Transfection is a powerful analytical tool enabling studies of gene products and functions in eukaryotic cells. Successful delivery of genetic material into cells depends on DNA quantity and quality, incubation time and ratio of transfection reagent to DNA, the origin, type and the passage of transfected cells, and the presence or absence of serum in the cell culture. So far a number of transfection methods that use viruses, non-viral particles or physical factors as the nucleic acids carriers have been developed. Among non-viral carriers, the cationic polymers are proposed as the most attractive ones due to the possibility of their chemical structure modification, low toxicity and immunogenicity. In this review the delivery systems as well as physical, biological and chemical methods used for eukaryotic cells transfection are described and discussed.

Osteogenesis Imperfecta (OI) is a group of connective tissue disorders with a broad range of phenotypes characterized primarily by bone fragility. The prevalence of OI ranges from about 1:15,000 to 1:20,000 births. Five types of the disease are commonly distinguished, ranging from a mild (type I) to a lethal one (type II). Types III and IV are severe forms allowing survival after the neonatal period, while type V is characterized by a mild to moderate phenotype with calcification of interosseous membranes. In most cases, there is a reduction in the production of normal type I collagen (col I) or the synthesis of abnormal collagen as a result of mutations in col I genes. Moreover, mutations in genes involved in col I synthesis and processing as well as in osteoblast differentiation have been reported. The currently available treatments try to prevent fractures, control symptoms and increase bone mass. Commonly used medications in OI treatment are bisphosphonates, Denosumab, synthetic parathyroid hormone and growth hormone for children therapy. The main disadvantages of these therapies are their relatively weak effectiveness, lack of effects in some patients or cytotoxic side effects. Experimental approaches, particularly those based on stem cell transplantation and genetic engineering, seem to be promising to improve the therapeutic effects of OI.

Fig. S1. Cryogenic transmission electron microscopy images obtained from PBS solutions of a) pDNA, b) STAR1.

Abdominal aortic aneurysm (AAA) is one of the most dangerous cardiovascular diseases, occurring mainly in men over the age of 55 years. As it is asymptomatic, patients are diagnosed very late, usually when they suffer pain in the abdominal cavity. The late detection of AAA contributes to the high mortality rate. Many environmental, genetic, and molecular factors contribute to the development and subsequent rupture of AAA. Inflammation, apoptosis of smooth muscle cells, and degradation of the extracellular matrix in the AAA wall are believed to be the major molecular processes underlying AAA formation. Until now, no pharmacological treatment has been implemented to prevent the formation of AAA or to cure the disease. Therefore, it is important that patients are diagnosed at a very early stage of the disease. Biomarkers contribute to the assessment of the concentration level, which will help to determine the level and rate of AAA development. The potential biomarkers today include homocysteine, cathepsins, osteopontin, and osteoprotegerin. In this review, we describe the major aspects of molecular processes that take place in the aortic wall during AAA formation. In addition, biomarkers, the monitoring of which will contribute to the prompt diagnosis of AAA patients over the age of 55 years, are described.

In this work, we studied the stability of matrices with temperature-dependent solubility and their interactions with water at physiological temperature for their application in cell culture in vitro. Gradient copolymers of 2-isopropyl- with 2-n-propyl-2-oxazoline (P(iPrOx-nPrOx)) were used to prepare the matrices. The comonomer ratio during polymerization was chosen such that the cloud point temperature (TCP) of the copolymer was below 37 °C while the glass transition (Tg) was above 37 °C. The role of the support for matrices in the context of their stability in aqueous solution was examined. Therefore, matrices in the form of both self-supported bulk polymer materials (fibrillar mats and molds) and polymer films supported on the silica slides were examined. All of the matrices remained undissolved when incubated in water at a temperature above TCP. For the self-supported mats and molds, we observed the loss of shape stability, but, in the case of films supported on silica slides, only slight changes in morphology were observed. For a more in-depth investigation of the origin of the shape deformation of self-supported matrices, we analyzed the wettability, thickness, and water uptake of films on silica support because the matrices remained undeformed under these conditions. It was found that, above the TCP of P(iPrOx-nPrOx), the wettability of the films decreased, but at the same time the films absorbed water and swelled. We examined how this specific behavior of the supported films influenced the culture of fibroblasts. The temperature-dependent solubility of the matrices and the possibility of noninvasive cell separation were also examined.

No abstract

Abdominal aortic aneurysm refers to abnormal, asymmetric distension of the infrarenal aortic wall due to pathological remodelling of the extracellular matrix. The distribution of enzymes remodelling the extracellular matrix and their expression patterns in the affected tissue are largely unknown. The goal of this work was to investigate the expression profiles of 20 selected genes coding for metalloproteinases and their inhibitors in the proximal to the distal direction of the abdominal aortic aneurysm. RNA samples were purified from four lengthwise fragments of aneurysm and border tissue obtained from 29 patients. The quantities of selected mRNAs were determined by real-time PCR to reveal the expression patterns. The genes of interest encode collagenases (MMP1, MMP8, MMP13), gelatinases (MMP2, MMP9), stromelysins (MMP3, MMP7, MMP10, MMP11, MMP12), membrane-type MMPs (MMP14, MMP15, MMP16), tissue inhibitors of metalloproteinases (TIMP1, TIMP2, TIMP3, TIMP4), and ADAMTS proteinases (ADAMTS1, ADAMTS8, and ADAMTS13). It was found that MMP, TIMP, and ADAMTS are expressed in all parts of the aneurysm with different patterns. A developed aneurysm has such a disturbed expression of the main participants in extracellular matrix remodelling that it is difficult to infer the causes of the disorder development. MMP12 secreted by macrophages at the onset of inflammation may initiate extracellular matrix remodelling, which, if not controlled, initiates a feedback loop leading to aneurysm formation.

Induced pluripotent stem cells (iPSCs) are defined as reprogrammed somatic cells exhibiting embryonic stem cell characteristics. Since their discovery in 2006, efforts have been made to utilize iPSCs in clinical settings. One of the promising fields of medicine, in which genetically patient-specific stem cells may prove themselves useful, is gene therapy. iPSCs technology holds potential in both creating models of genetic diseases and delivering therapeutic agents into the organism via auto-transplants, which reduces the risk of rejection compared to allotransplants. However, in order to safely administer genetically corrected stem cells into patients’ tissues, efforts must be made to establish stably pluripotent stem cells and reduce the risk of insertional tumorigenesis. In order to achieve this, optimal reprogramming factors and vectors must be considered. Therefore, in this review, the molecular bases of reprogramming safe iPSCs for clinical applications and recent attempts to translate iPSCs technology into the clinical setting are discussed.