Folia Biologica最新文献

Insulin-like growth factor binding protein 5 (IGFBP5) is broadly bioactive, but its role in osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) remains to be clarified. Here, we demonstrated that IGFBP5 expression was markedly increased during the early osteogenic differentiation of hMSCs. We then over-expressed and knocked down this gene in hMSCs and evaluated the impact of manipulation of IGFBP5 expression on osteogenic differentiation based upon functional assays, ALP staining, and expression of osteogenic markers. Together, these analyses revealed that IGFBP5 over-expression enhanced early osteogenic differentiation, as evidenced by increased ALP staining and osteogenic marker induction, whereas knocking down this gene impaired the osteogenic process. Over-expression of IGFBP5 also markedly bolstered the extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation level, while IGFBP5 knockdown suppressed this signalling activity. We additionally compared the impact of simultaneous IGFBP5 overexpression and ERK1/2 inhibitor treatment to the effect of IGFBP5 over-expression alone in these hMSCs, revealing that small molecule-mediated EKR1/2 inhibition was sufficient to impair osteogenic differentiation in the context of elevated IGFBP5 levels. These findings indicated that IGFBP5 drives the early osteogenic differentiation of hMSCs via the ERK1/2 signalling pathway. Our results offer value as a foundation for future efforts to study and treat serious bone-related diseases including osteoporosis.

Reperfusion therapies for ischaemic stroke can induce secondary injury accompanied by neuronal death. The Y-box binding protein 1 (YBX1), an oncoprotein, is critical for regulating tumour cell proliferation and apoptosis. Thus, we wanted to know whether YBX1 could regulate neuronal cell apoptosis caused by cerebral ischaemia/reperfusion (I/R). We established a model of cerebral I/R-induced injury in vitro by oxygen-glucose deprivation/reoxygenation (OGD/R) treatment and determined YBX1 expression using Western blot. Next, the effect of YBX1 on the apoptosis and viability of OGD/R-treated PC12 cells was evaluated by flow cytometry, MTT assay, and Western blot. Besides, the release of lactate dehydrogenase (LDH) and the activity of catalase (CAT) and superoxide dismutase (SOD) were detected to evaluate oxidative stress of PC12 cells induced by OGD/R. The regulatory roles of YBX1 in the AKT/GSK3β pathway were examined by Western blot. As a result, OGD/R treatment down-regulated YBX1 expression in PC12 cells. YBX1 over-expression attenuated the growth inhibition and apoptosis of PC12 cells induced by OGD/R. Besides, the increase of LDH release and the decrease of SOD and CAT activities caused by OGD/R were reversed by YBX1 over-expression. Moreover, YBX1 over-expression could activate the AKT/GSK3β pathway in OGD/ R-treated PC12 cells. Therefore, YBX1 could protect against OGD/R-induced injury in PC12 cells through activating the AKT/GSK3β signalling pathway, and thus YBX1 has the potential to become a therapeutic target for cerebral I/R-induced injury.

The crucial requirement of molecular genetic methods is high-quality input material. The key question is "how to preserve DNA during long-term storage." Biobanks are recommended to aliquot isolated DNA into provided volumes. The aim of this study was to analyse the effect of repeated freezing and thawing on the genomic DNA integrity, quality and concentration. The aliquoted DNA isolated from blood cells using the automatic MagNA system and manual salting out method underwent freeze/thaw cycles at different storage conditions (-20 °C, -80 °C and liquid nitrogen). The average initial concentrations were 270.6 ng/μl (salting out method) and 125.0 ng/μl (MagNA). All concentration deviations relative to the concentration after the first freeze/ thaw cycle were less than 5 % for -20 °C and -80 °C cycling with both isolation methods. The average percentage differences of liquid nitrogen samples were higher, and the MagNA isolation method showed significant differences. There were no significant changes in the DNA purity or quality. The repeating freeze/ thaw up to 100 cycles (through -20 °C and -80 °C, respectively) did not significantly influence the integrity, concentration, or purity of genomic DNA, suggesting that storage of samples in high-volume pools without multiple aliquoting is possible. Storage in a freezer seems to be the most suitable way of long-term DNA preservation, because liquid nitrogen storage leads to formation of DNA clumps.

