DNA Methylation Analysis Reveals Potential Mechanism in Takifugu rubripes Against Cryptocaryon irritans Infection.

Takifugu rubripes (T. rubripes) is a valuable commercial fish, and Cryptocaryon irritans (C. irritans) has a significant impact on its aquaculture productivity. DNA methylation is one of the earliest discovered ways of gene epigenetic modification and also an important form of modification, as well as an essential type of alteration that regulates gene expression, including immune response. To further explore the anti-infection mechanism of T. rubripes in inhibiting this disease, we determined genome-wide DNA methylation profiles in the gill of T. rubripes using whole-genome bisulfite sequencing (WGBS) and combined with RNA sequence (RNA-seq). A total of 4659 differentially methylated genes (DMGs) in the gene body and 1546 DMGs in the promoter between the infection and control group were identified. And we identified 2501 differentially expressed genes (DEGs), including 1100 upregulated and 1401 downregulated genes. After enrichment analysis, we identified DMGs and DEGs of immune-related pathways including MAPK, Wnt, ErbB, and VEGF signaling pathways, as well as node genes prkcb, myca, tp53, and map2k2a. Based on the RNA-Seq results, we plotted a network graph to demonstrate the relationship between immune pathways and functional related genes, in addition to gene methylation and expression levels. At the same time, we predicted the CpG island and transcription factor of four immune-related key genes prkcb and mapped the gene structure. These unique discoveries could be helpful in the understanding of C. irritans pathogenesis, and the candidate genes screened may serve as optimum methylation-based biomarkers that can be utilized for the correct diagnosis and therapy T. rubripes in the development of the ability to resist C. irritans infection.

Choline dehydrogenase interacts with SQSTM1 to activate mitophagy and promote coelomocyte survival in Apostichopus japonicus following Vibrio splendidus infection.

Previous studies have shown that Vibrio splendidus infection causes mitochondrial damage in Apostichopus japonicus coelomocytes, leading to the production of excessive reactive oxygen species (ROS) and irreversible apoptotic cell death. Emerging evidence suggests that mitochondrial autophagy (mitophagy) is the most effective method for eliminating damaged mitochondria and ROS, with choline dehydrogenase (CHDH) identified as a novel mitophagy receptor that can recognize non-ubiquitin damage signals and microtubule-associated protein 1 light chain 3 (LC3) in vertebrates. However, the functional role of CHDH in invertebrates is largely unknown. In this study, we observed a significant increase in the mRNA and protein expression levels of A. japonicus CHDH (AjCHDH) in response to V. splendidus infection and lipopolysaccharide (LPS) challenge, consistent with changes in mitophagy under the same conditions. Notably, AjCHDH was localized to the mitochondria rather than the cytosol following V. splendidus infection. Moreover, AjCHDH knockdown using siRNA transfection significantly reduced mitophagy levels, as observed through transmission electron microscopy and confocal microscopy. Further investigation into the molecular mechanisms underlying CHDH-regulated mitophagy showed that AjCHDH lacked an LC3-interacting region (LIR) for direct binding to LC3 but possessed a FB1 structural domain that binds to SQSTM1. The interaction between AjCHDH and SQSTM1 was further confirmed by immunoprecipitation analysis. Furthermore, laser confocal microscopy indicated that SQSTM1 and LC3 were recruited by AjCHDH in coelomocytes and HEK293T cells. In contrast, AjCHDH interference hindered SQSTM1 and LC3 recruitment to the mitochondria, a critical step in damaged mitochondrial degradation. Thus, AjCHDH interference led to a significant increase in both mitochondrial and intracellular ROS, followed by increased apoptosis and decreased coelomocyte survival. Collectively, these findings indicate that AjCHDH-mediated mitophagy plays a crucial role in coelomocyte survival in A. japonicus following V. splendidus infection.

ROS-mediated BNIP3-dependent mitophagy promotes coelomocyte survival in Apostichopus japonicus in response to Vibrio splendidus infection.

