Integration of hydrothermal liquefaction of Cyanophyta and supercritical water oxidation of its aqueous phase products: Biocrude production and nutrient removal.

Cyanophyta has the potential to produce biocrude via hydrothermal liquefaction (HTL). However, aqueous phase products (APs), as by-products of HTL, pose a risk of eutrophication for the high levels of carbon, nitrogen, and phosphorus. Supercritical water oxidation (SCWO) can efficiently convert organics into small molecules, offering a technique for the harmless treatment of APs. Effects of holding time, pressure, and moisture content on the biocrude yields from isothermal HTL (300 °C) and fast HTL (salt bath temperature of 500 °C) were comprehensively investigated. Biocrude properties were characterized by elemental analysis, FT-IR and GC-MS. Subsequently, the APs obtained under the conditions producing the highest biocrude yield were subjected to SCWO at 550 °C with different oxidation coefficients (n) from 0 to 2. Removal rates of chemical oxygen demand (COD), ammonia nitrogen (NH3-N), and total phosphorus (TP) were further explored. The results show that the highest biocrude yields from isothermal HTL and fast HTL were 24.2 wt% (300 °C, 1800 s, 25 MPa, and 80 wt% moisture content) and 21.9 wt% (500 °C, 40 s, 25 MPa, and 80 wt% moisture content), respectively. The biocrude primarily consisted of N-containing heterocyclic compounds, amides, and acids. SCWO effectively degraded the COD and TP in APs, while the NH3-N required further degradation. At n = 2, the highest removal rates of COD, NH3-N and TP were 98.5 %, 22.6 % and 89.1 %, respectively.

LncRNA GAS5 promotes spermidine­induced autophagy through the miRNA­31­5p/NAT8L axis in pulmonary artery endothelial cells of patients with CTEPH.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a leading cause of pulmonary hypertension. The present study investigated the mechanisms of long non­coding RNA growth arrest­specific transcript 5 (GAS5) on spermidine (SP)­induced autophagy. Pulmonary artery endothelial cells (PAECs) were collected from patients with CTEPH and the rat model. Immunofluorescence, Western blots, reverse transcription­quantitative polymerase chain reaction, bioinformatics, rapid amplification of cDNA ends assays, luciferase reporter assays, RNA­binding protein immunoprecipitation assays, GFP­LC3 adenoviruses, tfLC3 assays and transmission electron microscopy were performed. The results revealed that SP­induced autophagy increased GAS5 in PAECs. The upregulation of GAS5 enhanced and the downregulation of GAS5 reversed the roles of SP in PAECs. Furthermore, GAS5 promoted SP­induced autophagy in PAECs by targeting miRNA­31­5p. The miRNA­31­5p mimic suppressed and the inhibitor promoted SP­induced autophagy. Furthermore, N­Acetyltransferase 8 Like (NAT8L) was a target gene of miRNA­31­5p and knockdown of NAT8L inhibited the autophagic levels of PAECs. In vivo, SP treatment decreased miRNA­31­5p and increased NAT8L levels, which was reversed by the knockdown of GAS5. The downregulation of GAS5 abolished the stimulatory role of SP in PAECs of CTEPH rats. In conclusion, GAS5 promoted SP­induced autophagy through miRNA­31­5p/NAT8L signaling pathways in vitro and in vivo and GAS5 may be a promising molecular marker for therapies of CTEPH.1.

Silibinin augments the effect of clopidogrel on atherosclerosis in diabetic ApoE deficiency mice.

