bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of expression of HGF in BM‐MSCs by baculovirus‐mediated transduction
Authors: Tu Yi Ji , Ye Ai Fang , Pan Zhi Min , Zheng Chao , Wu Tian Long , Cheng Xi Gao , Guo Fei
Published: August 2013

Bone marrow‐derived mesenchymal stem cells (BM‐MSCs) transplantation is widely adopted for the curing of osteonecrosis of femoral head (ONFH) in recent years. Furthermore, it is known that introducing hepatocyte growth factor (HGF) into BM‐MSCs will greatly improve the therapeutic effect of stem‐cell therapy owing to the great angiogenic and anti‐fibrotic capabilities of HGF. However, continuing overexpression of HGF in vivo may cause sarcomas, such as Kaposi's sarcoma. Aiming at enhancing the therapeutic effect and preventing the side effects of HGF‐modified stem‐cell transplantation on ONFH, we sought to construct a gene regulation system to control HGF expression in BM‐MSCs rigorously and accurately. We selected baculovirus as the gene vector and introduced pTet‐on advanced system into that. Finally, a virus vector vAcrtTA2s‐Ptight‐HGF was successfully built and delivered into BM‐MSCs to regulate the accurate expression of HGF. As shown in the results, different levels of HGF expression were verified by ELISA and Western blot with different induction doses of doxycycline (DOX). There was a dose–response relationship between them, and the optimum dose of DOX to induce HGF expression in BM‐MSCs in vitro was 1 µg/mL. We conclude that it is feasible to regulate HGF expression in BM‐MSCs by baculovirus‐mediated one‐off transduction.
Differential activation and inhibition of RhoA by fluid flow induced shear stress in chondrocytes
Authors: Wan Qiaoqiao , Kim Seung Joon , Yokota Hiroki , Na Sungsoo
Published: August 2013

Physical force environment is a major factor that influences cellular homeostasis and remodelling. It is not well understood, however, as a potential role of force intensities in the induction of cellular mechanotransduction. Using a fluorescence resonance energy transfer‐based approach, we asked whether activities of GTPase RhoA in chondrocytes are dependent on intensities of flow‐induced shear stress. We hypothesized that RhoA activities can be either elevated or reduced by selecting different levels of shear‐stress intensities. The result indicates that C28/I2 chondrocytes have increased RhoA activities in response to high shear stress (10 or 20 dyn/cm2), whereas a decrease in activity was seen with an intermediate shear stress of 5 dyn/cm2. No changes were seen under low shear stress (2 dyn/cm2). The observed two‐level switch of RhoA activities is closely linked to the shear‐stress‐induced alterations in actin cytoskeleton and traction forces. In the presence of constitutively active RhoA (RhoA‐V14), intermediate shear stress suppressed RhoA activities, while high shear stress failed to activate them. In chondrocytes, expression of various metalloproteinases is, in part, regulated by shear and normal stresses through a network of GTPases. Collectively, the data suggest that intensities of shear stress are critical in differential activation and inhibition of RhoA activities in chondrocytes.
Effects of frozen timing on the spindle density, the angle between the polar body and spindle, and embryo development of intracytoplasmic sperm injection in mouse mature oocytes
Authors: Song Wen‐yan , Xin Zhi‐min , Jin Hai‐xia , Peng Zhao‐feng , Chen Xue‐mei , Shi Sen‐lin , Dai Shan‐jun , Sun Ying‐pu
Published: August 2013

Better pregnancy outcomes can be obtained by human mature oocyte vitrification, but many problems remain to be resolved in human mature oocyte vitrification. Since mature oocyte development possesses its own maturity cycle, there should be the optimal timing for mature oocyte vitrification. The purpose of this study was to observe the effects of frozen timing on the spindle density, the angle between the polar body and spindle, and embryo development of intracytoplasmic sperm injection (ICSI) in vitrified mouse mature oocytes and explore its possible mechanism. Mouse oocytes were randomly divided into three groups according to different frozen timing including Groups A, B, and C in which oocytes were vitrified within 2 h after ovum pick‐up, and 3–4 and 5–6 h after ovum pick‐up, respectively. Spindle‐related parameters were measured, ICSI was performed. The spindle occurrence rate of vitrified‐thawed oocytes was 98.4% in Group A, 82.3% in Group B, and 75.8% in Group C, without statistical differences between pre‐vitrification and post‐thawing and among the three groups (P > 0.05). The angles between the polar body and spindle were larger after thawing than before vitrification (P < 0.01). The spindle retardance values were lower after thawing than before vitrification in Groups B and C (P < 0.05), but higher in Group A (P < 0.05). The spindle retardance values before vitrification were higher in Group B than in Groups A and C (P < 0.05), but the spindle retardance value, oocyte survival and two‐cell rate after thawing were higher in Group A than in Groups B and C (P < 0.05). There were no statistical differences in ICSI fertility rate between the three groups (P > 0.05). The damage on the spindle is the slightest and embryo quality is the highest in the mouse oocytes vitrified within 2 h after ovum pick‐up. The spindle retardance value is more valuable than the spindle occurrence rate in the evaluation of vitrified‐thawed oocyte quality, and is positively correlated with embryo quality.
Integrin alpha v beta 6 contributes to maintaining corneal epithelial barrier function
Authors: Guo Huiling , Du Gaiping , Wang Liqiang , Wang Dajiang , Hu Lianna , Huang Yifei
Published: August 2013