Hepatitis B virus (HBV) infection is more likely to develop into chronic and persistent infection in China, which is the main cause of chronic liver disease. We examined the cytokine profiles of chronic hepatitis B (CHB) and CHB-caused liver cirrhosis (LC) to look for the predictor of progression from CHB to LC. Serum samples of 15 healthy controls (HC), 15 CHB patients and 15 LC patients were collected to detect the profiles of 48 cytokines by multiplex biometric ELISA-based immunoassay. Partial least squares discriminant analysis (PLS-DA) and random forest were used to analyse significant cytokines, which were further validated by ELISA using an independent cohort of 60 CHB patients, 60 LC patients and 35 HC samples. There were 18 differentially expressed cytokines of CHB and LC. Three cytokines were identified by PLS-DA and random forest, including interleukin (IL)-9, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-2 receptor subunit α (IL-2Rα), which displayed significant changes in serum levels. Differentially expressed cytokine networks between HC, CHB and LC also indicated particular cytokine co-expression network patterns of CHB and LC. The receiver-operator characteristic (ROC) analysis demonstrated that IL-9, GM-CSF, IL-2Rα and their logistic regression panel are potential predictors that significantly differentiate CHB from LC (P < 0.001) and CHB from Child class A LC (P < 0.001). The three cytokines and the panel showed significant correlation with the Child-Pugh score. IL-9, GM-CSF, IL-2Rα and their logistic panel may be predictors for monitoring the progression of CHB to LC.

Prematurely erupted teeth are rare in fullterm neonates and extremely rare in prematurely delivered infants. The aim of this study was to present macroscopic and scanning electron microscopy (SEM) investigations of prematurely erupted primary teeth of preterm very low birthweight (VLBW) and extremely low birthweight (ELBW) infants. Three preterm VLBW and ELBW infants with prematurely erupted lower incisors were examined. The dental examination assessed the type, location, clinical appearance, and degree of mobility of the prematurely erupted teeth. The structural appearance of enamel and dentin of three extracted and longitudinally sectioned prematurely erupted teeth was investigated with scanning electron microscopy (SEM). Lower incisors were rootless with hypermobility. The surface of enamel of the prematurely erupted primary teeth was hypoplastic and hypomineralized and had a typical "honeycomb" appearance in SEM. The aprismatic type of enamel was visible in some regions. The neonatal line separating the layer of prenatal enamel from postnatal enamel was observed. The enamel prisms were interconnected by interprismatic substances, and cross-striations of prisms were visible. Dentin presented a typical tubular character. The dentinal layer near the enamel dentin junction had Y-shaped branching of dentinal tubules. On the pulpal side, dentin had a globular character. The macroscopic and SEM investigations particularly revealed alterations in enamel, while the dentin of neonatal teeth had a nearly normal appearance.

Glucose is both the favourite carbon and energy source and acts as a hormone that plays a regulating role in many biological processes. Calorie restriction extends the lifespan in many organisms, including Schizosaccharomyces pombe, while uptake of high glucose leads to undesired results, such as diabetes and aging. In this study, sequence analysis of Schizosaccharomyces pombe ird5 and ird11 mutants was performed using next-generation sequencing techniques and a total of 20 different mutations were detected. ird11 is resistant to oxidative stress without calorie restriction, whereas ird5 displays an adaptive response against oxidative stress. We selected nine candidate mutations located in the non-coding (6) and coding (3) region among a total of 20 different mutations. The nine candidate mutations, which are thought to be responsible for ird5 and ird11 mutant phenotypes, were investigated via forward and backward mutations by using various cloning techniques. The results of this study provide report-like information that will contribute to understanding the relationship between glucose sensing/ signalling and oxidative stress response components.

It has been shown previously that oestradiol protects the vascular network, leading to increased skin flap viability associated with Bcl-2, VEGF and FGF-2 up-regulation. We have shown that genistein, a natural selective oestrogen receptor modulator, also increases skin flap viability in rats and induces Bcl-2 expression in human umbilical vein endothelial cells. In the present study we aimed to answer the question whether genistein increases expression of Bcl-2, a potent anti-apoptotic protein, in human dermal microvascular endothelial cells (HMVEC-d) as well. Our results showed that administration of genistein induces Bcl-2 expression in a concentration-dependent manner. Cell co-treatment with genistein and anti-ER compounds (MPP, PHTPP, ICI, G-15) diminished the observed positive effect of genistein on Bcl-2 expression. The decrease in Bcl-2 expression in HMVEC-d was most prominent after co-treatment with ICI (nuclear ER antagonist/ GPR30 agonist) and PHTPP (selective ER-β antagonist). In conclusion, genistein increases Bcl-2 expression in HMVEC-d, contributing to its protective effect on the skin flap viability. However, the question whether the mechanism is ER-specific (via ER-β) has to be answered in further studies using a model of gene silencing or genetically modified cells.