BACKGROUND: Diabetes mellitus (DM) abolishes the antithrombotic effect of Clopidogrel. Here, we investigated the synergistic effect of Silibinin on Clopidogrel-mediated atherosclerosis treatment in diabetic mice. METHODS: ApoE-/- mice were fed with high-fat diet (HFD) to establish the atherosclerotic model with diabetes. Animals were treated with Clopidogrel, Silibinin, or the combined to evaluate the protective effects on atherosclerosis and diabetes through Oil-red-O staining, qRT-PCR, Western blot, and metabolic measurements. Platelet activation and aggregation ex vivo assays were performed to detect the anti-thrombotic effect of different administrations. RESULTS: Silibinin significantly enhanced the inhibitory effect of Clopidogrel on atherosclerosis in DM mice. Co-administration of Silibinin with Clopidogrel remarkedly reduced the aortic lesion, inflammation, and endothelial dysfunction in aorta roots, and diabetic symptoms were significantly improved by the Silibinin-Clopidogrel treatment in HFD-fed ApoE-/- mice. Interestingly, the anti-thrombotic effect of Clopidogrel was further augmented by the Silibinin treatment in atherosclerotic mice. CONCLUSION: In atherosclerotic mouse model, Silibinin significantly improves the effect of Clopidogrel on atherosclerosis.

IRF2 contributes to myocardial infarction via regulation of GSDMD induced pyroptosis.

Interferon regulatory factor (IRF) 2 is a transcription factor belonging to the IRF family, which is essential for gasdermin D (GSDMD)­induced pyroptosis. Decreasing myocardial cell pyroptosis confers protection against heart damage and cardiac dysfunction caused by myocardial infarction (MI). The aim of the present study was to investigate the involvement of IRF2 in MI and the underlying mechanism of IRF2 in pyroptosis. To mimic MI, ligation of the left anterior descending coronary artery was performed to establish an in vivo mouse model and rat cardiomyocytes H9c2 cells were cultured under hypoxic conditions to establish an in vitro model. Transthoracic echocardiography was used to assess cardiac function. Hematoxylin and eosin staining was used to observe histopathological changes in the myocardial tissue. Immunohistochemistry and western blotting were performed to detect IRF2 expression levels. TUNEL staining and flow cytometry were used to detect apoptosis in myocardial tissue and cells. Chromatin immunoprecipitation and dual luciferase reporter assay were used to verify the effect of IRF2 on GSDMD transcription. IRF2 was upregulated in MI mice. MI induced pyroptosis, as evidenced by increased GSDMD, N­terminal GSDMD (GSDMD­N), and cleaved (c­) caspase­1 levels. MI increased IL­1ß and IL­18 levels. These alterations were alleviated by IRF2 silencing. Furthermore, in hypoxia­treated H9c2 cells, IRF2 silencing significantly decreased the elevated levels of IL­1ß and IL­18 and pyroptosis­associated proteins, including GSDMD, GSDMD­N and c­caspase1. Moreover, in hypoxia­treated H9c2 cells, IRF2 directly bound to the GSDMD promoter to drive GSDMD transcription and promote pyroptosis and IRF2 expression may be regulated via the hypoxia inducible factor 1 signaling pathway. In conclusion, the present results demonstrated that IRF2 is a key regulator of MI by mediating pyroptosis, which triggers GSDMD activation.

Study on pressurized upgradation of pyrolysis oil for high-value liquid products.

Upgradation of pyrolysis oil is a key process to achieve high-quality biofuel. In this study, the effects of different Ar pressures and H2/Ar ratios in the presence and absence of catalysts on deoxygenation of pyrolysis oil were investigated by autoclaving. When the initial pressure of the reaction is 6MPa and without catalyst addition, the content of carboxylic acid decreases from 51.52 to 41.54%, whereas with the addition of catalyst (10 % Ni/C), the deoxygenation and hydrocarbon content in the product were significantly improved. Hence, 6 MPa was found to be optimum and above which failed to induce such useful changes but can lead to lower high heating value (HHV). However, the presence of hydrogen affects the content of alkanes and olefins in the product.

Effect of Quercetin-Loaded Mesoporous Silica Nanoparticles on Myocardial Ischemia-Reperfusion Injury in Rats and Its Mechanism.

