About 4-day Rhythm of Proliferative Activity of Fibroblast-like Cell Cultures Depends on the Environmental Factor

A study of the 4-day rhythm of the proliferative activity of the embryonic fibroblast-like cells in the logarithmic growth phase was carried out. It was shown that in cell cultures obtained on different days from embryos of different ages, the phase of the 4-day rhythm coincides. In vitro the maxima of the proliferative activity were consistent with the minima of the motor activity of mice. Freezing the culture for 2 or 6 days does not cause a shift in the phase of the 4-day rhythm of cell proliferative activity compare with the unfreezing culture. That indicates the existence of an external synchronizer, which determines the 4-day infradian rhythm of the proliferative activity of embryonic cells. Then we daily thawed samples of single L-929 culture of mice fibroblast-like cells for 22 and 17 days and researched the dynamics of its proliferative activity. We also showed 4-day rhythm of the simultaneous increase in the number of cells for all thawed samples. Taking into account that deep freezing of a culture leads to the cessation of all life processes, the fact we obtained indicates an exogenous mechanism of the formation of about a 4-day rhythm of the proliferative activity of cell culture.

Downregulation of Rap1GAP Expression Activates the TGF-β/Smad3 Pathway to Inhibit the Expression of Sodium/Iodine Transporter in Papillary Thyroid Carcinoma Cells

Objective. Rap1GAP is considered a tumor suppressor gene, but its regulatory mechanism in papillary thyroid cancer (PTC) has not been clearly elucidated. The aim of this study was to explore whether the regulation between Rap1GAP and sodium/iodine transporter (NIS) in tumorigenesis of PTC is mediated by TGF-β1. Methods. Western blotting (WB) and quantitative reverse-transcription polymerase chain reaction were performed to analyze the relationships between TGF-β1 concentration and NIS expression. After transfecting BCPAP cells with siRNAs, the Rap1GAP interference model was successfully established. Then, the expression and nuclear localization of TGF-β1 and pathway-related proteins were detected. Flow cytometry was applied to analyze cell apoptosis and cycle. WB was performed to detect apoptotic-related proteins. Wound healing and transwell assays were used to measure cell migration and invasion. EDU was performed to detect cell proliferative activity. Results. The results suggested that TGF-β1 could significantly inhibit the expression of NIS in both mRNA and protein levels. In BCPAP cells transfected with siRNA-Rap1GAP, the expression levels of TGF-β1, Foxp3, and p-Smad3 were significantly increased. By applying immunofluorescence assay, the nuclear localizations of TβR-1 and p-Smad3 were found to be activated. Moreover, anti-TGF-β1 can reverse the decrease in NIS expression caused by downregulation of Rap1GAP. Additionally, the knockdown of Rap1GAP could alter the cell apoptosis, cycle, migration, invasion, and proliferation of BCPAP. Conclusion. The downregulation of Rap1GAP expression can activate the TGF-β/Smad3 pathway to inhibit NIS expression and alter the tumor cell functions of PTC.

Investigation of Cytotoxic Effects of Recombinant Human Interferon Lambda-1 and Its Pegylated Form on Human Conjunctival Epithelial Cells

Currently, there are no efficacious, all-purpose antiviral medicines for the treatment of ocular surface infections caused by viruses. At the same time, type III interferons demonstrate high potency for histological barriers, such as the conjunctiva. Modification of protein molecules in native products can significantly improve their pharmacodynamic properties. Thus, it seems reasonable to develop antiviral medicines based on interferon lambda (IFN-λ1) and its pegylated form (PEG IFN-λ1).The aim of the study was to evaluate the in vitro cytotoxic effect of recombinant human IFN-λ1 and its pegylated form on Chang conjunctiva clone 1-5c-4 human conjunctival cells.Materials and methods: PEG IFN-λ1 was obtained by the electron beam immobilisation method. A normal human conjunctival cell line Chang conjunctiva clone 1-5c-4 was used for cell cultivation. The MTT test was used to assess the cytotoxic effect. Cell proliferative activity was studied by measuring microelectrode impedance. Ultrastructural changes were assessed by electron microscopy. Statistical processing was performed using the Statistica 10.0 software package.Results: IFN-λ1 (37 μg/mL) and PEG IFN-λ1 (42 μg/mL) had no significant cytotoxic effect on the human conjunctiva cell culture and the cell proliferative activity. The analysis of ultrastructural changes demonstrated that IFN-λ1 activated metabolic processes in the cells, and PEG IFN-λ1 promoted differentiation and keratinisation of epithelial cells and led to modification of the cell membrane. A ten-fold increase in IFN-λ1 and PEG IFN-λ1 concentration (to 370 μg/mL and 420 μg/mL, respectively) reduced the cell viability by 15–20% as compared to the intact control.Conclusions: the study results demonstrated that IFN-λ1 and PEG IFN-λ1 could be used as active pharmaceutical ingredients in the development of medicines for the treatment of conjunctival viral infections.

