Detection of tumor stem cell markers in pancreatic carcinoma cell lines

BACKGROUND: Cancer of the pancreas is the fourth leading cause of cancer death in industrialized countries. In malignancy, actively proliferating cells may be effectively targeted and killed by anti-cancer therapies, but stem cells may survive and support re-growth of the tumor. Thus, new strategies for the treatment of cancer clearly will also have to target cancer stem cells. The goal of the present study was to determine whether pancreatic carcinoma cell growth may be driven by a subpopulation of cancer stem cells. Because previous data implicated ABCG2 and CD133 as stem cell markers in hematopoietic and neural stem/progenitor cells, we analyzed the expression of these two proteins in pancreatic carcinoma cell lines. METHODS: Five established pancreatic adenocarcinoma cell lines were analyzed. Total RNA was isolated and real- time RT-PCR was performed to determine the expression of ABCG2 and CD133. Surface expression of ABCG2 and CD133 was analyzed by flow cytometric analysis. RESULTS: All pancreatic carcinoma cell lines tested expressed significantly higher levels of ABCG2 than non-malignant fibroblasts or two other malignant non- pancreatic cell lines, i.e., SaOS2 osteosarcoma and SKOV3 ovarian cancer. Elevated CD133 expression was found in two out of five pancreatic carcinoma cell lines tested. Using flow cytometric analysis we confirmed surface expression of ABCG2 in all five lines. Yet, CD133 surface expression was detectable in the two cell lines, A818-6 and PancTu1, which exhibited higher mRNA levels.CONCLUSIONS: Two stem cell markers, ABCG2 and CD133 are expressed in pancreatic carcinoma cell lines. ABCG2 and/or CD133 positive cells may represent subpopulation of putative cancer stem cells also in this malignancy. Because cancer stem cells are thought to be responsible for tumor initiation and its recurrence after an initial response to chemotherapy, they may be a very promising target for new drug developments.

Germ cell markers in fishes-A review

Identification of germ cell markers in fishes is crucial to track the germ cell differentiation and migration for manipulation of the cells to study sexual differentiation as well as to carry out transgenic transplantation techniques.Several germ cell-specific markers such as vasa,cnbp,dnd,nanos3,cbx2,amh,dmrt1,Ly75/CD205 have been characterized so far in fishes using localization and expression analysis,which have highlighted the spatio-temporal pattern of expression in early gonadal development.Incidentally,seasonal breeders show dramatic changes during gonadal recrudescence,which might also influence germ cell differentiation and growth to entrain the reproductive cycle.Hence,an in-depth analysis of the gonadal cycle is required to delineate germ cell progress,differentiation,and maturation explicitly.In this context,fishes undergoing gonadal recrudescence for the seasonal cycle show germ cell proliferation differentially.Most of these germ cell markers belong to the DEAD-box protein family of ATP-dependent RNA helicases sharing consensus motifs and clustering in phylogenetic analysis.These markers were found to be well-conserved throughout evolution.In situ hybridization approaches confirmed the germ cell specific distribution of these molecular markers.In addition,several genes such as fgf and gsdf seem to facilitate germ cell development and differentiation.Hence,more detailed studies on these factors will facilitate a better understanding of germ cell development.This review highlights various germ cell markers in fishes and their immense potential to use these cells for germ cell transplantation.The extensive knowledge of the germ cell markers can also be exploited to carry out other biotechnological experiments aiming at the preservation of genetic information of endangered species or the analytical study of gonadogenesis.