Plasma levels of circulating platelet extracellular vesicles (PEVs) are an emerging marker of platelet activation, thrombosis, inflammation, and endothelial dysfunction. Analysis of PEVs in cord blood of preterm newborns may reflect the underlying pathology and possibly serve as a new diagnostic and prognostic tool. However, collection, preparation and analysis of cord blood samples in clinical settings is a logistically complex process. We have studied the effect of delay in sample preparation and sample freezing on the PEV analysis by flow cytometry. PEVs in the cord blood plasma were identified after double labelling with monoclonal antibodies CD36+CD41 or CD41+CD62. Both, the delay and the freezing significantly affected the count and often also fluorescence of the detected PEVs. Additionally, our pilot study utilizing fresh cord blood samples of term and preterm newborns demonstrated significantly decreased CD36 and CD62 PEV fluorescence in preterm newborns. Our data highlight the importance of pre-analytical steps in the analysis of cord blood PEVs and suggest that not only the count, but also the level of PEV fluorescence may have possible diagnostic potential.

Von Willebrand disease is a commonly inherited bleeding disorder caused by defects of von Willebrand factor (vWF). In the most common valve diseases, aortic valve stenosis (AVS) and mitral valve regurgitation (MVR), a bleeding tendency has been described in a number of patients. This has been associated to a high turbulence of blood flow through the compromised valve, promoting degradation of vWF with loss of high-molecular-weight multimers of vWF (HMWM), leading to an acquired von Willebrand syndrome (AvWS). We analysed three groups of patients, one affected by AVS, treated with transcatheter aortic valve implantation (TAVI), the second group of patients affected by MVR, treated with Mitraclip® mitral valve repair. The third group was represented by patients also affected by AVS, but not eligible for TAVI and treated with standard surgery. A fourth group of patients that underwent percutaneous coronary intervention (PCI) with stenting was used as a control. Our results demonstrated that the level of vWF measured as antigen concentration (vWF:Ag) increases in all cohorts of patients after treatment, while in control PCI patients, no modification of vWF:Ag has been registered. Western blot analysis showed only a quantitative loss of vWF in the pre-treatment time, but without significant HMWM modification. The monitoring of the vWF:Ag concentration, but not the quality of HMWM, can indicate the status of blood flow in the treated patients, thus introducing the possibility of using the vWF antigen detection in monitoring the status of replaced or repaired valves.

This study investigated whether kaempferol could inhibit ovarian cancer (OC) by activation of endoplasmic reticulum (ER) stress and autophagy, and tested its effect on the sensitivity of OC cells to cisplatin (cis-diamminedichloroplatinum, DPP). To study the effect of kaempferol on activation of ER stress and autophagy and find out whether its mechanism of action involves calcium (Ca2+), A2780 OC cells were cultured in DMEM/F12 for 24 h with or without kaempferol (40 μmol/l) in the presence or absence of autophagy or ER stress inhibitors or a calcium chelator. To study the effect of kaempferol on the sensitivity of OC cells to DPP and the potential involvement of modulation of protein kinase B (Akt) expression, A2780 OC were incubated with kaempferol and increasing concentrations of DPP (0-20 μmol/l) and then with kaempferol at its predetermined IC50 (6.8 μmol/l). Compared to control cells, kaempferol increased cell apoptosis (158 %) and decreased viability (53.17 %) and proliferation (49.17 %) of A2780 OC cells. Concomitantly, it increased the protein levels of GRP78, PERK, ATF6, IRE-1, LC3II, beclin 1, and caspase 4, thus suggesting activation of cytotoxic autophagy. This was mediated by increasing intracellular Ca+2 levels. In addition, kaempferol increased the sensitivity of A2780 cells to DPP (IC50 from 6.867 ± 0.99 to 3.73 ± 0.59 μmol/l) by decreasing the protein levels of p-Akt (0.31 ± 0.09 vs 0.12 ± 0.005). In conclusion, the findings of this study encourage the use of kaempferol alone or in combination with DPP to inhibit tumorigenesis of ovarian cells.