Analysis of the TID-I and TID-L Splice Variants' Expression Profile under In Vitro Differentiation of Human Mesenchymal Bone Marrow Cells into Osteoblasts.

Bone formation is a complex process regulated by a variety of pathways that are not yet fully understood. One of the proteins involved in multiple osteogenic pathways is TID (DNAJA3). The aim of this work was to study the association of TID with osteogenesis. Therefore, the expression profiles of the TID splice variants (TID-L, TID-I) and their protein products were analyzed during the proliferation and differentiation of bone marrow mesenchymal stromal cells (B-MSCs) into osteoblasts. As the reference, the hFOB1.19 cell line was used. The phenotype of B-MSCs was confirmed by the presence of CD73, CD90, and CD105 surface antigens on ~97% of cells. The osteoblast phenotype was confirmed by increased alkaline phosphatase activity, calcium deposition, and expression of ALPL and SPP1. The effect of silencing the TID gene on the expression of ALPL and SPP1 was also investigated. The TID proteins and the expression of TID splice variants were detected. After differentiation, the expression of TID-L and TID-I increased 5-fold and 3.7-fold, respectively, while their silencing resulted in increased expression of SPP1. Three days after transfection, the expression of SPP1 increased 7.6-fold and 5.6-fold in B-MSCs and differentiating cells, respectively. Our preliminary study demonstrated that the expression of TID-L and TID-I changes under differentiation of B-MSCs into osteoblasts and may influence the expression of SPP1. However, for better understanding the functional association of these results with the relevant osteogenic pathways, further studies are needed.

Fluorescence spectral analysis and logistic regression modeling for diagnosing basal cell carcinoma on head and neck.

The optical fluorescence method is distinguished by key features such as non-invasiveness, high sensitivity, and resolution, which are superior to traditional diagnostic approaches. Unlike histopathological examinations and biochemical analyses, optical diagnostic methods obviate the need for tissue sampling, enabling the analysis of virtually unlimited material. The research aims to examine the effectiveness of emission spectra analysis in the diagnosis of basal cell carcinoma (BCC) of the scalp and neck. The analysis was based on data provided by Specialized Hospital No. 2 in Bytom comprising a study sample of 10 patients. For each patient, fluorescence emission spectra were recorded from each of 512 points along a 5 mm line. The results obtained from the histopathological examination, the analysis and morphological evaluation of the tissue, and the diagnosis through microscopic observation were used to define a dichotomous variable (presence or absence of a cancerous lesion), adopted in the study as the modeled variable. The next step of the presented study involved constructing a logistic regression model for identifying cancerous lesions depending on the biochemical indicator's relative fluorescence value (RFV) and emission wavelength (ELW) within the 620 nm to 730 nm range. This wavelength range is often used in fluorescence diagnostics to detect various pathologies, including cancerous lesions. The resulting binary logistic regression model, logit(p)=-33.17+0.04ELW+0.01RFV, indicates a statistically significant relationship between wavelength and relative fluorescence values with the probability of detecting cancer. The estimated model exhibits a good fit and high predictive accuracy. The overall model accuracy is 84.8 %, with the correct classification rates at approximately 96 % for healthy individuals and 74 % for individuals with cancer. These findings underscore the potential of photodynamic diagnostics in cancer detection and monitoring.

Analysis of miRNAs in Osteogenesis imperfecta Caused by Mutations in COL1A1 and COL1A2: Insights into Molecular Mechanisms and Potential Therapeutic Targets.

Osteogenesis imperfecta (OI) is a group of connective tissue disorders leading to abnormal bone formation, mainly due to mutations in genes encoding collagen type I (Col I). Osteogenesis is regulated by a number of molecules, including microRNAs (miRNAs), indicating their potential as targets for OI therapy. The goal of this study was to identify and analyze the expression profiles of miRNAs involved in bone extracellular matrix (ECM) regulation in patients diagnosed with OI type I caused by mutations in COL1A1 or COL1A2. Primary skin fibroblast cultures were used for DNA purification and sequence analysis, followed by analysis of miRNA expression. Sequencing analysis revealed mutations of the COL1A1 or COL1A2 genes in all OI patients, including four previously unreported. Amongst the 40 miRNAs analyzed, 9 were identified exclusively in OI cells and 26 in both OI patients and the controls. In the latter case, the expression of six miRNAs (hsa-miR-10b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, has-miR-204-5p, has-miR-216a-5p, and hsa-miR-449a) increased, while four (hsa-miR-129-5p, hsa-miR-199b-5p, hsa-miR-664a-5p, and hsa-miR-30a-5p) decreased significantly in OI cells in comparison to their expression in the control cells. The identified mutations and miRNA expression profiles shed light on the intricate processes governing bone formation and ECM regulation, paving the way for further research and potential therapeutic advancements in OI and other genetic diseases related to bone abnormality management.