Ardisia gigantifolia ethanolic extract inhibits cell proliferation and targets cancer stem cells in gastric cancer

Objective:To evaluate the effects of ethanol extract from Ardisia gigantifolia leaves on cell proliferation and cancer stem cell(CSC)number in gastric cancer.Methods:The inhibitory effect of Ardisia gigantifolia extract on the proliferation of MKN45 and MKN74 gastric cancer cells was assessed using 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay.Non-adherent culture(3D)model was used to evaluate the effect of the extract on tumorsphere size and number.Moreover,the expression of CD44,ALDH,and p21 was determined by immunofluorescence analysis.Flow cytometric analysis was performed to evaluate cell cycle arrest and the expression of gastric CSC markers CD44 and ALDH.Real-time PCR analysis was also carried out to assess the effect of the extract on the expression of cell cycle-regulated genes.Results:Ardisia gigantifolia extract effectively inhibited cell proliferation with an IC_(50)of 55.7μg/m L in MKN45 cells and 123.6μg/m L in MKN74 cells.The extract also arrested cell cycle in the G_(0)/G_(1)phase as well as significantly reduced the size and number of tumorspheres.The markedly increased expression of p21 was observed at both m RNA and protein levels in the extract-treated adherent cells and tumorspheres.In addition,Ardisia gigantifolia extract significantly reduced the number of CD44-and/or ALDH-expressing gastric CSC.Conclusions:The development of gastric CSC can be inhibited by the ethanol extract of Ardisia gigantifolia.

Tissue-specific cancer stem/progenitor cells:Therapeutic implications

Surgical resection,chemotherapy,and radiation are the standard therapeutic modalities for treating cancer.These approaches are intended to target the more mature and rapidly dividing cancer cells.However,they spare the relatively quiescent and intrinsically resistant cancer stem cells(CSCs)subpopulation residing within the tumor tissue.Thus,a temporary eradication is achieved and the tumor bulk tends to revert supported by CSCs'resistant features.Based on their unique expression profile,the identification,isolation,and selective targeting of CSCs hold great promise for challenging treatment failure and reducing the risk of cancer recurrence.Yet,targeting CSCs is limited mainly by the irrelevance of the utilized cancer models.A new era of targeted and personalized anti-cancer therapies has been developed with cancer patient-derived organoids(PDOs)as a tool for establishing pre-clinical tumor models.Herein,we discuss the updated and presently available tissue-specific CSC markers in five highly occurring solid tumors.Additionally,we highlight the advantage and relevance of the threedimensional PDOs culture model as a platform for modeling cancer,evaluating the efficacy of CSC-based therapeutics,and predicting drug response in cancer patients.

Characterization of the osteogenic potential of mesenchymal stem cells from human periodontal ligament based on cell surface markers

Mesenchymal stem cell （MSC）-mediated therapy has been shown to be clinically effective in regenerating tissue defects. For improved regenerative therapy, it is critical to isolate homogenous populations of MSCs with high capacity to differentiate into appropriate tissues. The utilization of stem cell surface antigens provides a means to identify MSCs from various tissues. However, few surface markers that consistently isolate highly regenerative MSCs have been validated, making it challenging for routine clinical applications and making it all the more imperative to identify reliable surface markers. In this study, we used three surface marker combinations： CD51/CD140a, CD271, and STRO-1/CD146 for the isolation of homogenous populations of dental mesenchymal stem cells （DMSCs） from heterogeneous periodontal ligament cells （PDLCs）. Fluorescence-activated cell sorting analysis revealed that 24% of PDLCs were CD51＋/CD140a＋, 0.8% were CD271＋, and 2.4% were STRO-1＋/CD146＋. Sorted cell populations were further assessed for their multipotent properties by inducing osteogenic and chondrogenic differentiation. All three subsets of isolated DMSCs exhibited differentiation capacity into osteogenic and chondrogenic lineages but with varying degrees. CD271＋ DMSCs demonstrated the greatest osteogenic potential with strong induction of osteogenic markers such as DLX5, RUNX2, and BGLAP. Our study provides evidence that surface marker combinations used in this study are sufficient markers for the isolation of DMSCs from PDLCs. These results provide important insight into using specific surface markers for identifying homogenous populations of DMSCs for their improved utilization in regenerative medicine.