BACKGROUND: Quercetin has potential value in treating cardiovascular diseases, but it is not suitable for clinical application due to its own water solubility. The limitation of quercetin can be distinctly ameliorated by delivering it with nanocarriers. OBJECTIVE: To determine the effect of quercetin-loaded mesoporous silica nanoparticles (Q-MSNs) on myocardial ischemia-reperfusion injury in rats and its mechanism. METHODS: Q-MSNs were synthesized, and the morphology of Q-MSNs and MSNs was characterized by transmission electron microscopy and dynamic light scattering technique, respectively. Healthy rats were enrolled and randomly divided into a sham operation control group, an ischemia-reperfusion (IR) group, an IR+Q group, an IR+Q-MSNs group, and an MSNs group (each n = 10). Rats in the sham operation group were not treated with ischemia reperfusion, but given normal perfusion meantime. Rats in the sham operation control group, IR group, and MSNs group were given normal saline for 10 days before ischemia reperfusion, and rats in the IR+Q group and IR+Q-MSNs group were given drugs by gavage for 10 days before ischemia reperfusion. Primary myocardial cells were sampled from SD neonatal rats to construct hypoxia/reoxygenation myocardial cell models. The myocardial cells were assigned to a control group, IR group, quercetin (Q) group, Q-MSNs group, and MSNs group. Except for the control group, all the other groups were treated with hypoxia/reoxygenation. Cells in the Q group were treated with quercetin (10 µM, 20 µM, 40 µM) for 24 h in advance and then treated with measures to cause hypoxia-reoxygenation injury. Cells in the Q-MSNs group were treated with the same concentration of loaded quercetin and the same method used for the Q group. The myocardial apoptosis, myocardial infarction, ventricular remodeling, hemodynamic indexes, physiological and biochemical indexes, and JAK2/STAT3 pathway expression of each group were detected, and the apoptosis, viability, oxidative stress, and JAK2/STAT3 pathway expression of primary myocardial cells in each group were also detected. RESULTS: Quercetin significantly activated the JAK2/STAT3 pathway in vivo and in vitro, and MSNs intensified the activation. Compared with quercetin, Q-MSNs were more effective in inhibiting cell apoptosis and oxidative stress, reducing myocardial infarction size, improving ventricular remodeling and cardiac function-related biochemical indexes, and promoting the recovery of cardiac blood flow. CONCLUSION: Q-MSNs can significantly enhance the activation effect of quercetin on JAK2/STAT3 pathway, thus enhancing its protection on the heart of MIRI rats.

ASIC1a regulates miR-350/SPRY2 by N6 -methyladenosine to promote liver fibrosis.

As a reversible scar repair reaction, liver fibrosis can be blocked or even reversed by proper intervention during its formation. Our work suggests that acid-sensitive ion channel 1a (ASIC1a) participates in liver fibrosis and presents a novel mechanism involving m6 A modification and miR-350/SPRY2. We demonstrated that the expression of ASIC1a was significantly increased in liver tissue of patients with liver fibrosis and animal models of liver fibrosis, as well as PDGF-BB-induced activated HSC-T6. After downregulating the expression of ASIC1a, the degree of liver fibrosis is reduced and HSC activation was inhibited, the level of m6 A modification and miR-350 expression were also reduced. The results of dual luciferase reporter assay showed that miR-350 can bind to the target gene SPRY2 and inhibit its expression. We also found that METTL3 can regulate the extent of m6 A modification of pri-miR-350 by binding to DGCR8. In addition, silencing or blocking the expression of ASIC1a can reduce the expression of PI3K/AKT and ERK signaling pathway-related proteins in activated HSCs. Taken together, we demonstrated that ASIC1a regulates the processing of miR-350 through METTL3-dependent m6 A modification, and mature miR-350 targets SPRY2 and further promotes liver fibrosis through the PI3K/KT and ERK pathways.

Alterations of gut microbiota contribute to the progression of unruptured intracranial aneurysms.

Unruptured intracranial aneurysm (UIA) is a life-threatening cerebrovascular condition. Whether changes in gut microbial composition participate in the development of UIAs remains largely unknown. We perform a case-control metagenome-wide association study in two cohorts of Chinese UIA patients and control individuals and mice that receive fecal transplants from human donors. After fecal transplantation, the UIA microbiota is sufficient to induce UIAs in mice. We identify UIA-associated gut microbial species link to changes in circulating taurine. Specifically, the abundance of Hungatella hathewayi is markedly decreased and positively correlated with the circulating taurine concentration in both humans and mice. Consistently, gavage with H. hathewayi normalizes the taurine levels in serum and protects mice against the formation and rupture of intracranial aneurysms. Taurine supplementation also reverses the progression of intracranial aneurysms. Our findings provide insights into a potential role of H. hathewayi-associated taurine depletion as a key factor in the pathogenesis of UIAs.