The corneal epithelial barrier dysfunction is associated with a number of disorders of the cornea. This study aims to investigate the role of integrin alpha v beta 6 (avb6) in maintaining the corneal epithelial barrier function. In this study, the association of avb6 and endosome/lysosome in the human corneal epithelial cell line, HCE cells, was observed with immunocytochemistry. The corneal epithelial barrier function was assessed with HCE monolayers in Transwells. The results showed that avb6 was observed attaching to endosomes in HCE cells. Knockdown of avb6 resulted in disturbing the fusion of endosome/lysosome. Tumor necrosis factor (TNF)‐α could bind avb6 to form a complex that interfered with the fusion of endosome/lysosome in HCE cells. With the HCE monolayer as a corneal epithelial barrier model, the knockdown of avb6 or exposure to TNF‐α markedly increased the epithelial barrier permeability to a macomolecular protein, ovalbumin, in Transwell system. After passing through the deficient epithelial barrier, the ovalbumin molecules still conserved the antigenicity. We conclude that the integrin avb6 plays an important role in the fusion of endosome/lysosome in corneal epithelial cells; inhibition of avb6 results in corneal epithelial barrier dysfunction.
Effect of substrate stiffness on the osteogenic differentiation of bone marrow stem cells and bone‐derived cells
Authors: Witkowska‐Zimny Malgorzata , Walenko Katarzyna , Wrobel Edyta , Mrowka Piotr , Mikulska Agnieszka , Przybylski Jacek
Published: August 2013

There is a profound dependence of cell behaviour on the stiffness of its microenvironment. To gain a better understanding of the regulation of cellular differentiation by mechanical cues, we investigated the influence of matrix stiffness (E = 1.46 kPa and E = 26.12 kPa) on differentiated osteogenic cell lineage of bone marrow stem cells (BM‐MSCs) and bone‐derived cells (BDCs) using flexible collagen‐coated polyacrylamide substrates. Differentiation potential was determined by measuring alkaline phosphatase activity, expression of osteoblast‐specific markers including alkaline phosphatase, osteocalcin, Runx2 and collagen type I, as well as assessment of mineralisation (Alizarin Red S staining). We found that osteogenic differentiation can be regulated by the rigidity of the substrate, which may depend on the commitment in multi‐ or uni‐potent targeting cells. Osteogenic differentiation of BM‐MSCs was enhanced on a stiff substrate compared to a soft one, whereas BDCs osteogenic differentiation did not vary depending on the substrate stiffness. The data help in understanding the role of the external mechanical determinants in stem cell differentiation, and can also be useful in translational approach in functional tissue engineering.
Functional impact of cholesterol sequestration on actin cytoskeleton in normal and transformed fibroblasts
Authors: Chubinskiy‐Nadezhdin Vladislav I. , Efremova Tatiana N. , Khaitlina Sofia Y. , Morachevskaya Elena A.
Published: August 2013

Membrane cholesterol and lipid rafts are implicated in various signalling processes involving actin rearrangement in living cells. However, functional link between raft integrity and organisation of cytoskeleton remains unclear. We have compared the effect of cholesterol sequestration on F‐actin structures in normal and transformed fibroblasts in which microfilament system is developed to a different extent. The depletion of membrane cholesterol by methyl‐beta‐cyclodextrin (MbCD) resulted in a disruption of lipid rafts in plasma membrane as it was revealed by fluorescent labelling of GM1 ganglioside. In normal fibroblasts with highly developed microfilament system, the cholesterol depletion resulted in actin disassembly and reduction of stress fibres. However, in transformed cells containing low amount of fibrillar actin, MbCD treatment induced intensive formation of stress fibres and increased cell spreading. The results show that the effect of cholesterol depletion and lipid raft disruption on microfilament system is critically determined by the initial state of cytoskeleton, specifically, by the balance of polymerised and monomeric actin in the cell. We assume that uncapping of the microfilaments is the key step of cholesterol‐regulated actin remodelling.
Computational prediction and characterisation of ubiquitously expressed new splice variant of Prkaca gene in mouse
Authors: Banday Abdul Rouf , Azim Shafquat , Hussain Mohammed Aamir , Nehar Shamshun , Tabish Mohammad
Published: August 2013

Prkaca gene of mouse encodes for a cAMP dependent protein kinase catalytic alpha subunit. PKA occurs naturally as a 4‐membered structure having two regulatory (R) and two catalytic (C) subunits each encoded by separate gene. Alternatively spliced two transcript variants are known for the Prkaca gene, which encode for two isoforms of PKA C‐subunits, namely Cα1 and Cα2. These isoforms arise as a result of alternative splicing of the first coding exon with the internal exons. We have identified a new transcript variant using combinatorial approach of bioinformatics and molecular biology techniques involving RT‐PCR, semi‐nested PCR and sequencing. The new transcript variant encoding Cα3 isoform has N‐terminus that differs from Cα1 and Cα2 isoforms. Cα3 isoform also arise as a result of alternative splicing of first coding exon with the internal exon. Newly identified transcript is expressed ubiquitously in different tissues examined.
Evidence that TMEM67 causes polycystic kidney disease through activation of JNK/ERK‐dependent pathways
Authors: Du E , Li Hong , Jin Shunying , Hu Xuemei , Qiu Mengsheng , Han Ruifa
Published: August 2013

TMEM67 mutations are associated with severe autosomal recessive polycystic kidney disease (ARPKD) in both humans and animals. However, the molecular mechanisms underlying the pathogenesis of PKD caused by TMEM67 mutations remain to be determined. We have investigated the possible signalling pathways involved in the pathogenesis of PKD. Overexpression of TMEM67 in human embryonic kidney (HEK293) cells triggered the activation of overall tyrosine phosphorylated proteins, extracellular signal‐regulated kinase (ERK) and c‐jun N‐terminal KINASE (JNK). Activation was suppressed by pharmacological inhibitors of ERK or JNK. Activation of the mammalian target of rapamycin (mTOR) or p70s kinase (S6K) did not occur, although elevated phosphorylation of eIF4E‐binding protein 1 (4E‐BP1), a target of S6K, was seen. In animal studies, activation of a variety of signalling molecules was linked to ERK, JNK and 4E‐BP1. Significant induction of phosphorylation of tyrosine phosphorylated proteins, ERK and 4E‐BP1, at different postnatal ages was detected in mutant kidneys of B6C3Fe a/a‐bpck mice, a cystic renal disease mouse model caused by TMEM67 loss of function mutation. Based on these in vitro and in vivo observations, we propose that TMEM67 mutations cause PKD through ERK‐ and JNK‐dependent signalling pathways, which may provide novel insight into the therapy of polycystic kidney diseases.
Intratracheal transplantation of bone marrow‐derived mesenchymal stem cells reduced airway inflammation and up‐regulated CD4+CD25+ regulatory T cells in asthmatic mouse
Authors: Ge Xiahui , Bai Chong , Yang Jianming , Lou Guoliang , Li Qiang , Chen Ruohua
Published: August 2013