Effect of radiotherapy on tumor markers and serum immune-associated cells in patients with esophageal cancer

Objective This study aimed to investigate the effect of radiotherapy on serum immune-associated cells and tumor markers in patients with esophageal cancer.Methods A total of 87 patients with esophageal cancer admitted to our hospital between October 2016 and July 2020 were selected as the observation group,and all patients received radiotherapy.A total of 87 healthy volunteers who underwent physical examination at our hospital during the same period were selected as the control group in order to compare the changes in serum immune-associated cells and tumor markers between the two groups.Results The levels of carcinoembryonic antigen(CEA),cancer antigen(CA)125,CA72-4,C-terminus of cytokeratin(CYFRA)21-1,and squamous cell carcinoma(SCC)antigen in the observation group before radiotherapy were higher than those in the control group,and the differences were significant(P<0.05).The levels of CEA,CA125,CA72-4,CYFRA21-1,and SCC antigen in the research group after radiotherapy were significantly lower than those before radiotherapy,but were still significantly higher than those in the control group(P<0.05).The levels of CD3+,CD4+,CD4+/CD8+,and natural killer cells in the research group before and after radiotherapy were significantly lower,while the levels of Treg and CD8+cells were significantly higher than those in the control group(P<0.05).The levels of CD3+,CD4+,and CD4+/CD8+cells in the observation group after radiotherapy were lower,while the levels of CD8+cells were significantly higher than those before radiotherapy(P<0.05).Conclusion Radiotherapy can effectively reduce the level of serum tumor markers in patients with esophageal cancer;these antigens and cells can be used as tumor markers of esophageal cancer in order to determine its prognosis.However,radiotherapy has adverse effects on the immune function of the body.The reasons behind this need to be further studied and analyzed.

Aqueous Leaves Extract of Gongronema latifolium (Benth) Downregulates the Expression of IFN-γ, IL-10 and Cell Surface Markers in Rabbits

Background: The pathophysiology of the inflammatory process reveals intricate signaling which includes the IL-1β, IL-6, and TNFα pathways that could serve as drug targets. Aim: This study determined the effect of the aqueous extract of Gongronema latifolium (AEGL) leaves on the expression of IFNγ, IL-10, CD3, and CD56 in rabbits. Materials and Methods: ELISA tests were performed to determine the effect of the AEGL on the expression of a pro-inflammatory cytokine (IFNγ), an anti-inflammatory cytokine (IL-10), and CD3 and CD56 cell surface markers in rabbits. Twenty cross-bred male rabbits with an average weight range of 1.0 - 1.5 kg were selected. The rabbits were separated into four groups of four rabbits each treated as follows: Grp1 is the untreated control;Grp2 is the treated control;and Grp3, Grp4, and Grp5 were treated with 200, 400, and 600 mg/kg of AEGL respectively for 28 days. Results: The AEGL showed its greatest inhibitory effect in Group 4 on IL-10 (118.5 pg/ml), and IFNγ (332 pg/ml) on days 14 and 21 respectively. AEGL also showed the highest inhibition of CD3 expression on days 14 and 21 (0 pg/ml) in Group 3;and CD56 expression on day 21 (630.5 pg/ml) in Group 4. Conclusion: AEGL showed exhibited strong T cell mediated anti- inflammatory, and immunomodulatory activity in test rabbits within the 28-day period which can be confirmed by cell based assays. Specifically at 400 mg/kg, AEGL exhibited the greatest anti-inflammatory activity which is suggestive of its maximum effective dose.

