Activated Carbon-Enriched Electrospun-Produced Scaffolds for Drug Delivery/Release in Biological Systems.

To vectorize drug delivery from electrospun-produced scaffolds, we introduce a thin outer drug retention layer produced by electrospinning from activated carbon nanoparticles (ACNs)-enriched polycaprolacton (PCL) suspension. Homogeneous or coaxial fibers filled with ACNs were produced by electrospinning from different PCL-based suspensions. Stable ACN suspensions were selected by sorting through solvents, stabilizers and auxiliary components. The ACN-enriched scaffolds produced were characterized for fiber diameter, porosity, pore size and mechanical properties. The scaffold structure was analyzed by scanning electron microscopy and X-ray photoelectron spectroscopy. It was found that ACNs were mainly coated with a polymer layer for both homogeneous and coaxial fibers. Drug binding and release from the scaffolds were tested using tritium-labeled sirolimus. We showed that the kinetics of sirolimus binding/release by ACN-enriched scaffolds was determined by the fiber composition and differed from that obtained with a free ACN. ACN-enriched scaffolds with coaxial and homogeneous fibers had a biocompatibility close to scaffold-free AC, as was shown by the cultivation of human gingival fibroblasts and umbilical vein cells on scaffolds. The data obtained demonstrated that ACN-enriched scaffolds had good physico-chemical properties and biocompatibility and, thus, could be used as a retaining layer for vectored drug delivery.

A New Look at Causal Factors of Idiopathic Scoliosis: Altered Expression of Genes Controlling Chondroitin Sulfate Sulfation and Corresponding Changes in Protein Synthesis in Vertebral Body Growth Plates.

Background: In a previous report, we demonstrated the presence of cells with a neural/glial phenotype on the concave side of the vertebral body growth plate in Idiopathic Scoliosis (IS) and proposed this phenotype alteration as the main etiological factor of IS. In the present study, we utilized the same specimens of vertebral body growth plates removed during surgery for Grade III-IV IS to analyse gene expression. We suggested that phenotype changes observed on the concave side of the vertebral body growth plate can be associated with altered expression of particular genes, which in turn compromise mechanical properties of the concave side. Methods: We used a Real-Time SYBR Green PCR assay to investigate gene expression in vertebral body growth plates removed during surgery for Grade III-IV IS; cartilage tissues from human fetal spine were used as a surrogate control. Special attention was given to genes responsible for growth regulation, chondrocyte differentiation, matrix synthesis, sulfation and transmembrane transport of sulfates. We performed morphological, histochemical, biochemical, and ultrastructural analysis of vertebral body growth plates. Results: Expression of genes that control chondroitin sulfate sulfation and corresponding protein synthesis was significantly lower in scoliotic specimens compared to controls. Biochemical analysis showed 1) a decrease in diffused proteoglycans in the total pool of proteoglycans; 2) a reduced level of their sulfation; 3) a reduction in the amount of chondroitin sulfate coinciding with raising the amount of keratan sulfate; and 4) reduced levels of sulfation on the concave side of the scoliotic deformity. Conclusion: The results suggested that altered expression of genes that control chondroitin sulfate sulfation and corresponding changes in protein synthesis on the concave side of vertebral body growth plates could be causal agents of the scoliotic deformity.

Human ribosomal protein eS1 is engaged in cellular events related to processing and functioning of U11 snRNA.

Ribosomal proteins are involved in many cellular processes through interactions with various RNAs. Here, applying the photoactivatable-ribonucleoside-enhanced cross-linking and immunoprecipitation approach to HEK293 cells overproducing ribosomal protein (rp) eS1, we determined the products of RNU5A-1 and RNU11 genes encoding U5 and U11 snRNAs as the RNA partners of ribosome-unbound rp eS1. U11 pre-snRNA-associated rp eS1 was revealed in the cytoplasm and nucleus where rp eS1-bound U11/U12 di-snRNP was also found. Utilizing recombinant rp eS1 and 4-thiouridine-containing U11 snRNA transcript, we identified an N-terminal peptide contacting the U-rich sequence in the Sm site-containing RNA region. We also showed that the rp eS1 binding site on U11 snRNA is located in the cleft between stem-loops I and III and that its structure mimics the respective site on the 18S rRNA. It was found that cell depletion of rp eS1 leads to a decrease in the splicing efficiency of minor introns and to an increase in the level of U11 pre-snRNA with the unprocessed 3' terminus. Our findings demonstrate the engagement of human rp eS1 in events related to the U11 snRNA processing and to minor-class splicing. Contacts of rp eS1 with U5 snRNA in the minor pre-catalytic spliceosome are discussed.