Organisms produce high levels of reactive oxygen species (ROS) to kill pathogens or act as signaling molecules to induce immune responses; however, excessive ROS can result in cell death. To maintain ROS balance and cell survival, mitophagy selectively eliminates damaged mitochondria via mitophagy receptors in vertebrates. In marine invertebrates, however, mitophagy and its functions remain largely unknown. In the current study, Vibrio splendidus infection damaged mitochondrial morphology in coelomocytes and reduced mitochondrial membrane potential (ΔΨm) and mitophagosome formation. The colocalization of mitochondria and lysosomes further confirmed that lipopolysaccharide (LPS) treatment increased mitophagy flux. To explore the regulatory mechanism of mitophagy, we cloned Bcl2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), a common mitophagy receptor, from sea cucumber Apostichopus japonicus (AjBNIP3) and confirmed that AjBNIP3 was significantly induced and accumulated in mitochondria after V. splendidus infection and LPS exposure. At the mitochondrial membrane, AjBNIP3 interacts with microtubule-associated protein 1 light chain 3 (LC3) on phagophore membranes to mediate mitophagy. After AjBNIP3 interference, mitophagy flux decreased significantly. Furthermore, AjBNIP3-mediated mitophagy was activated by ROS following the addition of exogenous hydrogen peroxide (H2O2), ROS scavengers, and ROS inhibitors. Finally, inhibition of BNIP3-mediated mitophagy by AjBNIP3 small interfering RNA (siRNA) or high concentrations of lactate increased apoptosis and decreased coelomocyte survival. These findings highlight the essential role of AjBNIP3 in damaged mitochondrial degradation during mitophagy. This mitophagy activity is required for coelomocyte survival in A. japonicus against V. splendidus infection.

Identification of global alternative splicing and sex-specific splicing via comparative transcriptome analysis of gonads of Chinese tongue sole ( Cynoglossus semilaevis).

Chinese tongue sole ( Cynoglossus semilaevis) is an economically important marine fish species with a ZZ/ZW sex determination mechanism, which can be influenced by temperature. Alternative splicing (AS) is an important mechanism regulating the expression of genes related to sex determination and gonadal differentiation, but has rarely been reported in fish. In this study, to explore the molecular regulatory mechanisms of sex determination and gonadal differentiation, we combined isoform and RNA sequencing (Iso-Seq and RNA-Seq) to perform transcriptome profiling of male and female gonads in C. semilaevis. In total, 81 883 and 32 341 full-length transcripts were obtained in males and females, respectively. A total of 8 279 AS genes were identified, including 2 639 genes showing differential AS (DAS) between males and females. Many intersecting DAS genes and differentially expressed genes (DEGs) were enriched in the meiotic cell cycle pathway, and genes related to gonadal differentiation, such as esrrb and wt1a, were found to have sex-specific isoforms. Thus, this study revealed AS events in the gonadal transcriptomes of male and female C. semilaevis, described the characteristics of active transcription in the testes, and identified candidate genes for studying the regulatory mechanisms of AS during gonadal differentiation.

Genome-wide integrated analysis reveals functions of lncRNA-miRNA-mRNA interactions in Atlantic salmon challenged by Aeromonas salmonicida.

Aeromonas salmonicida (A. salmonicida) is a pathogenic bacterium that causes serious problems in the global Atlantic salmon aquaculture industry. In this study, we comprehensively analyzed the profiles of lncRNAs, miRNAs and mRNAs in gills of Atlantic salmon at high-dose A. salmonicida infection (3.06 × 108 CFU/mL), low-dose A. salmonicida infection (3.06 × 105 CFU/mL), and a PBS (100 µL) control. We identified 65 differentially expressed lncRNAs, 41 miRNAs, and 512 mRNAs between the control group and infection groups. Functional analysis showed that these genes were significantly enriched in the p53 signaling pathway, Wnt signaling pathway, mTOR signaling pathway, JAK-STAT signaling pathway, and Toll-like receptor signaling pathway. In addition, we predicted key genes in immune-related pathways and constructed a lncRNA-miRNA-mRNA network based on whole transcriptomic analysis. We further predicted three lncRNA-miRNA-mRNA axes as potential novel biomarkers in regulating the immune response of Atlantic salmon against A. salmonicida infection.

Long non-coding RNA MALAT1 enhances angiogenesis during bone regeneration by regulating the miR-494/SP1 axis.