Methods to produce induced pluripotent stem cell-derived mesenchymal stem cells: Mesenchymal stem cells from induced pluripotent stem cells

Mesenchymal stem cells(MSCs)have received significant attention in recent years due to their large potential for cell therapy.Indeed,they secrete a wide variety of immunomodulatory factors of interest for the treatment of immune-related disorders and inflammatory diseases.MSCs can be extracted from multiple tissues of the human body.However,several factors may restrict their use for clinical applications:the requirement of invasive procedures for their isolation,their limited numbers,and their heterogeneity according to the tissue of origin or donor.In addition,MSCs often present early signs of replicative senescence limiting their expansion in vitro,and their therapeutic capacity in vivo.Due to the clinical potential of MSCs,a considerable number of methods to differentiate induced pluripotent stem cells(iPSCs)into MSCs have emerged.iPSCs represent a new reliable,unlimited source to generate MSCs(MSCs derived from iPSC,iMSCs)from homogeneous and well-characterized cell lines,which would relieve many of the above mentioned technical and biological limitations.Additionally,the use of iPSCs prevents some of the ethical concerns surrounding the use of human embryonic stem cells.In this review,we analyze the main current protocols used to differentiate human iPSCs into MSCs,which we classify into five different categories:MSC Switch,Embryoid Body Formation,Specific Differentiation,Pathway Inhibitor,and Platelet Lysate.We also evaluate common and method-specific culture components and provide a list of positive and negative markers for MSC characterization.Further guidance on material requirements to produce iMSCs with these methods and on the phenotypic features of the iMSCs obtained is added.The information may help researchers identify protocol options to design and/or refine standardized procedures for large-scale production of iMSCs fitting clinical demands.

Biliary tract cancer stem cells-translational options and challenges

Management of biliary tract cancer remains challenging. Tumors show high recurrence rates and therapeutic resistance, leading to dismal prognosis and short survival. The cancer stem cell model states that a tumor is a heterogeneous conglomerate of cells, in which a certain subpopulation of cells-the cancer stem cells-possesses stem cell properties. Cancer stem cells have high clinical relevance due to their potential contributions to development, progression and aggressiveness as well as recurrence and metastasis of malignant tumors. Consequently, reliable identification of as well as pharmacological intervention with cancer stem cells is an intensively investigated and promising research field. The involvement of cancer stem cells in biliary tract cancer is likely as a number of studies demonstrated their existence and the obvious clinical relevance of several established cancer stem cell markers in biliary tract cancer models and tissues. In the present article, we review and discuss the currently available literature addressing the role of putative cancer stem cells in biliary tract cancer as well as the connection between known contributors of biliary tract tumorigenesis such as oncogenic signaling pathways, micro-RNAs and the tumor microenvironment with cancer stem cells.

Doublecortin-like kinase 1 exhibits cancer stem cell-like characteristics in a human colon cancer cell line

Objective： Colon cancer stem cells （CSCs） are implicated in colorectal cancer carcinogenesis, metastasis, and therapeutic resistance. The identification of these cells could help to develop novel therapeutic strategies. Doublecortin-like kinase 1 （DCLK1） has been viewed as a marker for gastrointestinal stem cells that fuel the self-renewal process, however others view them as a marker of Tuft cells or as an enteroendocrine subtype. The purpose of this study was to use a colon cancer cell line to identify and characterize the stem-like characteristics of the DCLKI＋ cell population. Methods： To enrich stem-like cells, HCT116 cells （derived from colon adenocarcinomas） were cultured using serum-free media to form spheres under both normal oxygen and hypoxia condition. DCLK1 transcript expression in the adherent parental cells and spheroids was quantified using quantitative real time reverse transcription- polymerase chain reaction [（q）RT-PCR]. DCLK1 protein expression was determined using flow cytometry. Self-renewal capability from adherent parental cells and spheroids was determined using extreme limiting dilution analysis （ELDA）. Results： Under both normal oxygen and hypoxia condition, the adherent parental cells were composed of cells that express low levels of DCLK1. However, spheroids exhibited an increased frequency of cells expressing DCLK1 on both mRNA and protein levels. Cells derived from spheroids also possess stronger self-renewal capability. Conclusions： The higher fraction of DCLK1 ＋ cells exhibited by spheroids and hypoxia reflects the stem- like characteristics of these cells. DCLK1 may represent an ideal marker to study and develop effective strategies to overcome chemo-resistance and relapse of colon cancer.