Mesenchymal stem cells attenuate the severity of lung injury due to their immunomodulatory properties. The effect of bone marrow‐derived mesenchymal stem cells on asthma is seldom reported. We have examined the effect of BMSCs on airway inflammation in asthma. Forty female BALB/c mice were equally randomised into PBS group, BMSCs treatment group, BMSCs control group and asthmatic group. Reactivity of the airway to acetylcholine was measured by barometric plethysmography. Cytokine profiles of bronchoalveolar lavage fluid and serum were determined by enzyme‐linked immunosorbent assay. Morphometric analysis was done with haematoxylin and periodic‐acid Schiff staining. Engraftment of BMSCs in asthmatic mice significantly decreased the number of eosinophils and mononuclear cells in bronchoalveolar lavage fluid and the airway (P < 0.05). Both goblet cell hyperplasia and responsiveness to acetylcholine were significantly reduced in BMSCs treatment groups. Moreover, BMSCs engraftment caused significant increases the ratio of Treg in pulmonary lymph node and interleukin‐10 (IL‐10) and interleukin‐12 levels in BALF and serum. We conclude that BMSCs engraftment ameliorated airway inflammation and improved lung function in asthmatic mouse and the protective effect might be mediated by upregulating Treg and partly involved with increasing IL‐10.
Cold exposure lowers energy expenditure at the cellular level
Authors: Park Seyeon , Chun Sohyun , Kim Danuh
Published: August 2013

Mitochondrial function is intimately involved in various metabolic processes and is therefore essential to maintain cell viability. Of particular importance is the fact that mitochondrial membrane potential (ΔΨm) is coupled with oxidative phosphorylation to drive adenosine triphosphate (ATP) synthesis. We have examined the effects of cold temperature stress on ΔΨm and the role of cold temperature receptor expression on ΔΨm. Human bronchial endothelial cell line, BEAS‐2B, and human embryonic kidney, HEK293, cell line were transfected with the gene for cold temperature responsive receptor protein TRPM8 or TRPA1, and exposed to cold temperature. ΔΨm was monitored using 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazoyl carbocyanine iodide derivative (JC‐10), a ΔΨm probe. While cold temperatures significantly increased ΔΨm and mitochondrial ATP levels in cells transfected with temperature responsive receptor TRPM8 or TRPA1, no change was noted in wild‐type cells. Moreover, the change in ΔΨm and ATP level was a dynamic process. ΔΨm was raised to peak levels within10 min of cold exposure, followed by a return to baseline levels at 30 min. Our findings suggest that cold temperature exposure increased mitochondrial ΔΨm via a mechanism involving cold temperature receptors.
Altered VEGF‐A and receptor mRNA expression profiles, and identification of VEGF144 in foetal rat calvaria cells, in coculture with microvascular endothelial cells
Authors: Weyand Birgit , von Schroeder Herbert P.
Published: August 2013

Cellular proliferation and differentiation during angiogenesis and osteogenesis require the communication of different cell types through growth factors and their receptors. Vascular endothelial growth factor (VEGF‐A) plays an important role in osteoblast and endothelial cell intercommunication. We have investigated the effect of monocultures and indirect coculture of foetal rat calvarial (FRC) osteoblasts and microvascular endothelial cells (ECs) on nodule formation, proliferation, and mRNA‐expression of VEGF‐A and its receptors during culturing. Despite increased nodule formation in the presence of dexamethasone (Dex) in monocultures, the number of nodules and alkaline phosphatise activity were decreased in cocultured FRCs. VEGF mRNA expression over the differentiation period showed the expression of most Vegf isoforms is biphasic in both FRCs and ECs, whereas receptor expression was quite variable; however, that of Np‐2 in FRCs increased steadily and significantly from 8 h to 14 days after an initial drop in expression. Significant changes in the proportion of Vegfa by Day 14 were noted mainly in the matrix‐bound variants Vegf144 and Vegf188 in ECs and osteoblasts, respectively. Less striking results were seen in the expression of the soluble isoforms in either cell type. These results have identified expression of Vegf144 in osteoblasts, suggesting a possible autocrine and/or paracrine role that is affecting osteoblast mineralisation along with Vegf188, as well as possible early roles of these isoforms in initial cell attachment. Further study of VEGF expression in coculture and Vegf144 will lead to better understanding of its role in cell–cell communication and bone development.
NF‐κBP65 promotes invasion and metastasis of oesophageal squamous cell cancer by regulating matrix metalloproteinase‐9 and epithelial‐to‐mesenchymal transition
Authors: Wang Feng , He Wei , Fanghui Peng , Wang Liuxing , Fan Qingxia
Published: August 2013