Exosomal miR-320d derived from adipose tissue-derived MSCs inhibits apoptosis in cardiomyocytes with atrial fibrillation (AF).

MicroRNAs (miRNAs) play a key role in various pathological processes like atrial fibrillation (AF), which is a common cardiac arrhythmia. Exosomes are essential information carrier in the intercellular communication. Therefore, this study aimed to investigate the effects of exosomal miR-320d on cardiomyocytes with AF and related mechanisms. To do this, AMSCs were transfected with miR-320d mimics, AMSCs-derived exosomes were co-cultured with cardiomyocytes with AF. MTT, TUNEL staining, flow cytometry, real-time PCR, western blots, and luciferase reporter assays were performed. The results revealed that miR-320d expression was decreased in AF cardiomyocytes. AF increased apoptosis and reduced cell viability in cardiomyocytes. By transfection with miR-320d mimics, the miR-320d level was increased in AMSCs, exosomes and cardiomyocytes, which reversed the effect of AF on cardiomyocytes. STAT3 was down-regulated in AF cardiomyocytes and was a direct target gene of miR-320d. Inhibition of STAT3 abolished the effect of modified exosomes in cardiomyocytes, causing decreased apoptosis and increased cell viability. Taken together, the results suggested that exosomal miR-320d was associated with AF cardiomyocytes apoptosis and cell viability and that the effect of miR-320d on cardiomyocytes is STAT3-dependent. Therefore, this study provides a novel understanding of the molecular basis of AF and provides insight into therapeutic strategies for AF.

Correlation between HCN4 gene polymorphisms and lone atrial fibrillation risk.

Background and objective: Atrial electrical remodelling (AER) was significantly associated with atrial fibrillation (AF) development. Polymorphisms in hyperpolarization activated cyclic nucleotide gated potassium channel 4 (HCN4) gene might be correlated with AER. In the present study, we explored the association of HCN4 polymorphisms (rs498005 and rs7164883) with lone AF risk in a Chinese Han population. Methods: In this case-control study, the Sanger sequencing method was utilized to genotype the HCN4 polymorphisms. Relative risk of AF was assessed by the χ2 test, and presented by odds ratios (ORs) and corresponding 95% confidence intervals (CIs). Logistic regression analysis was performed for multivariate analysis. The effects of HCN4 polymorphisms on AF clinical features were analyzed by the Mann-Whitney U test and adjusted by the Bonferroni method. Results: C allele of rs498005 was significantly correlated with increased risk of AF (OR = 1.412, 95%CI = 1.012-1.970), and the association still exited after adjustment by age, gender, the status of smoking and drinking, histories of diabetes, hyperlipidaemia and myocardial infarction (adjusted OR = 1.473, 95%CI = 1.043-2.081). G allele of rs7164883 SNP was marginally associated with enhanced AF risk after adjustment by the above clinical parameters (adjusted OR = 1.742, 95%CI = 1.019-2.980). Atrial late potential (ALP), including TP (P wave duration after filtering) and LP20 (the amplitude of superimposed potential in the final 20 ms of P wave) were significantly associated with rs498005 genotype (p < .001). Conclusion: HCN4 rs498005 and rs7164883 polymorphisms are significantly associated with AF risk.

PI3-kinase/Akt pathway-regulated membrane transportation of acid-sensing ion channel 1a/Calcium ion influx/endoplasmic reticulum stress activation on PDGF-induced HSC Activation.