Bone regeneration is a coordinated process involving connections between blood vessels and osteocytes. Angiogenesis and osteogenesis are tightly connected throughout the progression of bone regeneration. This study aimed to explore the underlying mechanism of metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)-regulated angiogenesis during bone regeneration. Gene and protein expression was detected by quantitative real-time PCR and western blot assay. Vascular endothelial growth factor (VEGFA) secretion was assessed by enzyme-linked immunosorbent assay. To evaluate the effect of osteogenic differentiation, alkaline phosphatase (ALP) and alizarin red staining assays were performed. Proliferation was detected by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Migration and angiogenesis were measured using Transwell and tube formation assays. A dual luciferase reporter assay was performed to confirm the binding relationship among MALAT1, miR-494, and specificity protein 1 (SP1). Expression levels of MALAT1, SP1, and VEGFA were elevated and miR-494 was suppressed in MC3T3-E1 cells after culture in osteogenic medium. MALAT1 knockdown suppressed the osteogenic differentiation of MC3T3-E1, since ALP activity, mineralized nodules, and expression of the osteodifferentiated markers runt-related transcription factor 2 and osterix were restrained. In addition, MALAT1 silencing inhibited angiogenesis during bone regeneration, as the proliferation, migration, and capillary tube formation of human umbilical vein endothelial cells were blocked. Furthermore, miR-494 was directly targeted by MALAT1 and regulated the SP1/Toll-like receptor 2 (TLR2)/bone morphogenetic protein 2 (BMP2) axis by targeting SP1. Furthermore, miR-494 overexpression inhibited angiogenesis and osteogenic differentiation. Moreover, SP1 overexpression or miR-494 inhibition rescued the regulatory effect of sh-MALAT1 on angiogenesis and osteogenic differentiation. Taken together, these findings indicate that MALAT1 promotes angiogenesis and osteogenic differentiation by targeting miR-494 and activating the SP1/TLR2/BMP2 pathway, suggesting a novel target for bone regeneration therapy by promoting angiogenesis.

Kushneria phosphatilytica sp. nov., a phosphate-solubilizing bacterium isolated from a solar saltern.

A Gram-stain-negative, motile, rod-shaped, non-endospore-forming, aerobic and halophilic bacterium, designated strain YCWA18T, was isolated from the sediment of Jimo-Daqiao saltern in China. This strain was able to grow at NaCl concentrations in the range 0.5-20 % (w/v) with optimum growth at 6 % (w/v) NaCl. Growth occurred at temperatures of 4-40 °C (optimum 28 °C) and pH 4.0-9.0 (optimum 7.0). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YCWA18T belonged to the genus Kushneria and shared the highest sequence similarity of 98.7 % with Kushneria sinocarnis DSM 23229T. Moreover, the phylogenetic analysis based on the 23S rRNA gene sequence also confirmed the phylogenetic position of this novel strain. The predominant fatty acids were C16 : 0, C17 : 0 cyclo and C12 : 0 3-OH. The major isoprenoid quinone was Q-9 (94.2 %) and the polar lipids were diphosphatidylglycerol (DPG), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), an unidentified aminolipid (AL), an unidentified phospholipids (PL) and two unidentified lipids (L). The complete genome of strain YCWA18T consisted of a single, circular chromosome of 3 624 619 bp, with an average G+C content of 59.1 mol%. A genome-based phylogenetic tree constructed using an up-to-date bacterial core gene set (UBCG) showed that strain YCWA18T formed a clade with K. sinocarnis DSM 23229T. However, the level of the ANI and dDDH values between strain YCWA18T and K. sinocarnis DSM 23229T were 82.3 and 24.6 %, respectively, which were low enough to distinguish strain YCWA18T from K. sinocarnis DSM 23229T. Overall, based on the phenotypic, chemotaxonomic, phylogenetic and genomic analyses, strain YCWA18T represents a novel species of genus Kushneria. The name Kushneria phosphatilytica sp. nov. is proposed, with the type strain YCWA18T (=CGMCC 1.9149T=NCCB 100306T).

M1 Macrophage-Derived Exosomal MicroRNA-326 Suppresses Hepatocellular Carcinoma Cell Progression Via Mediating NF-κB Signaling Pathway.