NF‐κB has been recognized as one of the factors responsible for the development of cancer; however, the mechanism by which high expression of NF‐κB contributes to the progression of human oesophageal squamous cell cancer (ESCC) is not fully understood. In our investigations, NF‐κBP65 was overexpressed in human ESCC tissues, especially in ESCC tissues with deep invasion and lymph node metastasis. Suppression of NF‐κBP65 by siRNA decreased the invasion and proliferation ability of EC9706 cells in vitro. Furthermore, siRNA‐mediated NF‐κBP65 knock‐down could lead to the downregulation of MMP‐9, a metastasis‐related gene. Reduced E‐cadherin is a hallmark of invasive carcinomas that have acquired epithelial–mesenchymal transition (EMT) phenotypes and Vimentin is another molecule that is used widely as a marker of the EMT. We found upregulation of E‐cadherin expression and downregulation of Vimentin was induced by NF‐κBP65 siRNA, which suggests that NF‐κBP65 siRNA could inhibit the invasion and proliferation ability of ECSS through attenuating the expression of MMP‐9 and EMT. Thus, ESCC NF‐κBP65 could be a useful target for cancer prevention and therapy.
Characteristics of Notch2+ pancreatic cancer stem‐like cells and the relationship with centroacinar cells
Authors: Zhou Zhu‐Chao , Dong Qiang‐Gang , Fu De‐liang , Gong Yi‐yi , Ni Quan‐Xing
Published: August 2013

Notch2, a surface marker in cell lines, is used to isolate, identify and localise pancreatic cancer stem‐like cells and is a target for therapy of these cells. Sphere formation was induced in Panc‐1 and Bxpc‐3 pancreatic cancer cell lines, and Notch2+ cells were separated from Bxpc‐3 and Panc‐1 cell lines by magnetic activated cell sorting (MACS). Expression of stem cell‐related markers, OCT4, Nanog and PDX1, were measured by immunofluorescent (IF) staining. Expression of Notch2 was also determined immunohistochemically in pancreatic tissues. Notch2+ cells were transplanted in subcutaneous of mice. AQP1 and AQP5 were also measured by IF in Bxpc‐3 cells. The Notch signal pathway inhibitor, Compound E (CE), was used to treat Notch2+Bxpc‐3 cells, and their vitalities were subsequently measured by the CCK‐8 method. Positive expression of OCT4, Nanog and PDX1 was observed in Notch2+ cells. Notch2+ cells at centroacinar cell (CAC) and terminal ductal locations expressed AQP1 and AQP5. They were strongly tumourigenic in mice, and CE inhibited proliferation of Notch2+ Bxpc‐3 cells to some degree. OCT4 and Nanog can be used as markers of self‐renewal in pancreatic cancer stem cells. Notch2+ cells in human pancreatic cancer Bxpc‐3 and Panc‐1 cell lines had the properties of cancer stem cells. The results suggest that Notch2+ pancreatic cancer stem‐like cells had a close relationship with CAC.
RNA interference targeting CD147 inhibits the invasion of human cervical squamous carcinoma cells by downregulating MMP‐9
Authors: Fan Xiaobin , Wu Weiguang , Shi Haixia , Han Jianqiu
Published: August 2013

Cervical squamous carcinoma is a highly invasive tumour that has a great capacity to metastasise. Extracellular matrix metalloproteinase inducer (EMMPRIN or CD147), a member of the immunoglobulin superfamily, is a widely distributed cell surface glycoprotein. It is highly expressed on malignant tumour cell surfaces, including human cervical squamous carcinoma. It also plays a critical role in the invasive and metastatic activity of malignant cells by stimulating the expression of matrix metalloproteinases (MMPs). The anti‐invasive effect of small interfering RNA (siRNA) against CD147 on human cervical squamous carcinoma cells and its possible pathways has been investigated. The downregulation of CD147 by transfection with siRNA resulted in MMP‐9 expression and decreased activity in the cervical squamous carcinoma cell line SiHa. In vitro analysis showed that the invasive capacity of SiHa cells decreased. Thus CD147 inhibition and subsequent MMP‐9 deletion may have anti‐tumour effects by inhibiting the invasiveness of human cervical squamous carcinoma cells.
RNAi‐mediated MMP‐9 silencing inhibits mouse melanoma cell invasion and migration in vitro and in vivo
Authors: Tang Zhao‐Yong , Liu Yang , Liu Long‐Xing , Ding Xiao‐Yan , Zhang Hong , Fang Liao‐Qiong
Published: August 2013

MMP‐9 participates in tumour growth, invasion, metastasis and vascularisation. Thus, inhibition of MMP‐9 may be involved in the process of tumourigenesis. We have investigated the effect of RNAi‐mediated MMP‐9 silencing on inhibiting invasion and migration of mouse melanoma cell B16. A specific and optimised siRNA vector was used to target MMP‐9 mRNA synthesis in B16 cells. Changes of invasion and migration capability of B16 cell were examined after transfection at different time, and a footpad tumour model was performed to measure the effect of MMP‐9 siRNA on melanoma tumourigenesis in vivo. In vitro, down‐regulation of MMP‐9 expression significantly inhibited B16 cell invasion and migration. In vivo, intratumoural injection of plasmid DNA expressing MMP‐9 siRNA every 3 days for three times remarkably inhibited melanoma growth and also suppressed tumour metastasis. The results indicate that RNA‐mediated targeting of MMP‐9 may have promising applications for the treatment of melanoma.
New strategies in cord blood cells transplantation
Authors: Yu Xin , Gu Zongjiang , Wang Yunliang , Wang Hongwei
Published: August 2013