The roles of mechanotransduction, vascular wall cells, and blood cells in atheroma induction.

BACKGROUND: This paper describes and analyzes the cellular and molecular mechanisms underlying atherosclerosis development. In particular, the roles of monocytes/macrophages, smooth muscle cells, and vascular endothelium in the formation of stable/unstable atheromatous plaques, and the contributions of some processes to atheroma formation. METHODS AND RESULTS: In this study we analyzed endothelium: function, dysfunction, and involvement into atherogenesis; cell proteins mediating mechanotransduction; proatherogenic role of monocytes; the role of macrophages in the development of unstable atheromatous plaques and smooth muscle cell origin in atherosclerosis. Smooth muscle cell phenotypic switching; their functioning; the ability to retain cholesterol and lipoproteins as well as secretion of pro- and anti-inflammatory molecules and extracellular matrix proteins, their response to extracellular stimuli secreted by other cells, and the effect of smooth muscle cells on the cells surrounding atheromatous plaques are fundamentally important for the insight into atherosclerosis molecular basis. CONCLUSION: Atheromatous plaque transcriptome studies will be helpful in the identification of the key genes involved in atheroma transformation and development as well as discovery of the new targets for diagnosis and therapy.

MicroRNA-guided gene expression in prostate cancer: Literature and database overview.

Insight into the aberrant expression of microRNAs (miRNAs) and the genes that they regulate during the progression of cancer in general and prostate cancer (PCa) in particular is one of the most important issues in current molecular biomedicine and allows for the discovery of therapeutic or diagnostic miRNA targets. The present study aimed to analyze the available data regarding the direct or indirect effects of miRNAs on the expression of the mRNAs involved in carcinogenesis and to enable updating and optimizing the selection of the corresponding targets. The present review focuses on the data related to the genes with miRNA-dependent expression during the development of PCa. The data used in this review have been extracted from research papers and the databases STRING, PANTHER and TargetScan, with a special focus on the genes directly associated with cell transformation and the maintenance of the transformed genotype, as well as tumor invasion and spread. The search for miRNA markers of PCa and therapeutically active molecules should rely on bioinformatics resources, such as data from recent experimental studies, as well as meta-analysis and cross-analysis of the data on the state of the tumor, patient status, histological/immunohistological data and data on mRNA-miRNA coexpression.

A New Look at Etiological Factors of Idiopathic Scoliosis: Neural Crest Cells.

Idiopathic scoliosis is one of the most common disabling pathologies of children and adolescents. Etiology and pathogenesis of idiopathic scoliosis remain unknown. To study the etiology of this disease we identified the cells' phenotypes in the vertebral body growth plates in patients with idiopathic scoliosis. Materials and methods: The cells were isolated from vertebral body growth plates of the convex and concave sides of the deformity harvested intraoperatively in 50 patients with scoliosis. Cells were cultured and identified by methods of common morphology, neuromorphology, electron microscopy, immunohistochemistry and PCR analysis. Results: Cultured cells of convex side of deformation were identified as chondroblasts. Cells isolated from the growth plates of the concave side of the deformation showed numerous features of neuro- and glioblasts. These cells formed synapses, contain neurofilaments, and expressed neural and glial proteins. Conclusion: For the first time we demonstrated the presence of cells with neural/glial phenotype in the concave side of the vertebral body growth plate in scoliotic deformity. We hypothesized that neural and glial cells observed in the growth plates of the vertebral bodies represent derivatives of neural crest cells deposited in somites due to alterations in their migratory pathway during embryogenesis. We also propose that ectopic localization of cells derived from neural crest in the growth plate of the vertebral bodies is the main etiological factor of the scoliotic disease.

Mechanisms Underlying Atheroma Induction: The Roles of Mechanotransduction, Vascular Wall Cells, and Blood Cells.