Gene-repaired iPS cells as novel approach for patient with osteogenesis imperfecta.

Introduction: The benefits of patient's specific cell/gene therapy have been reported in relation to numerous genetic related disorders including osteogenesis imperfecta (OI). In osteogenesis imperfecta particularly also a drug therapy based on the administration of bisphosphonates partially helped to ease the symptoms. Methods: In this controlled trial, fibroblasts derived from patient diagnosed with OI type II have been successfully reprogrammed into induced Pluripotent Stem cells (iPSCs) using Yamanaka factors. Those cells were subjected to repair mutations found in the COL1A1 gene using homologous recombination (HR) approach facilitated with star polymer (STAR) as a carrier of the genetic material. Results: Delivery of the correct linear DNA fragment to the osteogenesis imperfecta patient's cells resulted in the repair of the DNA mutation with an 84% success rate. IPSCs showed 87% viability after STAR treatment and 82% with its polyplex. Discussion: The use of novel polymer Poly[N,N-Dimethylaminoethyl Methacrylate-co-Hydroxyl-Bearing Oligo(Ethylene Glycol) Methacrylate] Arms (P(DMAEMA-co-OEGMA-OH) with star-like structure has been shown as an efficient tool for nucleic acids delivery into cells (Funded by National Science Centre, Contract No. UMO-2020/37/N/NZ2/01125).

Osteogenesis Imperfecta: Current and Prospective Therapies.

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.

mRNA Expression of thrombospondin 1, 2 and 3 from proximal to distal in human abdominal aortic aneurysm - preliminary report.

Abdominal aortic aneurysm is a process involving the disruption and reconstruction of the extracellular matrix and the apoptosis of smooth muscle cells under the strong influence of the immune system. Thrombospondins are proteins that influence a wide range of cell-matrix interactions. While THBS1 and THBS2 are widely studied, the effects of THBS3 on extracellular matrix and vascular cells are poorly understood. Additionally, it is not known whether expression of these genes' changes along the aneurysm tissue. Here we analyzed the expression of THBSs mRNA isolated from the harvested tissues along the aneurysm divided into three zones based on their morphology. Total mRNA was isolated from 13 male patients undergoing scheduled open aortic repair, with each aneurysm divided into a proximal part, an aneurysm bag, and a distal part with border tissue as a control. Two step real-time PCR analysis with random hexamers was performed, which allowed the detection of significantly increased expression of all analyzed thrombospondins, especially THBS3, at the control tissue. Overexpression of THBSs may have a destabilizing effect on the structure of the extracellular matrix by affecting both the matrix producing cells and by inhibiting the activity of matrix proteins.

Electromagnetic Fields Modify Redox Balance in the Rat Gastrointestinal Tract.

Objective: The aim of the study was to assess the influence of electromagnetic fields with divergent physical properties on the prooxidative and antioxidative balances in homogenates of the tongue, salivary glands, esophagus, stomach, and small and large intestines of rats. Material and Methods: Forty rats were randomly divided into four equal groups, namely, a control group, a group exposed to low-frequency electromagnetic fields (LF-EMFs; frequency: 50 Hz; intensity: 10 kV/m; magnetic induction: 4.3 pT), a group exposed to radiofrequency electromagnetic fields (RF-EMFs) emitted by mobile phones (frequency: 900 MHz), and a group exposed simultaneously to LF-EMFs and RF-EMFs emitted by mobile phones. After 28 consecutive days of the experiment, the following pro- and antioxidative markers were assessed in the gastrointestinal tract homogenates: superoxide dismutase (SOD) and its two isoenzymes (Mn-SOD, Cu,Zn-SOD) catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), total antioxidative capacity (TAC), total oxidative status (TOS), and malondialdehyde (MDA). Results: In rats exposed to LF-EMFs, higher concentrations of the markers of prooxidant processes, MDA or TOS, were observed in the salivary glands, esophagus, and small intestine homogenates in comparison with the control group. Additionally, in the group of rats opposite to the control, antioxidant activity was observed. The main differences included a higher activity of Cu,Zn-SOD in homogenates of the tongue, salivary glands, and esophagus as well as decreased activity of CAT in homogenates of the tongue, esophagus, and small intestine. In animals exposed to RF-EMFs, the concentration of TOS was higher in the large intestine than in control rats. The main difference of antioxidant activity was presented by decreased Cu,Zn-SOD in homogenates of the salivary glands, stomach, small and large intestine as well as CAT in homogenates of the tongue, esophagus, stomach, and small and large intestine. Moreover, in rats exposed simultaneously to LF-EMFs and RF-EMFs, a lower concentration of TOS was observed. Antioxidant activity was presented by a decreased activity of CAT in homogenates of the tongue, esophagus, stomach, and small and large intestine in comparison to the control group. Conclusion: Among those applied in the study, electromagnetic fields of a low-frequency caused the most significant disturbances of oxidative stress in the rat gastrointestinal tract.