Current Biomarkers of Apoptosis Process in Chronically Inflamed Nasal Sinus Epithelium: Representatives of BAX, Bcl-2 and miRNA Group

In chronic upper respiratory tract diseases, increased cell proliferative activity is observed, which is coordinated by Bcl-2 proteins as well as by small non-coding RNAs.The aim of this study was to determine the expression of critical apoptosis markers at the mRNA and miRNA level in patients with chronic rhinosinusitis with nasal polyps (CSRwNP).The study group consisted of 10 patients with CSRwNP and 10 healthy controls. TUNEL staining was performed to detect in situ apoptosis in the maxillary sinus mucosa. The levels of selected mRNA transcripts associated with cell survival and apoptosis: BAX, p53, p21, CASP3, CASP9, c-MYC, CCND1, BRIC5 and APAF1 and miRNAs: miR-17-5p, miR-145-5p, miR-146a-5p and miR-203a-3p were determined by RT-qPCR. CSRwNP patients showed increased apoptosis determined by TUNEL assay accompanied by increased expression of BAX, P21, P53, CASP3, CASP9, c-MYC, APAF-1 transcripts and decreased mRNA levels of BCL-2 and BIRC5. There were increased expression levels of miR-203a-3p and decreased expression levels of miR-17-5p and miR-145-5p. These findings appear to be characteristic features of apoptosis in CRSwNP. The proapoptotic effect of miR-203a-3p may be crucial for future treatment strategies for CRSwNP.

Design of epidermal growth factor immobilization on 3D biocompatible scaffolds to promote tissue repair and regeneration

Exogenous application of human epidermal growth factor (hEGF) stimulates epidermal wound healing. The aim of this study was to develop bioconjugates based on hEGF mimicking the protein in its native state and thus suitable for tissue engineering applications, in particular for treating skin-related disorders as burns. Ribonuclease A (RNase A) was used to investigate a number of different activated-agarose carriers: cyanogen bromide (CNBr)-activated-agarose and glyoxyl-agarose showed to preserve the appropriate orientation of the protein for receptor binding. EGF was immobilized on these carriers and immobilization yield was evaluated (100% and 12%, respectively). A peptide mapping of unbound protein regions was carried out by LC–MS to take evidence of the residues involved in the immobilization and, consequently, the flexibility and surface accessibility of immobilized EGF. To assess cell proliferative activities, 10, 25, 50, and 100 ng/mL of each immobilized EGF sample were seeded on fibroblast cells and incubated for 24, 48 and 72 h. The immobilized growth factor showed significantly high cell proliferative activity at 50 and 100 ng/mL compared to control and soluble EGF. Although both of the immobilized samples show dose-dependency when seeded with high number of fibroblast cells, CNBr-agarose-EGF showed a significantly high activity at 100 ng/mL and 72 h incubation, compared to glyoxyl-agarose-EGF.

Chitosan Nanoparticles as a Carrier for Indigofera intricata Plant Extract: Preparation, Characterization and Anticancer Activity

Background: Cancer is one of the major causes of the death and affects people of all ages throughout the world. The drugs that are currently available to treat cancer have many side effects. Hence, there is considerable scientific interest in the continuing discovery of new anticancer agents from natural sources. The aim of this study was to prepare and characterize nanoparticles combining Indigofera intricata crude alcoholic extract and chitosan and to evaluate the anticancer cell proliferative activity for both extract and nanoparticles. Methods: Dried alcoholic extract was prepared and characterized for its phenolic and flavonoid contents. Chitosan extract nanoparticles was prepared by ionic gelation method and characterized by thin layer chromatography (TLC), Fourier-transform infrared spectroscopy (FTIR), particle size and zeta-potential analysis. The anticancer cell proliferative activities of both plant extract and nanoparticles at different concentrations were evaluated using breast cancer cell line (MCF 7). Results: The alcoholic extract showed high contents from both phenolic and flavonoid constituents (15 % and 22 % respectively). The interaction of polyphenolic compounds of the extract with chitosan was confirmed by the TLC and FTIR results. The particle size and zeta-potential of nanoparticles found to be 400.6nm ± 101.8 nm and +42.1 mV ± 9.27 mV respectively. The plant extract showed the lowest cell viability of 45.21% ± 4.8% at the highest dose (250 mg) tested in this investigation. Almost 500-fold reduction (from 250 mg to 0.5 mg) in the extract concentration required to achieve same anticancer cell proliferative activity when formulated as nanoparticles. Also 2.5 mg extract containing nanoparticles showed similar anticancer cell proliferative activity as 5 mg 5-FU. Conclusion: Our results revealed that traditional medicinal plants could be an excellent source of natural anticancer agents and the chitosan-extract nanoparticles is a promising formulation strategy to enhance their clinical effectiveness.