Globose basal cells for spinal cord regeneration

Spinal cord injury （SCI） is a devastating condition with loss of motor and sensory functions below the injury level. Cell based therapies are experimented in pre-clinical studies around the world. Neural stem cells are located intra-craniafly in subventricular zone and hippocampus which are highly invasive sourc- es. The olfactory epithelium is a neurogenic tissue where neurogenesis takes place throughout the adult life by a population of stem/progenitor cells. Easily accessible olfactory neuroepithelial stem/progenitor cells are an attractive cell source for transplantation in SCI. Globose basal cells （GBCs） were isolated from rat olfactory epithelium, characterized by flow cytometry and immunohistochemically. These ceils were further studied for neurosphere formation and neuronal induction. T10 laminectomy was done to create drop-weight SCI in rats. On the 9th day following SCI, 5 × 105 cells were transplanted into injured rat spinal cord. The outcome of transplantation was assessed by the Basso, Beattie and Bresnahan （BBB） locomotor rating scale, motor evoked potential and histological observation. GBCs expressed neural stem cell markers nestin, SOX2, NCAM and also mesenchymal stem cell markers （CD29, CD54, CD90, CD73, CD105）. These cells formed neurosphere, a culture characteristics of NSCs and on induction, differentiated cells expressed neuronal markers ~III tubulin, microtubule-associated protein 2, neuronal nuclei, and neurofilament. GBCs transplanted rats exhibited hindlimb motor recovery as confirmed by BBB score and gastrocnemius muscle electromyography amplitude was increased compared to controls. Green fluorescent protein labelled GBCs survived around the injury epicenter and differentiated into βⅢ tubulin-immunoreactive neuron-like cells. GBCs could be an alternative to NSCs from an accessible source for autologous neurotransplantation after SCI without ethical issues.

Reporter gene systems for the identification and characterization of cancer stem cells

Cancer stem cells(CSCs)are tumor cells that share functional characteristics with normal and embryonic stem cells.CSCs have increased tumor-initiating capacity and metastatic potential and lower sensitivity to chemo-and radiotherapy,with important roles in tumor progression and the response to therapy.Thus,a current goal of cancer research is to eliminate CSCs,necessitating an adequate phenotypic and functional characterization of CSCs.Strategies have been developed to identify,enrich,and track CSCs,many of which distinguish CSCs by evaluating the expression of surface markers,the initiation of specific signaling pathways,and the activation of master transcription factors that control stemness in normal cells.We review and discuss the use of reporter gene systems for identifying CSCs.Reporters that are under the control of aldehyde dehydrogenase 1A1,CD133,Notch,Nanog homeobox,Sex-determining region Y-box 2,and POU class 5 homeobox can be used to identify CSCs in many tumor types,track cells in real time,and screen for drugs.Thus,reporter gene systems,in combination with in vitro and in vivo functional assays,can assess changes in the CSCs pool.We present relevant examples of these systems in the evaluation of experimental CSCs-targeting therapeutics,demonstrating their value in CSCs research.