For patients lacking a human leucocyte antigen‐matched donor, umbilical cord blood (UCB) is an ideal, alternative source of haematopoietic stem cells (HSCs) for transplantation purposes. UCB has many advantages over bone marrow or peripheral blood taken from volunteer donors. UCB is also an important source of other stem cells, including endothelial progenitors, mesenchymal stem cells, very small embryonic/epiblast‐like (VSEL) stem cells, and unrestricted somatic stem cells, which are potentially suitable for regenerative medicine. However, a significant clinical problem is that the number of HSCs in one cord‐blood unit is not enough for an adult transplantation. The development of new approaches including use of multiple donors, ex vivo expansion, increasing efficiency of homing and engraftment, retrieving more cells from the placenta and cord blood is of crucial importance for the delayed engraftment after UCB transplantation. In the future, UCB will emerge as a source of cells for cellular therapies associated with tissue repair and regeneration.
3D Arrays for high throughput assay of cell migration and electrotaxis
Authors: Zhao Sanjun , Gao Runchi , Devreotes Peter N. , Mogilner Alex , Zhao Min
Published: August 2013

Cell behaviour in 3D environments can be significantly different from those in 2D cultures. With many different 3D matrices being developed and many experimental modalities used to modulate cell behaviour in 3D, it is necessary to develop high throughput techniques to study behaviour in 3D. We report on a 3D array on slide and have adapted this to our electrotaxis chamber, thereby offering a novel approach to quantify cellular responses to electric fields (EFs) in 3D conditions, in different matrices, with different strains of cells, under various field strengths. These developments used Dictyostelium cells to illustrate possible applications and limitations.
Correlation analysis between gene expression profile of high‐fat emulsion‐induced non‐alcoholic fatty liver and liver regeneration in rat
Authors: Wang Gaiping , Li Bing , Hao Yunpeng , Zhi Jia , He Chunxiao , Xu Cunshuan
Published: August 2013

To explore the relevance of non‐alcoholic fatty liver disease (NAFLD) to liver regeneration (LR), rat models of non‐alcoholic steatohepatitis (NASH) and LR were established, respectively, then Rat Genome 230 2.0 Array was used to detect the gene expression abundance of them, and the reliabilities of the array data were confirmed by real‐time RT‐PCR. As a result, the expression of 93 genes was significantly changed during NAFLD occurrence and 948 genes in LR. Hierarchical clustering indicated that the expression profiles of the above two events were quite different. K‐means cluster classified their expression patterns into four clusters, and gene expression trends of clusters 1, 2 were similar in NAFLD and LR, while clusters 3, 4 were contrary with the gene expression changes of LR more abundant. DAVID classifications and functional enrichment analysis found that lipid metabolism and carbohydrate metabolism were stronger in NAFLD than in LR, but some other physiological activities including inflammation/immune response, cell adhesion, and migration, cell proliferation and differentiation in NAFLD were weaker than in LR. IPA further indicated that lipid metabolism, inflammation response, and cellular development were highly associated with NAFLD, and thus identified some potential biomarkers for NAFLD.
Sphere‐forming cells from peripheral cornea demonstrate polarity and directed cell migration
Authors: Yoon Jinny Jung , Wang Ellen F. , Ismail Salim , McGhee Jennifer J. , Sherwin Trevor
Published: August 2013

Sphere‐forming cells from peripheral cornea represent a potential source of progenitor cells for treatment of corneal degenerative diseases. Control of cellular repopulation on transplantable substrates is important to prevent uncontrolled growth in unfavourable directions. The coordination of cellular outgrowth may be in response to environmental cues and/or cellular signals from other spheres. To investigate this, cell migration patterns were observed following placement of spheres on an adhesive surface. Human peripheral corneal cells were maintained using a sphere‐forming assay and their behaviour on collagen substrate recorded by time‐lapse imaging. Immunocytochemistry and proliferation assays were used to detect protein expression and cell division. Proliferation assays showed that spheres formed by a combination of cell division and aggregation. Cell division continued within spheres for up to 4 months and was up‐regulated when exposed to differentiation medium and collagen substrate. The spheres expressed both epithelial and stromal cell markers. When exposed to collagen; (1) 25% of the spheres showed spontaneous polarised outgrowth. (2) One sphere initially showed polarised outgrowth followed by collective migration with discrete morphological changes to form leading and trailing compartments. (3) A sphere which did not show polarised outgrowth was also capable of collective migration using cell protrusion and retraction. (4) Active recruitment of cells into spheres was observed. (5) Placement of spheres in close proximity led to production of a cell exclusion area adjacent to spheres. Thus peripheral corneal cell spheres are dynamic entities capable of developing polarity and modifying migration in response to their environment.
Effects of oleic acid and chicken serum on the expression of adipogenic transcription factors and adipogenic differentiation in hen preadipocytes
Authors: Regassa Alemu , Kim Woo Kyun
Published: August 2013

We have examined the effect of oleic acid (OA) concentrations and incubation time, along with chicken serum (CS), on adipogenic differentiation and expression of adipogenic transcripts in hen preadipocytes. Preadipocytes were treated with (i) an adipogenic cocktail (DMI) containing 500 nM dexamethasone, 0.5 mM 3‐isobutyl‐1‐methylxanthine and 20 µg/mL insulin alone and DMI + 75, 150, 300 or 600 µM OA for 48 h; (ii) DMI + 300 µM OA (DMIOA) for 6, 12, 24 or 48 h; and (iii) foetal bovine serum (FBS), CS, DMI + FBS, DMI + CS, DMIOA + FBS and DMIOA + CS. While FABP4 was significantly expressed with increasing concentrations of OA, the expression of C/EBPβ, LEPR and FAS were unchanged compared with the control. PPARγ2 expression was unchanged across all time‐points. A significantly higher level of C/EBPα was measured at 48 h, but the levels of C/EBPβ increased after 12 h. Levels of FABP4 significantly increased with the time of incubation after 12 h, but that of LPL was reduced (P < 0.05) at 6, 24 and 48 h. FABP4 was highly expressed in cells treated with CS, DMI + CS and DMIOA + CS compared to cells treated with FBS, DMI + FBS and DMIOA + FBS. In conclusion, increased concentrations of OA and incubation time increases lipid accumulation; FABP4 and C/EBPβ are potential transcription factors regulating OA induced adipogenesis of fat cells obtained from laying hen. CS is a potent inducer of adipogenic differentiation in hen preadipocytes.
Mesostoma ehrenbergii spermatocytes – A unique and advantageous cell for studying meiosis
Authors: Ferraro‐Gideon Jessica , Hoang Carina , Forer Arthur
Published: August 2013

