltage electrical stimulation. The electrical stimulation was applied 40 min postmortem for 120 s. The pH values of m. longissimus thoracis et lumborum were analyzed in the function of electrical current intensity changes and its change during electrical stimulation. Mathematical linear correlations of the y = ax ± b type were demonstrated between pH values at 2, 6, and 24 h postmortem and the initial (I_i) and ultimate (I_u) electrical current intensity values, the difference between them and the initial pH values determined before electrical stimulation. High multiple correlation coefficients (R² = 0.416, α ≤ 0.001) between I_u and pH values 24 h post-slaughter enabled concluding that there is a possibility to predict a pH value of stimulated carcass with high accuracy, and thus also beef quality, based merely on the ultimate electrical current intensity values.