Acid-sensing ion channel 1a (ASIC1a) allows Na+ and Ca2+ flow into cells. It is expressed during inflammation, in tumour and ischaemic tissue, in the central nervous system and non-neuronal injury environments. Endoplasmic reticulum stress (ERS) is caused by the accumulation of misfolded proteins that interferes with intracellular calcium homoeostasis. Our recent reports showed ASIC1a and ERS are involved in liver fibrosis progression, particularly in hepatic stellate cell (HSC) activation. In this study, we investigated the roles of ASIC1a and ERS in activated HSC. We found that ASIC1a and ERS-related proteins were up-regulated in carbon tetrachloride (CCl4 )-induced fibrotic mouse liver tissues, and in patient liver tissues with hepatocellular carcinoma with severe liver fibrosis. The results show silencing ASIC1a reduced the expression of ERS-related biomarkers GRP78, Caspase12 and IREI-XBP1. And, ERS inhibition by 4-PBA down-regulated the high expression of ASIC1a induced by PDGF, suggesting an interactive relationship. In PDGF-induced HSCs, ASIC1a was activated and migrated to the cell membrane, leading to extracellular calcium influx and ERS, which was mediated by PI3K/AKT pathway. Our work shows PDGF-activated ASIC1a via the PI3K/AKT pathway, induced ERS and promoted liver fibrosis progression.

A comparative study on the quality of bio-oil derived from green macroalga Enteromorpha clathrata over metal modified ZSM-5 catalysts.

The green macroalga Enteromorpha clathrata was pyrolyzed with or without catalysts at the temperature of 550 °C for producing high-quality bio-oil. The ZSM-5 and 1,2,3 mmol Mg-Ce/ZSM-5 catalysts were introduced to investigate the yields and components distribution of bio-oil. Increase of bio-oil production was obtained with the use of ZSM-5 and 1,2,3 mmol Mg-Ce/ZSM-5 catalysts. The 1 mmol Mg-Ce/ZSM-5 catalyst exhibited more promising property for promoting the relative content of C5-C7 compounds, and decreasing the relative content of acids in bio-oil. The results suggested that E. clathrata had potential as pyrolysis feedstocks for producing the high-quality bio-oil with large amounts of C5-C7 compounds and low relative content of acids when the 1 mmol Mg-Ce/ZSM-5 catalyst was used. Furthermore, the physicochemical properties of ZSM-5 and 1 mmol Mg-Ce/ZSM-5 catalysts were investigated by scanning electron microscopy, transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy and temperature-programmed desorption of ammonia.

Co-pyrolysis mechanism of seaweed polysaccharides and cellulose based on macroscopic experiments and molecular simulations.

Co-pyrolysis conversion of seaweed (Enteromorpha clathrat and Sargassum fusiforme) polysaccharides and cellulose has been investigated. From the Py-GC/MS results, Enteromorpha clathrata (EN) polysaccharides pyrolysis mainly forms furans; while the products of Sargassum fusiforme (SA) polysaccharides pyrolysis are mainly acid esters. The formation mechanisms of H2O, CO2, and SO2 during the pyrolysis of seaweed polysaccharides were analyzed using the thermogravimetric-mass spectrometry. Meanwhile the pyrolysis of seaweed polysaccharide based on the Amber and the ReaxFF force fields, has also been proposed and simulated respectively. The simulation results coincided with the experimental results. During the fast pyrolysis, strong synergistic effects among cellulose and seaweed polysaccharide molecules have been simulated. By comparing the experimental and simulation value, it has been found that co-pyrolysis could increase the number of molecular fragments, increase the pyrolysis conversion rate, and increase gas production rate at the middle temperature range.

Control of hydroxyapatite coating by self-assembled monolayers on titanium and improvement of osteoblast adhesion.

Self-assembly technique was applied to introduce functional groups and form hydroxyl-, amine-, and carboxyl-terminal self-assembled monolayers (SAMs). The SAMs were grafted onto titanium substrates to obtain a molecularly smooth functional surface. Subsequent hydrothermal crystal growth formed homogeneous and crack-free crystalline hydroxyapatite (HA) coatings on these substrates. AFM and XPS were used to characterize the SAM surfaces, and XRD, SEM, and TEM were used to characterize the HA coatings. Results show that highly crystalline, dense, and oriented HA coatings can be formed on the OH-, NH2 -, and COOH-SAM surfaces. The SAM surface with -COOH exhibited stronger nucleating ability than that with -OH and -NH2 . The nucleation and growth processes of HA coatings were effectively controlled by varying reaction time, pH, and temperature. By using this method, highly crystalline, dense, and adherent HA coatings were obtained. In addition, in vitro cell evaluation demonstrated that HA coatings improved cell adhesion as compared with pristine titanium substrate. The proposed method is considerably effective in introducing the HA coatings on titanium surfaces for various biomedical applications and further usage in other industries. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 124-135, 2017.