Mesostoma ehrenbergii have a unique male meiosis: their spermatocytes have three large bivalents that oscillate for 1–2 h before entering into anaphase without having formed a metaphase plate, have a precocious (‘pre‐anaphase’) cleavage furrow, and have four univalents that segregate between spindle poles without physical interaction between them, that is via ‘distance segregation’. These unique and unconventional features make Mesostoma spermatocytes an ideal organism for studying the force produced by the spindle to move chromosomes, and to study cleavage furrow control and ‘distance segregation’. We review the literature on meiosis in Mesostoma spermatocytes and describe our current research with Mesostoma spermatocytes, rearing the animals in the laboratory using methods that described in our companion article [Hoang et al. (2013); Cell Biol Int].
Corticotropin releasing hormone activates CD14+ cells to induce endothelial barrier dysfunction
Authors: Song Jiang‐Ping , Chen Xiao , Yang Gui , Geng Xiao‐Rui
Published: August 2013

Endothelial barrier dysfunction is associated with the pathogenesis of a number of disorders in the body, but the aetiology is unclear. We have investigated the mechanism of the psychological stress mediator, corticotropin releasing hormone (CRH), on compromising the endothelial barrier function. Human endothelial cell line, Hmvec cells, was cultured in monolayers as a model of endothelial barrier. Human peripheral CD14+ cells were collected to be used as effector immune cells. The transepithelial resistance (TER) and permeability of horseradish peroxidase (HRP) were used as indicators of endothelial barrier function. Apoptosis in Hmvec cells were analysed by flow cytometry. Human CD14+ cells expressed both receptors (R) of CRH, the CRH‐R1 and CRH‐R2. Exposure to CRH induced CD14+ cells to release tumour necrosis factor (TNF)‐α. CRH‐activated CD14+ cells decreased TER and increased the permeability to HRP in co‐cultured Hmvec monolayers. Co‐culture with CRH‐activated CD14+ cells increased the apoptosis in Hmvec cells. We conclude that CRH can activate CD14+ cells to produce TNF‐α and compromise endothelial barrier function by inducing apoptosis of the endothelial cells.
Stem cells conditioned medium: a new approach to skin wound healing management
Authors: Jayaraman Pukana , Nathan Prakash , Vasanthan Punitha , Musa Sabri , Govindasamy Vijayendran
Published: August 2013

Stem cell biology has gained remarkable interest in recent years, driven by the hope of finding cures for numerous diseases including skin wound healing through transplantation medicine. Initially upon transplantation, these cells home to and differentiate within the injured tissue into specialised cells. Contrariwise, it now appears that only a small percentage of transplanted cells integrate and survive in host tissues. Thus, the foremost mechanism by which stem cells participate in tissue repair seems to be related to their trophic factors. Indeed, stem cells provide the microenvironment with a wide range of growth factors, cytokines and chemokines, which can broadly defined as the stem cells secretome. In in vitro condition, these molecules can be traced from the conditioned medium or spent media harvested from cultured cells. Conditioned medium now serves as a new treatment modality in regenerative medicine and has shown a successful outcome in some diseases. With the emergence of this approach, we described the possibility of using stem cells conditioned medium as a novel and promising alternative to skin wound healing treatment. Numerous pre‐clinical data have shown the possibility and efficacy of this treatment. Despite this, significant challenges need to be addressed before translating this technology to the bedside.
The effect of ambroxol on chloride transport, CFTR and ENaC in cystic fibrosis airway epithelial cells
Authors: Varelogianni Georgia , Hussain Rashida , Strid Hilja , Oliynyk Igor , Roomans Godfried M. , Johannesson Marie
Published: August 2013

Ambroxol, a mucokinetic anti‐inflammatory drug, has been used for treatment of cystic fibrosis (CF). The respiratory epithelium is covered by the airway surface liquid (ASL), the thickness and composition of which is determined by Cl− efflux via the cystic fibrosis transmembrane conductance regulator (CFTR) and Na+ influx via the epithelial Na+ channel (ENaC). In cells expressing wt‐CFTR, ambroxol increased the Cl‐ conductance, but not the bicarbonate conductance of the CFTR channels. We investigated whether treatment with ambroxol enhances chloride transport and/or CFTR and ENaC expression in CF airway epithelial cells (CFBE) cells. CFBE cells were treated with 100 µM ambroxol for 2, 4 or 8 h. mRNA expression for CFTR and ENaC subunits was analysed by real‐time polymerase chain reaction (RT‐PCR); protein expression was measured by Western blot. The effect of ambroxol on Cl− transport was measured by Cl− efflux measurements with a fluorescent chloride probe. Ambroxol significantly stimulated Cl− efflux from CFBE cells (a sixfold increase after 8 h treatment), and enhanced the expression of the mRNA of CFTR and α‐ENaC, and of the CFTR protein. No significant difference was observed in β‐ENaC after exposure to ambroxol, whereas mRNA expression of γ‐ENaC was reduced. No significant effects of ambroxol on the ENaC subunits were observed by Western blot. Ambroxol did not significantly affect the intracellular Ca2+ concentration. Upregulation of CFTR and enhanced Cl− efflux after ambroxol treatment should promote transepithelial ion and water transport, which may improve hydration of the mucus, and therefore be beneficial to CF‐patients.
Insulin facilitates osteoblast differentiation
Authors: Wang Lin , Wang Hongwei , Xu Xia , Wang Dongshen , Liu Hongchen
Published: August 2013

Tissue engineering of bone has been increasingly used in the bone defect repair. To generate osteoblasts is a major approach, and here we have examined ways of improving the efficiency of producing osteoblasts. Adipose stem cells (ADSC) were prepared from rat mesentery tissue, and transfected with Cbfa1 gene vector or/and IGF‐1R gene vector. The cells were stimulated with insulin. Osteocalcin expression by the ADSCs was assessed by quantitative RT‐PCR (qRT‐PCR), Western blotting and enzyme‐linked immunobosorbent assay.