Canine Splenic Nodular Lymphoid Lesions: Immunophenotyping, Proliferative Activity, and Clonality Assessment

Canine splenic lymphoid nodules are currently classified as indolent lymphomas (marginal zone lymphoma [MZL], mantle cell lymphoma [MCL]) or nodular hyperplasia (lymphoid [LNH] or complex [CNH] type). Their differentiation can be difficult on morphology, because of similar histologic appearance and poorly defined diagnostic criteria. Thirty-five surgical samples of splenic lymphoid nodules were reviewed in order to assess the diagnostic contribution of immunophenotyping, proliferative activity and clonality (PARR) in differentiating between hyperplastic and neoplastic lesions. Proliferative activity was evaluated by double immunolabeling for Ki-67 and CD79a, in order to separately assess the proliferative activity of B cells and non-B cells. Definitive diagnoses were MZL ( n = 11), MCL ( n = 4), LNH ( n = 10), and CNH ( n = 10). The overall concordance between histology and PARR was above 90%. Lymphomas had a significantly higher percentage of CD79a-positive areas (mean, 36.30%; P = .0004) and a higher B-cell proliferative activity (median Ki-67 index, 5.49%; P = .0012). The threshold value most accurately predicting a diagnosis of lymphoma was ≥28% of B-cell areas, with a Ki-67 index above 3%. Dogs were monitored for a median follow-up time of 870 days (IQR, 569-1225), and no relapses were documented. Overall median survival time was 1282 days. The combination of histology, immunohistochemistry and PARR can improve the diagnostic accuracy for canine splenic lymphoid nodules, although the long-term behavior of these lesions appears similar.

Cho/Cr ratio at MR spectroscopy as a biomarker for cellular proliferation activity and prognosis in glioma: correlation with the expression of minichromosome maintenance protein 2

Background Magnetic resonance (MR) spectroscopy (1H-MRS) has been demonstrated to be useful in grading glioma, but the utility in assessing cellular proliferation activity and prognosis correlated with the expression of minichromosome maintenance protein 2 (MCM2) has not been reported. Purpose To explore the correlation between proton MR spectroscopy parameters (including choline [Cho]/creatine [Cr], N-acetyl aspartate [NAA]/Cr, and Cho/NAA ratios) and the expression of MCM2 and to further evaluate whether 1H-MRS can predict cell proliferative activity and provide prognostic information in high-grade gliomas (HGGs). Material and Methods Forty-three patients with histopathologically confirmed gliomas were involved in this study. All patients underwent 1H-MRS examination before surgery. Proliferative activity of gliomas was evaluated by MCM2 labeling index (LI). Pearson correlation analysis and empiric receiver operating characteristic (ROC) curves were performed. The Kaplan–Meier method and Cox regression were used for survival analysis. Results Significant correlation was observed between the Cho/Cr ratio and MCM2 LI ( r = 0.522, P < 0.01); however, there was no correlation between MCM2 LI and the Cho/NAA or NAA/Cr ratios ( r = 0.295, P = 0.55 and r = −0.042, P = 0.788, respectively). According to ROC analysis, MCM2 LI of 50% and Cho/Cr ratio of 2.68 represented the optimized cut-off values, respectively, to distinguish longer or shorter survival than 15 months in HGGs patients. Multivariate analysis revealed that both the Cho/Cr ratio and MCM2 expression were independent prognostic markers. Conclusion Cho/Cr ratio has a potential in predicting the expression of MCM2 and can evaluate cell proliferative activity noninvasively. Both the Cho/Cr ratio and MCM2 expression are independent prognostic markers in patients with HGGs.