C23 promotes tumorigenesis via suppressing p53 activity.

C23 is an abundant and multi-functional protein, which plays an important role in various biological processes, including ribosome biogenesis and maturation, cell cycle checkpoints and transcriptional regulation [1, 2]. However, the role of C23 in controlling tumorigenesis has not been well defined. Here we report that C23 is highly expressed in cancer cells and the elevated expression of C23 facilitates cancer cell proliferation in vitro and tumor xenograft growth in vivo. Notably, C23 binds to p53 through its GAR domain and suppresses the transcriptional activity of p53 under DNA damage and hypoxia. Moreover, the GAR domain is critical for C23-mediated tumor cell proliferation both in vitro and in vivo. Our findings reveal a novel role of C23 in tumorigenesis and suggest that C23 may represent a potential therapeutic target for treating malignancy.

Leaf Chlorophyll Content Estimation of Winter Wheat Based on Visible and Near-Infrared Sensors.

The leaf chlorophyll content is one of the most important factors for the growth of winter wheat. Visual and near-infrared sensors are a quick and non-destructive testing technology for the estimation of crop leaf chlorophyll content. In this paper, a new approach is developed for leaf chlorophyll content estimation of winter wheat based on visible and near-infrared sensors. First, the sliding window smoothing (SWS) was integrated with the multiplicative scatter correction (MSC) or the standard normal variable transformation (SNV) to preprocess the reflectance spectra images of wheat leaves. Then, a model for the relationship between the leaf relative chlorophyll content and the reflectance spectra was developed using the partial least squares (PLS) and the back propagation neural network. A total of 300 samples from areas surrounding Yangling, China, were used for the experimental studies. The samples of visible and near-infrared spectroscopy at the wavelength of 450,900 nm were preprocessed using SWS, MSC and SNV. The experimental results indicate that the preprocessing using SWS and SNV and then modeling using PLS can achieve the most accurate estimation, with the correlation coefficient at 0.8492 and the root mean square error at 1.7216. Thus, the proposed approach can be widely used for winter wheat chlorophyll content analysis.

Reciprocal actions of microRNA-9 and TLX in the proliferation and differentiation of retinal progenitor cells.

Recent research has demonstrated critical roles of a number of microRNAs (miRNAs) in stem cell proliferation and differentiation. miRNA-9 (miR-9) is a brain-enriched miRNA. Whether miR-9 has a role in retinal progenitor cell (RPC) proliferation and differentiation remains unknown. In this study, we show that miR-9 plays an important role in RPC fate determination. The expression of miR-9 was inversely correlated with that of the nuclear receptor TLX, which is an essential regulator of neural stem cell self-renewal. Overexpression of miR-9 downregulated the TLX levels in RPCs, leading to reduced RPC proliferation and increased neuronal and glial differentiation, and the effect of miR-9 overexpression on RPC proliferation and differentiation was inhibited by the TLX overexpression; knockdown of miR-9 resulted in increased TLX expression as well as enhanced proliferation of RPCs. Furthermore, inhibition of endogenous TLX by small interfering RNA suppressed RPC proliferation and promoted RPCs to differentiate into retinal neuronal and glial cells. These results suggest that miR-9 and TLX form a feedback regulatory loop to coordinate the proliferation and differentiation of retinal progenitors.

Bone marrow mesenchymal stem cells stimulate proliferation and neuronal differentiation of retinal progenitor cells.