Both genes Cbfa1 and IGF‐1R were transfected in ADSCs, as shown by qRT‐PCR and Western blotting. Stimulation by insulin in the culture increased osteocalcin expression in ADSCs transfected by both Cbfa1 and IGF‐1R, but not in those transfected with only one of these two genes. Osteocalcin in the culture supernatant was also increased by stimulation with insulin.

Thus IGF‐1R gene transfer together with insulin stimulation can markedly increase the efficiency of generation of osteoblasts.
Suppression of adipogenic differentiation by muscle cell‐induced decrease in genes related to lipogenesis in muscle and fat co‐culture system
Authors: Park Sungkwon , Baek Kyunghoon , Choi Changbon
Published: August 2013

Intercellular signalling communication between adipose and muscle tissue has been investigated. To test the effect of muscle cells on adipogenic gene expression, we utilised an in vitro co‐culture system, in which fat (3T3‐L1) and muscle (L‐6) cells were physically separated but chemically exposed each other via insert with 0.4 µm porous membrane. When 3T3‐L1 and L‐6 cells reached at 80 and 40% confluence, respectively in separate wells, L‐6 cells grown in insert were transferred onto 6‐well plates where 3T3‐L1 cells were being grown. When both cells were fully differentiated in co‐culture plates, morphology of 3T3‐L1 was examined by staining with Oil‐red‐O. Activity of glycerol‐3‐phosphate dehydrogenase (GPDH) and adipogenic gene expression including lipoprotein lipase (LPL), adipsin, GPDH, peroxisome proliferator‐activated receptor‐γ (PPARγ) and CCAAT/enhancer binding protein (C/EBPα) were analysed. The presence of muscle cells during preadipocyte differentiation inhibited (P < 0.05) lipogenesis by suppressing lipogenic gene expression including LPL, adipsin and GPDH. Furthermore, GPDH activity was also decreased (P < 0.05) in 3T3‐L1 cells by the presence of L‐6 cells. These results suggest that presence of muscle cells suppresses adipogenic differentiation by inhibiting the adipogenic gene expression and GPDH activity in the muscle and fat cell co‐culture system.
Modulation of physical environment makes placental mesenchymal stromal cells suitable for therapy
Authors: Mathew Suja Ann , Rajendran Sowmya , Gupta Pawan Kumar , Bhonde Ramesh
Published: August 2013

Low level of oxygen at the site of injury is likely to affect the viability and proliferation of the transplanted mesenchymal stromal cells (MSCs). Hence there is a need to understand the effect of the physical environment on transplanted stromal cells. Therefore, we have studied the effect of the duration of hypoxic exposure alone or in combination with normoxia on placenta derived mesenchymal stem cell (PDMSCs). PDMSCs and bone marrow MSCs (BMMSCs) were analysed under four different culture conditions, exposure to direct normoxia (N), direct hypoxia (H) and intermittent normoxia followed by hypoxia (NH) and intermittent hypoxia followed by normoxia (HN). The effect on morphology, proliferation, metabolic activity by MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide) and viability by 7AAD (7‐amino‐actinomycin D) were assayed, along with markers for MSCs and HLADR. No change in morphology, marker expression or HLADR was detected in N, H, NH or HN. An increase in proliferation rate, decrease in population doubling‐time (PDT) and a relative increase in metabolic activity was strongly noted in the order: NH, N/HN and H. No significant difference was observed in the viability between N, H, NH or HN. A similar pattern was also observed in BMMSCS, indicating comparable suitability of PDMSCs in therapeutic applications. Thus we conclude that intermittent exposure to normoxia prior to hypoxic exposure is a better option than direct exposure to hypoxia. This may have clinical relevance in that they probably mirror the in vivo scenario of systemic delivery (NH) of cells as opposed to local delivery (H), thereby suggesting that systemic delivery is better than local delivery.
Design of polyethylene glycol–polyethylenimine nanocomplexes as non‐viral carriers: mir‐150 delivery to chronic myeloid leukemia cells
Authors: Biray Avcı Çığır , Özcan İpek , Balcı Tuğçe , Özer Özgen , Gündüz Cumhur
Published: August 2013

MicroRNAs (miRNAs) are acknowledged as indispensable regulators relevant in many biological processes, and they have been pioneered as therapeutic targets for curing disease. miRNAs are single‐stranded, small (19–22 nt) regulatory non‐coding RNAs whose deregulation of expression triggers human cancers, including leukemias, mainly through dysregulation of expression of leukemia genes. miRNAs can function as tumour suppressors (suppressing malignant potential) or oncogenes (activating malignant potential) like actors of complex diseases. To address the issue of overcoming instability and low transfection efficiency in vitro, the polyethylene glycol–polyethyleneimine (PEG–PEI) nanoparticle was used as non‐viral vector carrier for miR‐150 transfection, which is downregulated in chronic myeloid leukemia. PEG–PEI [PEG(550)3‐g‐PEI(1800)]/miRNA nanocomplexes were synthesised and characterised by particle size distribution (PSD), polydispersity index (PDI) and zeta potential, surface charge, their cytotoxicity, and transfection efficiency. Interaction with human leukemia cells (K‐562 and KU812) and control cells NCI‐BL2347 with them has been investigated. The transfection efficiency of PEG–PEI/miRNA at N/P 26 rose 6.7‐fold above the control by qRT‐PCR. The size of homogenous nanocomplexes (PBI < 0.5) was 160.8 ± 11 nm. The data indicate that PEG–PEI may be an encouraging non‐viral carrier for altering miRNA expression in the treatment of chronic myeloid leukemia, with many advantages such as relatively high miRNA transfection efficiency and low cytotoxicity.
Neuropeptide SP activates the WNT signal transduction pathway and enhances the proliferation of bone marrow stromal stem cells
Authors: Mei Gang , Xia Liheng , Zhou Jian , Zhang Yongtao , Tuo Yonghua , Fu Su , Zou Zhenlv , Wang Zhao , Jin Dan
Published: August 2013