BACKGROUND: The objective of this article is to review cellular mechanism of atherosclerosis (AS) development. The pathogenesis of AS comprises a sequence of biological events leading to build up of a dense or loose atheromatous plaque (AP). METHODS: In this review, we tried to attempt to analyze the cellular mechanisms underlying AS development, including the roles of monocytes/macrophages and smooth muscle cells in the formation of stable/unstable APs. RESULTS: As a rule, APs are formed in the regions with irregular blood flow; both mechanical perturbations of the vascular wall and several biological events contribute to plaque formation. Blood lipid/lipoprotein deposition, recruitment of monocytes/macrophages, foam cell formation, migration and proliferation of smooth muscle cells, secretion of extracellular matrix, and formation of the connective tissue in plaques are among the latter events. CONCLUSIONS: The review briefs the contributions of different processes to atheroma formation and describes the molecular mechanisms involved in AS development. AP transcriptome studies will be helpful in the identification of the key genes involved in atheroma transformation and development as well as discovery of the new targets for diagnosis and therapy.

Searching for the Novel Specific Predictors of Prostate Cancer in Urine: The Analysis of 84 miRNA Expression.

The aim of this study was to investigate miRNA profiles of clarified urine supernatant and combined urine vesicle fractions of healthy donors and patients with benign prostatic hyperplasia and prostate cancer (PCa). The comparative analysis of miRNA expression was conducted with a custom miRCURY LNA miRNA qPCR panel. Significant combinations of miRNA pairs were selected by the RandomForest-based feature selection algorithm Boruta; the difference of the medians between the groups and a 95% confidence interval was built using the bootstrap approach. The Asymptotic Wilcoxon-Mann-Whitney Test was performed for miRNA combinations to compare different groups of donors. Benjamini-Hochberg correction was used to adjust the statistical significance for multiple comparisons. The most diagnostically significant miRNAs pairs were miR-107-miR-26b.5p and miR-375.3p-miR-26b.5p in the urine supernatant fraction that discriminated the group of healthy patients and PCa patients, as well as miR-31.5p-miR-16.5p, miR-31.5p-miR-200b, miR-31.5p-miR-30e.3p and miR-31.5p-miR-660.5p in the fraction extracellular vesicles that were different between healthy men and benign prostate hyperplasia patients. Such statistical criteria as the occurrence of individual significant miRNA pairs in the total number of comparisons, median ΔCt difference, and confidence interval can be useful tools for determining reliable markers of PCa.

Protocol for miRNA isolation from biofluids.

MicroRNAs (miRNAs) have been identified as promising biomarkers in cancer and other diseases. Packaging of miRNAs into vesicles and complexes with proteins ensures their stability in biological fluids but also complicates their isolation. Conventional protocols used to isolate cell-free RNA are generally successful in overcoming these difficulties; however, they are costly, labor-intensive, or heavily reliant on the use of hazardous chemicals. Here we describe a protocol that is suitable for isolating miRNAs from biofluids, including blood plasma and urine. The protocol is based on precipitation of proteins, denaturation of miRNA-containing complexes with octanoic acid and guanidine isothiocyanate, and subsequent purification of miRNA on spin columns. The efficacy of miRNA extraction by phenol-chloroform extraction, miRCURY RNA isolation kit--biofluids (Exiqon), and the proposed protocol was compared by quantitative reverse-transcription PCR of miR-16 and miR-126. The proposed protocol was slightly more effective for isolating miRNA from plasma and significantly superior to the other two methods for miRNA isolation from urine. Spectrophotometry and SDS-PAGE data suggest that the disparity in performance between miRCURY Biofluids and the proposed protocol can be attributed to differences in precipitation mechanisms, as confirmed by the retention of different proteins in the supernatant.

Biological evaluation of tetracationic compounds based on two 1,4-diazabicyclo[2.2.2]octane moieties connected by different linkers.

A series of 1,4-diazabicyclo[2.2.2]octane derivatives differing by linker moiety was evaluated for activity against several strains of both Gram-positive and Gram-negative bacteria including drug-resistant strains, one strain of fungus and influenza virus A/Puerto Rico/8/34 (H1N1). All compounds exhibited high antibacterial activity against all bacteria except Proteus vulgaris. The minimum inhibitory concentrations (MICs) of compound 1c with an o-phenylenebismethyl linker and compound 1e with a propylene aliphatic linker were found to be low and were comparable or better to the reference drug ciprofloxacin for Pseudomonas aeruginosa and Staphylococcus aureus. Additionally, a time-kill assay was performed to examine the bactericidal kinetics. Compounds 1c and 1e displayed rapid killing effects against St. aureus and Ps. aeruginosa after 2h. Furthermore, compounds 1a-c with aromatic linkers and compound 1e showed the highest antiviral activity.

Dynamic changes in circulating miRNA levels in response to antitumor therapy of lung cancer.