The effect of compression therapy on quality of life in patients with chronic venous disease: a comparative 6-month study.

INTRODUCTION: Chronic venous diseases (CVD), because of its chronic and progressive nature, impairs patients' quality of life (Qol). AIM: To compare the QoL in patients with primary superficial venous insufficiency at different stages before and after compression therapy (CT). MATERIAL AND METHODS: We compared the change in the QoL parameters from baseline to the end of a 6-month compression therapy. 180 subjects were enrolled. They were subdivided into 6 equal subgroups according to CEAP classes. The QoL was assessed using questionnaires, the general SF-36v2 and the disease-specific CIVIQ-20. At the beginning and after the completion of the study intervention, the severity of CVD was assessed in each patient using CEAP and VCSS. The pain intensity was assessed using the numerical rating scale. RESULTS: The CT reduced the severity of CVD, which translated into the increased size of C2 an d C5 subgroups, and reduced size of C3 and C6 subgroups. Another marker of reduced severity of CVD after CT was a significant reduction in VCSS scores in C1, and C3-C6 subgroups. A 6-month CT was associated with a significant QoL improvement in all CEAP class-based subgroups, across all individual and composite domains of SF-36v2, as well as dimensions and GIS of CIVIQ-20. Similarly, there was a significant pain reduction reported in all CEAP class-based subgroups. CONCLUSIONS: Compression therapy using ready-made compression hosiery significantly affects the quality of life in patients with chronic venous disease at all its stages, CEAP classes C1-C6.

An Overview of Methods and Tools for Transfection of Eukaryotic Cells in vitro.

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.

Evaluation of the therapeutic efficacy of active specialistic medical dressings in the treatment of decubitus.

INTRODUCTION: Treatment of decubitus ulcers is a grave medical problem. In many cases, it is difficult to cure a pressure ulcer, especially when it is deep and extensive, and prognosis is usually unfavourable. Treatment of decubitus ulcers requires new specialist dressings, which play an important role in the healing process. AIM: To evaluate therapeutic efficacy of active specialist medical dressings in the treatment of decubitus. MATERIAL AND METHODS: Research involved 40 patients - 18 (45%) women and 22 (55%) men, suffering from decubitus ulcers of different size and depth, localized in the sacral region, lasting from 1.5 to 30 months. Patients were randomly assigned to two research groups (20 people each), were treated for 4 weeks with 2 different specialist dressings. ATRAUMAN Ag, which contains silver ions, was used in the first group, while paraffin gauze of BACTIGRAS type was used in the second group. An assessment of pressure ulcers' healing progress was done with a planimetric method, which evaluates the wound surface area. RESULTS: The analysis results showed a significant statistical decrease in an average decubitus ulcer surface area in both research groups: in the first group by 60.2% (p = 0.001), and in the second group by 32.95% (p < 0.001), which speaks in favour of dressings with silver ions as having better therapeutic effectiveness. CONCLUSIONS: Using specialist dressings results in a significant decrease in the decubitus ulcer surface area, depending on the type of dressing and active substances contained within, while silver ions support curative effectiveness of the dressing used.

Searching for new molecular markers for cells obtained from abdominal aortic aneurysm.

The aim of the study was to investigate specific potential markers for cells obtained from three layers of human AAA divided into three segments along the AAA based on morphological differences. The isolated cells were compared to control commercial cell types from healthy human abdominal aortas. For each type of aortic layer, three specimens from 6 patients were compared. Total RNA was isolated from 36 cell cultures for gene expression profiling and potential new cytometry markers were typed. Isolated cells were analyzed by flow cytometry by using fluorochrome-conjugated antibodies to markers: CNN1, MYH10, ENG, ICAM2, and TEK. The relative expression of 45 genes in primary cell cultures and control lines was analyzed. Statistically significant differences were found in the expression of most of the analyzed genes between individual layers and control lines. Based on relative expression, antibodies were selected for flow cytometry. Gene expression profiles allowed to select new potential cytometry markers: CNN1, MYH10, MYOCD, ENG, ICAM2, TEK. However, none of the tested markers seems to be optimal and characteristic for a specific layer of AAA.

Expression gradient of metalloproteinases and their inhibitors from proximal to distal segments of abdominal aortic aneurysm.

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.

Potential of Induced Pluripotent Stem Cells for Use in Gene Therapy: History, Molecular Bases, and Medical Perspectives.

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.

The role of autofluorescence, photodynamic diagnosis and Photodynamic therapy in malignant tumors of the duodenum.