Substance P (SP) mediates multiple activities in various cell types, such as proliferation, anti‐apoptotic response, and inflammation. We have investigated the effects of SP, NK1 antagonist and DKK1 on proliferation of bone marrow stromal stem cells (BMSCs), as well as the underlying mechanism. Isolated BMSCs were exposed to SP (10−8 M) (group A), SP + NK1 antagonist (1 µM) (group B), SP + DKK1 (0.2 µg/mL) (group C), or the same amount of PBS (group D). Expression of gene and protein of Wnt/β‐catenin signalling was detected using quantitative PCR and western blotting. SP (10−8 M) significantly enhanced the proliferation of BMSCs and the number of viable cells was reduced by treatment with NK1 antagonist (1 µM) or DKK1 (0.2 µg/mL). SP also significantly increased the expression of C‐myc mRNA, Lef1, β‐catenin protein and C‐myc protein, but decreased the expression of Tcf7 and p‐β‐catenin protein compared to group D. These roles of SP were inhibited by the NK1 antagonist and DKK1. Expression of CyclinD1 and β‐catenin mRNAs, however, was not significantly influenced by SP, NK1 antagonist and DKK1. These findings suggest that SP enhances BMSC proliferation via regulation of the Wnt/β‐catenin signalling pathway.
Hydrogen Production from Bio‐Char via Steam Gasification in a Fluidized‐Bed Reactor
Authors: Ma Z. , Zhang S. , Xie D. , Yan Y. , Ren Z.
Published: August 2013

Bio‐char was used as feedstock to investigate hydrogen production via steam gasification in a fluidized‐bed reactor. The effects of reaction conditions such as reaction temperature and steam‐to‐char ratio on hydrogen yield, potential hydrogen yield, and carbon conversion were studied. Finally, 83.3 mol hydrogen was generated per kilogram of bio‐char through steam gasification of bio‐char in a fluidized‐bed reactor.
Impact of SO2 in the Presence of Steam on Carbonation and Sulfation of Calcium Sorbents
Authors: Pawlak‐Kruczek H. , Baranowski M. , Tkaczuk‐Serafin M.
Published: August 2013

The effect of the presence of steam and SO2 on CO2 sorption capacity during multicycle carbonation‐calcination tests on three limestone types from Germany, Greece, and Poland was studied. The behavior of carbonation conversion under realistic process atmospheres containing SO2 and additionally steam as well as of sulfation under similar temperature conditions was investigated. The deactivation effect of SO2 on CO2 sorption capacity in two cases with and without steam was analyzed. The negative impact of the competitive sulfation reaction towards carbonation increased in the subsequent cycles for both cases, but to a less extent in the presence of steam. Sulfation conversion changed quite monotonically in the subsequent cycle in dry gas, whereas in the presence of steam sulfation conversion decreased slightly while carbonation conversion increased. It was found for the tested sorbents that carbonation conversion decreased to varying degrees in the presence of SO2. The Polish and Greek limestone showed the largest loss of sorption capacity in the presence of SO2. The quantitative influence of SO2 on carbonation conversion in dry and wet gas was determined.
Genetic algorithm for CoMFA setting optimization: 3D‐QSAR study on α‐aminosuberic acid derivatives as anti‐cancer compounds
Authors: Ebrahimi S. , Azimi G. , Akhlaghi Y. , Kompany‐Zareh M.
Published: August 2013

In most three‐dimensional quantitative structure–activity relationship studies, default SYBYL parameters for comparative molecular field analysis (CoMFA) have been used to derive the models. In this work, a genetic algorithm has been employed for the first time to select the best set of parameters. Three‐dimensional quantitative structure–activity relationship analysis of a set of 33 analogues of α‐aminosuberic acid as a new generation of histone deacetylase inhibitors was performed. Contrary to the ordinary and region focusing CoMFA models, in genetic algorithm optimized model, H‐bond was the preferred field type. Genetic algorithm optimized model showed a better predictive ability (r2pred = 0.982, q2LOO = 0.828, and q2LMO = 0.795) compared with ordinary (r2pred = 0.937, q2LOO = 0.629, and q2LMO = 0.537) and region focusing (r2pred = 0.954, q2LOO = 0.665, and q2LMO = 0.564) models derived by CoMFA default set of parameters. Copyright © 2013 John Wiley & Sons, Ltd.
Analytic Approach for Phase Equilibrium Measurements of the System Non‐Ionic Surfactant/Water/Olefine
Authors: Schrader Philipp , Enders Sabine
Published: August 2013

Die analytischen Herausforderungen bei der Messung von komplexen Phasengleichgewichten über einen großen Temperaturbereich für das System nichtionisches Tensid + Wasser + Olefin werden diskutiert. In diesem Stoffsystem treten diverse Zwei‐ und Dreiphasenzustände auf. Die Herausforderung besteht einerseits in der sehr geringen Löslichkeit von Öl in Wasser bzw. von Wasser in Öl, so dass die zu messenden Konzentrationen nahe an der Nachweisgrenze von analytischen Standardmethoden liegen. Eingesetzt werden die Hochdruckflüssigkeitschromatographie und zwei Verfahren der Karl‐Fischer‐Titration.