PURPOSE: Expression levels of cancer-associated microRNAs were reported to be altered in serum/plasma samples from lung cancer patients compared with healthy subjects. The purpose of this study was to estimate the value of five selected miRNAs plasma levels as markers of response to antitumor therapy in lung cancer patients. MATERIALS AND METHODS: Expression levels of miR-19b, miR-126, miR-25, miR-205, and miR-125b have been evaluated by quantitative reverse transcription PCR versus control miR-16 in blood plasma samples from 23 lung cancer (LC) patients. Plasma samples were obtained from LC patients before treatment (untreated-UT), within 30 days after completing two courses of chemotherapy (postchemotherapy-PC) and 15 days after surgery (postoperative-PO). RESULTS: Repeated Measures ANOVA demonstrated that miR-19b expression levels were decreased in PC and increased in PO samples. These changes were characterized by a significant quadratic trend (p = 0.03). Expression levels of miR-125b increased both after chemotherapy and again after surgery and demonstrated a significant linear trend (p = 0.03). The miR-125b/miR-19b ratio changed during the course of the antitumor treatment with a significant linear trend (p = 0.04). Individual analysis in the groups of patients with partial response to chemotherapy and patients with stable or progressive disease showed different trends for miR-19b, miR-125b, and miR-125b/miR-19b ratio between the groups. The Kaplan-Meier survival curves demonstrated an association of miR-125b/miR-19b ratio value with the survival time without the tumor relapse (p < 0.1). CONCLUSIONS: Dynamic change of trends for miR-19b and miR-125b expression levels and miR-125b/miR-19b ratio in the blood plasma have shown a potentiality to discriminate types of response to antitumor therapy in lung cancer patients. Further in-depth investigation is needed to establish a direct link the miRNAs expression levels in blood plasma with therapy response and patient's survival.

Oligodeoxynucleotide Analogues of Circulating DNA Inhibit dsRNA-Induced Immune Response at the Early Stages of Signal Transduction Cascade in a Cell Type-Dependent Manner.

Oligodeoxynucleotide (ODN) analogues of cell-surface-bound circulating DNA inhibit the dsRNA-induced production of pro-inflammatory interleukin 6, interferon beta and antibacterial peptide beta-defensin 2 not only in human gingival fibroblasts, but also in human primary endothelial and transformed cells (Hela and A431). ODN analogues do not effect dendritic cells activation by poly(I:C). The data obtained indicate that the early stages of the signal transduction cascade are violated by ODN analogues and the effects depend on the cell type.

Features of Circulating DNA Fragmentation in Blood of Healthy Females and Breast Cancer Patients.

Size and termini of cell-free DNA molecules circulating in blood plasma and being bound with blood cell surface of healthy females and untreated breast cancer patients were investigated. The size and concentration of circulating blood DNA were analyzed by Agilent 2100 Bioanalyser TM and TaqMan PCR. The termini of circulating DNA were examined by ligation using biotinylated double-stranded oligonucleotide adapters with random 1-3 b overhangs of both chains and subsequent quantification by PCR. Short (180 bp) and longer (>8 kbp) DNA fragments were found in cell free DNA from both groups, but short were less represented in primary breast cancer patient plasma. Predominantly high molecular weight DNA was found in cell surface bound DNA both in healthy females and breast cancer patients with a minor fraction of short fragments. Heterogeneous DNA molecules with diverse 5'- and 3'- protruding as well as blunt ends were found both in plasma DNA and cell bound DNA from healthy individuals. Cell surface bound DNA from breast cancer patients mainly contains 5'-protruding ends, whereas 5'- and 3'-protruding ends are equally presented in cell free DNA from these patients. The data obtained obviously reflect over-representation of specific nucleases in breast cancer.

Cell-Free miRNA-141 and miRNA-205 as Prostate Cancer Biomarkers.

Expression levels of five miRNAs (miR-19b, miR-21, miR-126, miR-141, miR-205) were measured in the plasma of healthy donors and prostate cancer patients. It was shown that miR-141 expression level efficiently discriminates early stage prostate cancer patients and correlates with the Gleason score.

Protein Content of Circulating Nucleoprotein Complexes.

In the current study we have investigated the protein content of blood plasma deoxyribonucleoprotein complexes. The complexes were isolated using affinity chromatography with immobilized polyclonal anti-histone antibodies. Proteins were separated by SDS PAAGE and identified by MALDI-TOF mass-spectrometry. 111 and 56 proteins (excluding histones), respectively, were identified with a good score in deoxyribonucleoprotein complexes of healthy females and breast cancer patients. However, only four of these proteins were found in 30 % of all samples. Fourteen proteins previously described as tumor specific proteins were found in cancer patients whereas not one of them was found in healthy individuals. The data obtained demonstrate the involvement of different cellular and extracellular proteins in circulating cell-free DNA.