Accumulating evidence has shown that microRNA (miR) derived from M1 macrophage-derived exosomes can regulate the progression of hepatocellular carcinoma (HCC). However, the effect of miR-326 derived from M1 macrophage-derived exosomes on HCC has not been reported. Therefore, the objective of the present study was to explore the mechanism of exosomal miR-326 from M1 macrophages in regulating HCC cell progression. RT-qPCR detected miR-326 expression in HCC cell lines. miR-326 expression in HCC was altered by transfection, and the effect of miR-326 on CD206 and NF-κB expression, cell proliferation, colony formation, migration, apoptosis and invasion was detected. Subsequently, exosomes were isolated from M1 macrophages. RT-qPCR identified miR-326 expression in M1 macrophage-derived exosomes. miR-326 expression in M1 macrophage-derived exosomes was changed by transfection. M1 macrophage-derived exosomes were co-cultured with HCC cells to figure out their effects on the biological progress of HCC cells. Finally, in vivo experiments were performed to verify the in vitro results. MiR-326 was decreased in HCC cells and enriched in M1 macrophage-derived exosomes. Up-regulating miR-326 would inhibit HCC cell proliferation, colony formation, migration, invasion, and CD206 and NF-κB expression and promoted apoptosis, and inhibited the growth of HCC tumors in vivo, while down-regulating miR-326 showed opposite effects. M1 macrophage-derived exosomes inhibited HCC cell proliferation, colony formation, migration, invasion, and CD206 and NF-κB expression and enhanced apoptosis, while overexpression of miR-326 enhanced the effect of M1 macrophage-derived exosomes on HCC cells. It is revealed that M1 macrophages-derived exosomal miR-326 suppresses proliferation, migration and invasion as well as advances apoptosis of HCC through down-regulating NF-κB expression.

The NK-1R Antagonist Aprepitant Prevents LPS-Induced Oxidative Stress and Inflammation in RAW264.7 Macrophages.

BACKGROUND: The macrophage is one of the most important types of immune cells that protect against harmful stimuli. Macrophage activation plays a pivotal role in the progression and development of various inflammatory diseases. The neurokinin 1 receptor (NK-1R) is a G protein-coupled receptor that plays an important role in inflammatory diseases. Aprepitant is a kind of NK-1R antagonist. The purpose of this study is to determine the protective effect of aprepitant in lipopolysaccharide (LPS)-induced inflammatory responses in macrophages. METHODS: We examined the anti-inflammatory and anti-oxidant effects of aprepitant in LPS-treated RAW264.7 macrophages by using real-time PCR, ELISA, and Western blot analysis. We also assessed cellular oxidative stress signaling by measuring the levels of cellular MDA, total ROS, and NADPH oxidase expression. Cellular NO production was measured by DAF-FM DA staining. The inhibitory effect of aprepitant against NF-κB signaling was evaluated by luciferase assay and Western blot analysis. RESULTS: The expression of NK-1R is increased in LPS-induced macrophages, suggesting a potential role of the receptor in the inflammatory response. We show that aprepitant protects macrophages against oxidative stress by reducing the generation of ROS and the expression of NOX-4. Furthermore, aprepitant inhibits the secretion of pro-inflammatory cytokines and chemotactic factors by mediating the NF-κB signaling pathway. CONCLUSION: The NK-1R receptor antagonist aprepitant acts as an anti-inflammatory agent, indicating that the blockage of the NK-1R pathway in macrophages has the potential to suppress inflammation.

Characterization of microRNAs by deep sequencing in red claw crayfish Cherax quadricarinatus haematopoietic tissue cells after white spot syndrome virus infection.

White spot syndrome virus (WSSV) is one of the most prevalent and widespread viruses in both shrimp and crayfish aquaculture. MicroRNAs (miRNAs) are crucial post-transcriptional regulators and play critical roles in cell differentiation and proliferation, apoptosis, signal transduction and immunity. In this study, miRNA expression profiles were identified via deep sequencing in red claw crayfish Cherax quadricarinatus haematopoietic tissue (Hpt) cell cultures infected with WSSV at both early (i.e., 1 hpi) and late (i.e., 12 hpi) infection stages. The results showed that 2 known miRNAs, namely, miR-7 and miR-184 play key roles in immunity. Meanwhile, 106 novel miRNA candidates were predicted by software in these combined miRNA transcriptomes. Compared with two control groups, 36 miRNAs showed significantly different expression levels after WSSV challenge. Furthermore, 10 differentially expressed miRNAs in WSSV-exposed Hpt cells were randomly selected for expression analysis by quantitative real-time RT-PCR. Consistent with the expression profiles identified by deep sequencing, RT-PCR showed a significant increase or decrease in miRNA expression in Hpt cells after WSSV infection. Prediction of targets of miRNAs such as miR-7, cqu-miR-52, cqu-miR-126 and cqu-miR-141 revealed that their target genes have diverse biological roles, including not only immunity but also transcriptional regulation, energy metabolism, cell communication, cell differentiation, cell death, autophagy, endocytosis and apoptosis. These results provide insight into the molecular mechanism of WSSV infection and highlight the function of miRNAs in the regulation of the immune response against WSSV infection in crustaceans.