During retina development, retinal progenitor cell (RPC) proliferation and differentiation are regulated by complex inter- and intracellular interactions. Bone marrow mesenchymal stem cells (BMSCs) are reported to express a variety of cytokines and neurotrophic factors, which have powerful trophic and protective functions for neural tissue-derived cells. Here, we show that the expanded RPC cultures treated with BMSC-derived conditioned medium (CM) which was substantially enriched for bFGF and CNTF, expressed clearly increased levels of nuclear receptor TLX, an essential regulator of neural stem cell (NSC) self-renewal, as well as betacellulin (BTC), an EGF-like protein described as supporting NSC expansion. The BMSC CM- or bFGF-treated RPCs also displayed an obviously enhanced proliferation capability, while BMSC CM-derived bFGF knocked down by anti-bFGF, the effect of BMSC CM on enhancing RPC proliferation was partly reversed. Under differentiation conditions, treatment with BMSC CM or CNTF markedly favoured RPC differentiation towards retinal neurons, including Brn3a-positive retinal ganglion cells (RGCs) and rhodopsin-positive photoreceptors, and clearly diminished retinal glial cell differentiation. These findings demonstrate that BMSCs supported RPC proliferation and neuronal differentiation which may be partly mediated by BMSC CM-derived bFGF and CNTF, reveal potential limitations of RPC culture systems, and suggest a means for optimizing RPC cell fate determination in vitro.

Effects of a miR-31, Runx2, and Satb2 regulatory loop on the osteogenic differentiation of bone mesenchymal stem cells.

Recently, a cohort of miRNAs, including miR-31, was reported to be downregulated during osteogenic induction by miR microarray analysis. It remains unclear how changes in miR-31 expression collaborate with bone transcription factors to activate the biological pathways that regulate the differentiation of bone mesenchymal stem cells (BMSCs). Here the effects of miR-31, Runx2, and Satb2 on the osteogenic differentiation of BMSCs were investigated using mimics and inhibitors of miR-31, small interfering RNA for knockdown of Runx2 and plasmids for overexpression of Runx2. Our results showed that miR-31 expression decreased progressively in BMSC cultures during differentiation. Inhibition of miR-31 dramatically increased the alkaline phosphatase activity and mineralization in BMSC cultures. Additionally, miR-31 diminished the levels of the Satb2 protein without significantly affecting Satb2 mRNA levels, and Runx2 directly repressed miR-31 expression. Overexpression of miR-31 significantly reduced expression of the osteogenic transcription factors OPN, BSP, OSX, and OCN, but not Runx2. Furthermore, the high expression of miR-31 in BMSCs cultured in the proliferation medium repressed Satb2 protein levels, which may contribute to the maintenance of BMSCs in an undifferentiated state. In conclusion, our results suggest that a Runx2, Satb2, and miR-31 regulatory mechanism may play an important role in inducing BMSC osteogenic differentiation. The results of this study provide us with a better understanding of the molecular mechanisms that govern the BMSC fate.

An in vitro comparison study: the effects of fetal bovine serum concentration on retinal progenitor cell multipotentiality.

Retinal progenitor cells (RPCs) are an excellent resource for retinal replacement therapy, because they show enormous potential to differentiate into retinal-specific cell types. While the differentiating influence of serum has long been appreciated, the effects of serum concentration on RPC differentiation into specified retinal neural cells have not been investigated. Using cultured murine RPCs, this study compared the effects of different levels of fetal bovine serum (FBS) (1%, 5%, 10% and 20%) on RPC differentiation in vitro. RPC multipotentiality was assessed by using quantitative polymerase chain reaction (qPCR) to determine the relative expression levels of 10 genes involved in retinal development. In addition, analyses of cell morphology and retinal development-related protein expression were performed using microscopy and immunocytochemistry. The data revealed that 1% FBS-induced cultures preferentially generated rhodopsin- and PKC-α-positive cells. Calbindin and AP2α expression levels were greater in 5% FBS-induced cultures. Brn3a was expressed at similar levels in 1%, 5% and 10% FBS treatment conditions but diminished in 20% FBS conditions. Twenty percent FBS induced more glial fibrillary acid protein (GFAP)-immunoreactive cells corresponding to glia populations. These findings suggest that the concentration of FBS plays an important role in RPC differentiation in vitro. Treatment with low levels of FBS favors differentiation of rhodopsin-positive photoreceptors, interneurons and retinal ganglion cells (RGCs), while high FBS concentrations preferentially induce differentiation of glia cells. These results are expected to facilitate research in the treatment of neurodegenerative retinal diseases.