A phenol-free method for isolation of microRNA from biological fluids.

MicroRNAs (miRNAs) found in biological fluids such as blood and urine have been identified as promising biomarkers for many human disorders, including cancer, cardiopathies, and neurodegenerative diseases. However, circulating miRNAs are either encapsulated into vesicles or found in complexes with proteins and lipoproteins and, thus, require a special approach to their isolation. Acid phenol-chloroform extraction can solve this problem, but it is a labor-intensive procedure that relies heavily on the use of hazardous chemicals. Here we describe a fast and simple phenol-free protocol for miRNA isolation from biofluids. MiRNA is extracted from complexes with biopolymers by a high concentration of guanidine isothiocyanate combined with water/organic composition of solvents. Purification is finished using silica-based spin columns. Comparison of miRNA isolation from blood plasma and urine using the single-phase method and acid phenol-chloroform extraction by means of radioisotope spike-ins and quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) showed similar performance of the two methods.

Effect of Sterilization Methods on Electrospun Scaffolds Produced from Blend of Polyurethane with Gelatin.

Fibrous polyurethane-based scaffolds have proven to be promising materials for the tissue engineering of implanted medical devices. Sterilization of such materials and medical devices is an absolutely essential step toward their medical application. In the presented work, we studied the effects of two sterilization methods (ethylene oxide treatment and electron beam irradiation) on the fibrous scaffolds produced from a polyurethane-gelatin blend. Scaffold structure and properties were studied by scanning electron microscopy (SEM), atomic force microscopy (AFM), infrared spectroscopy (FTIR), a stress-loading test, and a cell viability test with human fibroblasts. Treatment of fibrous polyurethane-based materials with ethylene oxide caused significant changes in their structure (formation of glued-like structures, increase in fiber diameter, and decrease in pore size) and mechanical properties (20% growth of the tensile strength, 30% decline of the maximal elongation). All sterilization procedures did not induce any cytotoxic effects or impede the biocompatibility of scaffolds. The obtained data determined electron beam irradiation to be a recommended sterilization method for electrospun medical devices made from polyurethane-gelatin blends.

Triterpenoid saponins from the roots of Acanthophyllum gypsophiloides Regel.

Two new triterpenoid saponins 1 and 2 were isolated from the methanol extract of the roots of Acanthophyllum gypsophiloides Regel. These saponins have quillaic acid or gypsogenin moieties as an aglycon, and both bear similar sets of two oligosaccharide chains, which are 3-O-linked to the triterpenoid part trisaccharide α-L-Arap-(1→3)-[α-D-Galp-(1→2)]-ß-D-GlcpA and pentasaccharide ß-D-Xylp-(1→3)-ß-D-Xylp-(1→3)-α-L-Rhap-(1→2)-[ß-D-Quip-(1→4)]-ß-D-Fucp connected through an ester linkage to C-28. The structures of the obtained saponins were elucidated by a combination of mass spectrometry and 2D NMR spectroscopy. A study of acute toxicity, hemolytic, anti-inflammatory, immunoadjuvant and antifungal activity was carried out. Both saponins 1 and 2 were shown to exhibit immunoadjuvant properties within the vaccine composition with keyhole limpet hemocyanin-based immunogen. The availability of saponins 1 and 2 as individual pure compounds from the extract of the roots of A. gypsophiloides makes it a prospective source of immunoactive agents.

Structural Aspects of Electrospun Scaffolds Intended for Prosthetics of Blood Vessels.

Electrospinning is a popular method used to fabricate small-diameter vascular grafts. However, the importance of structural characteristics of the scaffold determining interaction with endothelial cells and their precursors and blood cells is still not exhaustively clear. This review discusses current research on the significance and impact of scaffold architecture (fiber characteristics, porosity, and surface roughness of material) on interactions between cells and blood with the material. In addition, data about the effects of scaffold topography on cellular behaviour (adhesion, proliferation, and migration) are necessary to improve the rational design of electrospun vascular grafts with a long-term perspective.

Correction: Gostev et al. In Vivo Stability of Polyurethane-Based Electrospun Vascular Grafts in Terms of Chemistry and Mechanics. Polymers 2020, 12, 845.

The authors wish to make a change to the published paper [...].