Single CD271 marker isolates mesenchymal stem cells from human dental pulp

Mesenchymal stem cells （MSCs） are a promising tool in regenerative medicine due to their capacity to differentiate into multiple lineages. In addition to MSCs isolated from bone marrow （BMSCs）, adult MSCs are isolated from craniofacial tissues including dental pulp tissues （DPs） using various stem cell surface markers. However, there has been a lack of consensus on a set of surface makers that are reproducibly effective at isolating putative multipotent dental mesenchymal stem cel^s （~M^Cs）. II1 ~his stucly, we used clif^et（~nt combinations of surface markers （CD51/CD140a, CD271, and STRO-1/CD146） to isolate homogeneous populations of DMSCs from heterogeneous dental pulp cells （DPCs） obtained from DP and compared their capacity to undergo multilineage differentiation. Fluorescence-activated cell sorting revealed that 27.3% of DPCs were CD51＋/CD140a＋, 10.6% were CD271＋, and 0.3% were STRO-1＋/CD146＋. Under odontogenic conditions, all three subsets of isolated DMSCs exhibited differentiation capacity into odontogenic lineages. Among these isolated subsets of DMSCs, CD271＋ DMSCs demonstrated the greatest odontogenic potential. While all three combinations of surface markers in this study successfully isolated DMSCs from DPCs, the single CD271 marker presents the most effective stem cell surface marker for identification of DMSCs with high odontogenic potential. Isolated CD271＋ DMSCs could potentially be utilized for future clinical applications in dentistry and regenerative medicine.

Effect of senescence marker protein 30 on the proliferation and apoptosis of human lens epithelial cells SRA01/04

AIM： To study the effect of senescence marker protein 30（SMP30） on the proliferation and apoptosis of human lens epithelial cell（HLEC） SRA01/04.METHODS： SMP30 overexpression（OE） and knock down（KD） type cell lines were cultivated by using two groups regucalcin（RGN; SMP30） lentiviral vectors（LVRGN, LV-RGN-RNAi） and the respective negative control virus infect SRA01/04 cells. Western blot and real-time quantitative polymerase chain reaction（q-PCR） analysis were used to determine RGN overexpression and knock down efficiency. We use cell counting kit-8（CCK8） assay to measure cell viability and 5-bromodeoxyuridine（Brd U） assay to test cell proliferation. Cell cycle was measured by PI FACS assay and cell apoptosis was tested by Annexin V-APC assay through flow cytometry. We use Western blot to measure the content of caspase-3 in SRA01/04.RESULTS： We used PCR and Western blot techniques to determine the successful transfection of SMP30 OE and KD SRA01/04 cell lines. By CCK8, Brdu and PI FACS cell cycle assay, it was found that the SMP30 OE group promoted cell proliferation（P〈0.05） compared with the control group, and the KD group inhibited cell proliferation（P〈0.05）. The results of Annexin V-APC signal staining detection indicated that compared with respective control group, the cell apoptosis rate was higher in KD group（P〈0.05） but lower in OE group（P〈0.01）. The expression of caspase-3 was down-regulated in OE group through Western blot assay and up-regulated in KD group compared with respective control group. CONCLUSION： Proliferation of SRA01/04 was promoted by SMP30 OE and apoptosis was suppressed. Increasing the expression of SMP30 may protect HLEC SRA01/04 against apoptosis in cataract.

Human dental pulp stem cells express many pluripotency regulators and differentiate into neuronal cells

Stem cells were isolated from human dental pulp using an optimized method, in which pulp pieces were digested by enzymes and immobilized to enhance cell outgrowth. Stem cell marker expression was detected by reverse transcription-PCR （RT-PCR）, and differentiation markers were detected by real-time quantitative RT-PCR and immunocytochemistry. Results showed that dental pulp stem cells actively expressed nanog, oct4, nucleostemin slain-l, jmjdla, jmjd2c, and cyclin DI. When stem cells were induced to differentiate into neurons, nucleostemin, nanog, and cyclin D1 expression significantly decreased, whereas expression of neuronal markers, such as microtubule associated protein-2 and neurofilament-heavy, significantly increased. These results suggested that stem cells exited a pluripotent state and entered a neuronal differentiation pathway. In addition, results demonstrated that human dental pulp serves as a reservoir of stem cells that express defined stem cell markers; these cells were easily isolated and were induced to differentiate towards a desired cell lineage.