White spot syndrome virus entry is dependent on multiple endocytic routes and strongly facilitated by Cq-GABARAP in a CME-dependent manner.

White spot syndrome virus (WSSV) is a lethal pathogen of shrimp and many other crustaceans, including crayfish. However, the molecular mechanism underlying its cellular entry remains elusive due to the lack of shrimp cell lines for viral propagation. Crayfish hematopoietic tissue (Hpt) cell culture was recently established as a good model for WSSV infection study. Here, we showed that multiple endocytic routes, including clathrin-mediated endocytosis (CME), macropinocytosis and caveolae-mediated endocytosis, were indispensably employed for the viral entry into Hpt cell of the crayfish Cherax quadricarinatus. Intriguingly, cellular autophagic activity was positively correlated with efficient viral entry, in which a key autophagy-related protein, γ-aminobutyric acid receptor-associated protein (Cq-GABARAP), that not only localized but also co-localized with WSSV on the Hpt cell membrane, strongly facilitated WSSV entry by binding to the viral envelope VP28 in a CME-dependent manner that was negatively regulated by Cq-Rac1. Furthermore, cytoskeletal components, including Cq-ß-tubulin and Cq-ß-actin, bound to both recombinant rCq-GABARAP and WSSV envelope proteins, which likely led to viral entry promotion via cooperation with rCq-GABARAP. Even under conditions that promoted viral entry, rCq-GABARAP significantly reduced viral replication at an early stage of infection, which was probably caused by the formation of WSSV aggregates in the cytoplasm.

miRNA-133 augments coelomocyte phagocytosis in bacteria-challenged Apostichopus japonicus via targeting the TLR component of IRAK-1 in vitro and in vivo.

In this study, we explored the potential roles of miRNA-133 in regulating TLR pathways in the sea cucumber Apostichopus japonicus. Target screening of RNA-Seq data successfully identified interleukin-1 receptor-associated kinase (AjIRAK-1) as a putative target of miR-133. This result was further validated by negative expression profiles in Vibrio splendidus-challenged coelomocytes and lipopolysaccharide (LPS)-exposed cell cultures. HEK-293T cells transfected with a dual-luciferase reporter fused to the 3'UTR of wild-type or mutant AjIRAK-1 exhibited a 52.9% reduction in luciferase activity (p < 0.01) compared to controls. Co-infection with a miR-133 mimics or a specific siRNA targeting AjIRAK-1 significantly repressed the mRNA and protein expression levels of AjIRAK-1 and its downstream molecules, such as AjTRAF6 and Ajp105, in primary coelomocytes. In contrast, a miR-133 inhibitor significantly increased the expression of these TLR pathway members. The injection of miR-133 agomir or AjIRAK-1 siRNA into sea cucumbers not only decreased the expression of AjIRAK-1 and its downstream molecules but also significantly increased V. splendidus coelomocyte phagocytosis. All of the present data provide direct evidence that miR-133 is involved in TLR cascade modulation through AjIRAK-1 targeting to promote V. splendidus coelomocyte phagocytosis in these non-model invertebrates.

The influence of astragalus polysaccharide and ß-elemene on LX-2 cell growth, apoptosis and activation.