This article presents the current state of knowledge and a review of the literature in terms of the prevalence, etiopathogenesis, differential diagnosis, management, prognosis, and treatment of malignant tumors of the duodenum. The role of autofluorescence and photodynamic diagnosis as an emerging treatment method for rarely o ccurring duodenal malignant neoplasms .. We selected publications which can be found in databases such as The National Center for Biotechnology Information, U.S. National Library of Medicine (PubMed), The American Chemical Society, The American Association of Pharmaceutical Sciences and The American Society for Photobiology and The Canada Institute for Scientific and Technical Information.

Spectral analysis of basal cell cancer in vivo.

One of the most important features of optical biopsy methods is its non-invasive nature, high sensitivity and resolution in comparison to traditional diagnostic methods. Optical diagnostic methods, contrary to histopathological tests and biochemical analysis, do not require the collection of tissue samples for analysis, and the amount of analyzed material is practically unlimited. The aim of this study was to evaluate the usefulness of spectral studies in the diagnostics of skin basal cell cancer using a protoptive device in clinical conditions. Each of the subjects was measured 4 times. The measurements were performed on the skin of a patient without a photosensitizer and on the healthy skin on the opposite side of the change in relation to the middle line, also without a photosensitizer. Patients were then given photosensitizer in form of ointment from ALA (Sigma Aldrich, USA) and lubricated with this ointment with occlusal dressing. The patient was examined again 3 h after the application. In each case the measurement time was 1.2 s. Significant differences were observed for the measurements of the sick patient's skin with photosensitizer. Significant differences in spectral curve between 570 and 780 nm were observed.

Methods of spectral diagnostics in modern prevention of oncological diseases.

This paper contains structured information on photodynamic diagnostics. Photodynamic diagnostics is a young diagnostic modality used in the detection of pre-neoplastic and very early neoplastic lesions. A characteristic feature of the presented method is its completely non-invasive nature and thus the possibility of multiple repetitions at the same patient. This is very important in modern health care and in preventive measures. Aim of the paper: The article aims to present technical and diagnostic possibilities of a photodynamic method as one of the possible modalities of screening diagnostics in patients with ambiguous clinical picture of early neoplastic lesions.

Star polymer-based nanolayers with immobilized complexes of polycationic stars and DNA for deposition gene delivery and recovery of intact transfected cells.

We designed a novel thermoresponsive system of nanolayers composed of star poly[oligo(ethylene glycol) methacrylate]s (S-POEGMA) covalently bonded to a solid support and covered with polyplexes of cationic star polymers and plasmid DNA (pDNA). S-POEGMA stars were attached to the solid support via a UV-mediated "grafting to" method. To the best of our knowledge, for the first time, the conformational changes of obtained star nanolayers, occurring with changes in temperature, were studied using a quartz crystal microbalance technique. Next, the polyplexes of star poly[N,N'-dimethylaminoethyl methacrylate-ran-di(ethylene glycol) methacrylate] (S-P(DMAEMA-DEGMA)) with pDNA, exhibiting a phase transition temperature (TCP) in culture medium DMEM, were deposited on S-POEGMA layers when the temperature increased above the TCP of polyplex. The thermoresponsivity of the system was then the main mechanism for controlling the adhesion, proliferation, transfection and detachment of HT-1080 cells. The nanolayers promoted the effective cell culture and delivered nucleic acids into cells, with a transfection efficiency several times higher than that of the control. The detachment of the transfected cells was regulated only by the change of temperature. The studies demonstrated that we obtained a novel and effective system, based on a star polymer architecture, useful for gene delivery and tissue engineering applications.

Monitoring PDT effects in basal cell carcinoma treatment using thermal imaging.

BACKGROUND: One of the most frequent type of malignant skin lesion (almost 95 percent of all skin tumours) is basal cell carcinoma (BCC). It is often treated by radiotherapy using ionizing radiation as well by photodynamic therapy (PDT) which is a selective method directed only on cancer cells and well tolerated by patients. MATERIALS AND METHODS: Eight male patients of the Department and Clinic of Internal Diseases, Angiology and Physical Medicine in Bytom, Medical University of Silesia, in Katowice, Poland suffering from basal cell carcinoma were monitored by thermovision during the photodynamic therapy. All lesions were diagnosed as superficial were confirmed by histopathological examination. RESULTS: The dynamics of changes observed in the isotherm area during the therapy can provide physicians with additional information. The significant increase of observed isotherm area in comparison to the lesion area diagnosed by a physician was confirmed, which may be connected with the increased metabolism processes occurring in the tissue surrounded the lesion. CONCLUSION: The obtained results based on the temperature gradient changes in the lesion vicinity area may bring some new information describing the range of biochemical and physiological processes occurring during photodynamic therapy.

Poly(2-oxazoline) Matrices with Temperature-Dependent Solubility-Interactions with Water and Use for Cell Culture.

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.