A cell marker-based clustering strategy(cmCluster)for precise cell type identification of scRNA-seq data

Background:The precise and efficient analysis of single-cell transcriptome data provides powerful support for studying the diversity of cell functions at the single-cell level.The most important and challenging steps are cell clustering and recognition of cell populations.While the precision of clustering and annotation are considered separately in most current studies,it is worth attempting to develop an extensive and flexible strategy to balance clustering accuracy and biological explanation comprehensively.Methods:The cell marker-based clustering strategy(cmCluster),which is a modified Louvain clustering method,aims to search the optimal clusters through genetic algorithm(GA)and grid search based on the cell type annotation results.Results:By applying cmCluster on a set of single-cell transcriptome data,the results showed that it was beneficial for the recognition of cell populations and explanation of biological function even on the occasion of incomplete cell type information or multiple data resources.In addition,cmCluster also produced clear boundaries and appropriate subtypes with potential marker genes.The relevant code is available in GitHub website(huangyuwei301/cmCluster).Conclusions:We speculate that cmCluster provides researchers effective screening strategies to improve the accuracy of subsequent biological analysis,reduce artificial bias,and facilitate the comparison and analysis of multiple studies.

Large Eddy Simulation for Plunge Breaker and Sediment Suspension

Breaking waves are a powerful agent for generating turbulence that plays an important role in many fluid dynamical processes, particularly in the mixing of materials. Breaking waves can dislodge sediment and throw it into suspension, which will then be carried by wave-induced steady current and tidal flow. In order to investigate sediment suspension by breaking waves, a numerical model based on large-eddy-simulation (LES) is developed. This numerical model can be used to simulate wave breaking and sediment suspension. The model consists of a free-surface model using the surface marker method combined with a two-dimensional model that solves the flow equations. The turbulence and the turbulent diffusion are described by a large-eddy-simulation (LES) method where the large turbulence features are simulated by solving the flow equations, and a subgrid model represents the small-scale turbulence that is not resolved by the flow model , A dynamic eddy viscosity subgrid scale stress model has been used for the present simulation. By applying this model to Stokes' wave breaking problem in the surf zone, we find that the model results agree very well with experimental data. By use of this model to simulation of the breaking process of a periodic wave, it can be found that the model can reproduce the complicated flow phenomena, especially the plunging breaker. It reflects the dynamic structures of roller or vortex in the plunging breaker, and when the wave breaks, many strong vortex structures will be produced in the inner surf zone where the concentration of suspended sediment can thereby become relatively high.

Large Eddy Simulation for Wave Breaking in the Surf Zone

In this paper, the large eddy simulation method is used combined with the marker and cell method to study the wave propagation or shoaling and breaking process. As wave propagates into shallow water, the shoaling leads to the increase of wave height, and then at a certain position, the wave will be breaking. The breaking wave is a powerful agent for generating turbulence, which plays an important role in most of the fluid dynamic processes throughout the surf zone, Such as transformation of wave energy, generation of near-shore current and diffusion of materials. So a proper numerical model for describing the turbulence effect is needed. In this paper, a revised Smagorinsky subgrid-scale model is used to describe the turbulence effect. The present study reveals that the coefficient of the Smagorinsky model for wave propagation or breaking simulation may be taken as a varying function of the water depth and distance away from the wave breaking point. The large eddy simulation model presented in this paper has been used to study the propagation of the solitary wave in constant water depth and the shoaling of the non-breaking solitary wave on a beach. The model is based on large eddy simulation, and to track free-surface movements, the Tokyo University Modified Marker and Cell (TUMMAC) method is employed. In order to ensure the accuracy of each component of this wave mathematical model, several steps have been taken to verify calculated solutions; with either analytical solutions or experimental data. For non-breaking waves, very accurate results are obtained for a solitary wave propagating over a constant depth and on a beach. Application of the model to cnoidal wave breaking in the surf zone shows that the model results are in good agreement with analytical solution and experimental data. From the present model results, it can be seen that the turbulent eddy viscosity increases from the bottom to the water surface in surf zone. In the eddy viscosity curve, there is a turn-point obviously, dividing water depth into two parts, in the upper part, the eddy viscosity becomes very large near the wave breaking position.