BACKGROUND: Activated hepatic stellate cells are the main source of excessive collagen deposition in liver fibrosis. Here we report the inhibitory effects of the combinational treatment of two natural products, astragalus polysaccharide (APS) and ß-elemene (ELE) on the activation of human liver hepatic stellate cell line LX-2 cells. METHODS: Cultured LX-2 cells were treated with different concentrations of APS or ELE for 24 or 48 hours. Cell viability/apoptosis was measured by MTT assay and Annexin V/PI staining , activation related genes including α-SMA and CD44 expressions were measured by real-time PCR and western blot respectively. RESULTS: The majority of LX-2 cells showed morphological change in the presence of APS or ELE for 24 hours. Treatment with APS + ELE for 24 or 48 hours significantly inhabited the cell proliferation compared with APS or ELE treatment alone on LX-2 cells. APS + ELE may block the up-regulation of α-SMA and CD44 both in mRNA and protein levels through TGF-ß pathway in LX-2 cells. CONCLUSION: APS or ELE treatment alone on LX-2 cells could inhibit cell proliferation and induce apoptosis. The combinational treatment using APS + ELE significantly increased the killing efficiency on LX-2 cells. α-SMA and CD44 expressions was inhibited upon APS + ELE treatment through TGF-ß pathway in LX-2 cells. The results indicated a novel treatment using natural products for liver diseases with anti-fibrotic effect.

[Effects of basic fibroblast growth factor and vascular endothelial growth factor on the proliferation, migration and adhesion of human periodontal ligament stem cells in vitro].

OBJECTIVE: To evaluate the effects of basic fibroblast growth factor (FGF-2) and vascular endothelial growth factor (VEGF) on the proliferation, migration, and adhesion of human periodontal ligament stem cells (PDLSC) in vitro. METHODS: Human PDLSC were cultured in vitro using tissue culture method.The cells were cultured and incubated with various concentrations of FGF-2 and VEGF [A:α-MEM with 2% fetal bovine serum (FBS) (control 1); B:A supplemented with 20 µg/L FGF-2; C:A supplemented with 10 µg/L VEGF; D:A supplemented with 20 µg/L FGF-2 and 10 µg/L VEGF; E:α-MEM with 10% FBS (control 2); F:E supplemented with 20 µg/L FGF-2; G:E supplemented with 10 µg/L VEGF; H:E supplemented with 20 µg/L FGF-2 and 10 µg/L VEGF]. Soluble tetrazolium salts assay was used to evaluate the proliferative capacity on the 1st, 3rd, 5th and 7th d. Then the groups were changed according to result of the proliferation assay (control:α-MEM with 2% FBS; FGF-2 group:control supplemented with 20 µg/L FGF-2; VEGF:control supplemented with 10 µg/L VEGF; Combination group:control supplemented with 20 µg/L FGF-2 and 10 µg/L VEGF). The cell cycle, migration and adhesion capacities were evaluated using flow cytometer, soluble tetrazolium salts assay, cell adhesion assay and scratch wound-healing motility assay. RESULTS: In 2% volume fraction serum containing medium, FGF-2 and VEGF did not stimulate the cell proliferation. However, in 10% serum condition, in groups treated with FGF-2 for 3,5 or 7 d, the A value was (1.22 ± 0.17, 2.15 ± 0.19, 2.72 ± 0.11) respectively, which were significantly higher than that in the control group (0.76 ± 0.16, 1.25 ± 0.06, 1.64 ± 0.09) (P < 0.01) while lower than that in the group treated with FGF-2 and VEGF in combination on the 5 th and 7 th d (2.46 ± 0.17, 3.18 ± 0.27) ( P < 0.05). The A value in the VEGF group on the 5 th and 7 th d is higher than the control group while lower than the FGF-2 group (1.66 ± 0.05, 2.13 ± 0.13) (P < 0.05). Flow cytometer showed that the proliferation index in VEGF group [(34.3 ± 2.0)% ] were significantly lower than those in FGF-2 [(46.8 ± 3.2)%] group and (FGF-2+ VEGF) group [(45.0 ± 4.0)%] but higher than in the control group [(14.5 ± 1.7)%] (P < 0.01). The cell migration assay indicated that the group stimulated with FGF-2 showed no migration promoted effect. Cell adhesion assay showed that the ratio of the adhesive cells number to the original cells number is greater in the FGF-2 group (79 ± 4) than in the VEGF group (62 ± 4) (P < 0.05). Light microscope identified a better cellular morphology on the adhesive surface in the group with FGF-2 than groups without FGF-2. CONCLUSIONS: Both FGF-2 and VEGF could simulate the proliferation of PDLSC in a dose dependent manner, and showed an synergistic effect. FGF-2 was more effective to promote the adhesive capacity of PDLSC compared with VEGF. VEGF could facilitate the migration of PDLSC to the wound side.