Methodical and pre-analytical characteristics of a multiplex cancer biomarker immunoassay

AIM: To test the methodical and pre-analytical performance of a new multiplex cancer biomarker panel using magnetic beads. METHODS: The MILLIPLEX? MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 1 comprises the tumor markers carcinoembryonic antigen, alpha-fetoprotein, total prostate-specific antigen, cancer antigen 15-3, cancer antigen 19-9, cancer antigen 125, cytokeratine 19-fragment, β-human chorionic gonadotropin, human epididymis protein 4, osteopontin, prolactin, the cell death and angiogenesis markers soluble Fas, soluble Fas-ligand, tumor necrosis factor related apoptosisinducing ligand, vascular endothelial growth factor andthe immunological markers interleukin-6(IL-6), IL-8, tumor necrosis factor-α, transforming growth factor α, fibroblast growth factor-2, macrophage migration inhibitory factor, leptin, hepatocyte growth factor, and stem cell factor. We determined intra- and inter-assay imprecision as well as dilution linearity using quality controls and serum pools. Furthermore, the stability of the 24 biomarkers examined in this panel was ascertained by testing the influence of different storage temperatures and time span before centrifugation.RESULTS: For all markers measured in the synthetic internal quality controls, the intra-assay imprecision ranged between 2.26% and 9.41%, while for 20 of 24 measured markers in the physiological serum pools, it ranged between 1.68% and 12.87%. The inter-assay imprecision ranged between 1.48%-17.12% for 23 biomarkers in synthetic, and between 4.59%-23.88% for 18 biomarkers in physiological quality controls. Here, single markers with very low concentration levels had increased imprecision rates. Dilution linearity was acceptable(70%-130% recovery) for 20 biomarkers. Regarding pre-analytical influencing factors, most markers were stable if blood centrifugation was delayed or if serum was stored for up to 24 h at 4 ℃ and 25 ℃ after centrifugation. Comparable results were obtained in serum and plasma for most markers. However, great changes were observed for single markers.CONCLUSION: MILLIPLEX? MAP Human Circulating Cancer Biomarker Magnetic Bead Panel 1 assay is a stable and precise method for detection of most biomarkers included in the kit. However, single markers have to be interpreted with care.

Expression of endothelial cell IgG Fc receptors and markers on various cultures

Objective To determine and compare the expression of endothelial cell IgG Fc receptors (FcγR) and markers on various kinds of cultures.Methods Human breast microvascular endothelial cells (HMVEC), human aortic endothelial cells (HAEC), human umbilical vein endothelial cells (HUVEC) and canine aortic endothelial cells (CAEC) were stimulated with cytokines tumor necrosis factor α (TNF α) and interferon γ (IFN γ). The binding of anti Fcγ receptor (FcγR) type Ⅰ, Ⅱ and Ⅲ antibodies was measured using an enzyme linked immunosorbent assay (ELISA). The constitutive expression of endothelial cell markers was examined using anti von Willebrand factor antibodies, DiI low density lipoprotein (Dil Ac LDL) and fluorescein isothiocyanate (FITC) labeled ulex europaeus agglutinin 1. Results The binding of anti FcγRⅡ was significantly increased by the simultaneous stimulation with TNF α and IFN γ on all three types of human endothelial cells (ECs), but not on canine endothelial cells. Enhanced FcγRⅡ expression was most significant when human ECs were cultured in endothelial cell basal medium (ECBM). However, the expression of FcγRⅡ on CAECs could not be induced by human cytokines even after they were cultured in ECBM for 3 passages. Endothelial cells also showed diversity for the constitutive expression of classic markers. Conclusions This study demonstrates that cytokines TNF α and IFN γ enhance low affinity FcγR expression on human endothelial cells in vitro. The results indicate that heterogeneity of endothelial cells exists not only on constitutive expression but also on stimulative expression.