Clinical significance of serum transforming growth factor-ß1 in lung cancer.

BACKGROUND: Transforming growth factor-ß1 (TGF-ß1) plays a critical role in human cancer development. Present study aimed to explore the clinical significance of serum TGF-ß1 levels in patients with lung cancer and analyze the relationship between TGF-ß1 and existing tumor markers for lung cancer. METHODS: Serum was collected from 118 patients with lung cancer and 40 healthy volunteers. Serum TGF-ß1 levels were measured by enzyme-linked immunosorbent assay (ELISA), and the association with various clinical characteristics was analyzed. The diagnostic value of TGF-ß1 was assessed alone and in combination with existing tumor markers for lung cancer. RESULTS: Serum TGF-ß1 levels were significantly higher in patients with lung cancer compared to healthy volunteers [0.6 × 10(5) (0.4 × 10(5), 0.9 × 10(5))pg/ml vs 0.5 × 10(5) (0.3 × 10(5), 0.7 × 10(5))pg/ml, P=0.040]. Although there was a positive correlation between serum TGF-ß1 levels and advanced stages, the significant difference was not found between early stages and advanced stages (P=0.116). The ability of serum TGF-ß1 to discriminate lung cancer at a cutoff value of 79,168 pg/ml exhibited sensitivity of 30.6% and specificity of 97.5%. Serum TGF-ß1 levels were correlated to cytokeratin fragment 21-1 (CYFRA21-1; R=0.308, P=0.020) and neuron-specific enolase (NSE; R=0.558, P=0.003). The diagnostic accuracy rates for the existing lung-tumor markers, as SCC, CYFRA21-1, and NSE, were increased from 20.0%, 34.6%, and 45.9% to 48.9%, 51.7%, and 54.5%, respectively by the inclusion of serum TGF-ß1 levels. CONCLUSION: Quantification of serum TGF-ß1 levels by ELISA may provide a novel complementary tool for the clinical diagnosis of lung cancer.

Quantification of plasma hTERT DNA in hepatocellular carcinoma patients by quantitative fluorescent polymerase chain reaction.

PURPOSE: To investigate the levels of human telomerase reverse transcriptase (hTERT) DNA in the plasma of patients with hepatocellular carcinoma (HCC), and to evaluate the diagnostic value and correlation of hTERT DNA with clinical parameters in HCC. METHODS: A real-time quantitative fluorescent polymerase chain reaction (FQ-PCR) system was designed and evaluated. Plasma samples were collected from 60 HCC patients, 21 patients with hepatitis B virus (HBV) and 29 healthy controls. Plasma DNA was extracted and quantified by FQ-PCR. The diagnostic value of plasma hTERT DNA levels and their relationships with clinical characteristics were analyzed statistically. RESULTS: Plasma levels of hTERT DNA in HCC patients were significantly higher than in HBV patients (4.18×104±4.94×104 copies/µl vs 1.21×104±6.63×103 copies/µl, P=0.003) and healthy controls (4.18×104±4.94×104 copies/µl vs 1.44×104±6.61×103 copies/µl, P < 0.001). Receiver operating characteristic curve analysis indicated a sensitivity of 64% and a specificity of 90% for the ability of hTERT DNA levels to detect malignancy at a cutoff value of 1.87×104 copies/µl. Association analysis revealed that plasma hTERT DNA levels were closely related to tumor size, portal vein cancer embolus and TNM stage (P=0.013, P=0.010, and P=0.029, respectively), but were not associated with lymph node metastasis, hepatitis B surface antigen, or α-fetoprotein (AFP) (all P > 0.05). The levels of plasma hTERT DNA in HCC patients with AFP ≤20 ng/ml were significantly higher than in HBV patients (4.59×104±4.98×104 copies/µl vs 1.44×104±6.63×103 copies/µl, P=0.016) and in healthy controls (4.59×104±4.98×104 copies/µl vs 1.21×104±6.63×103 copies/µl, P=0.001). CONCLUSIONS: Quantitation of plasma hTERT DNA by FQ-PCR may provide a novel complementary tool with potential clinical applications for the screening and detection of HCC. Plasma hTERT DNA has the potential to be a broad tumor marker for common cancers.