Plant-on-chip: Core morphogenesis processes in the tiny plant Wolffia australiana.

A plant can be thought of as a colony comprising numerous growth buds, each developing to its own rhythm. Such lack of synchrony impedes efforts to describe core principles of plant morphogenesis, dissect the underlying mechanisms, and identify regulators. Here, we use the minimalist known angiosperm to overcome this challenge and provide a model system for plant morphogenesis. We present a detailed morphological description of the monocot Wolffia australiana, as well as high-quality genome information. Further, we developed the plant-on-chip culture system and demonstrate the application of advanced technologies such as single-nucleus RNA-sequencing, protein structure prediction, and gene editing. We provide proof-of-concept examples that illustrate how W. australiana can decipher the core regulatory mechanisms of plant morphogenesis.

Wogonin affects proliferation and the energy metabolism of SGC-7901 and A549 cells.

Many studies have focused on the identification of therapeutic targets for the treatment of certain types of cancer. Wogonin is a natural flavonoid compound that exhibits a potent anti-cancer effect. The underlying mechanism of wogonin may therefore reveal an effective way to identify novel therapeutic targets. In the current study, growth curves and MTT assays were performed to determine the effects of wogonin in human gastric cancer cells (SGC-7901) and human lung adenocarcinoma cells (A549), respectively. Changes in morphology were observed using hematoxylin and eosin (H&E) staining. The activities of key enzymes in the glycolysis and tricarboxylic acid cycle were measured using spectrophotometry. Western blot analysis was performed to determine the expression levels of hypoxia inducible factor-1α (HIF-1α) and monocarboxylate transporter-4 (MCT-4). Wogonin inhibited cell proliferation in a time- and dose-dependent manner in SGC-7901 and A549 cells. H&E staining suggested that wogonin induced cell morphology changes. In SGC-7901 cells, lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) activities and adenosine triphosphate (ATP) generation were decreased significantly by wogonin treatment compared with the untreated control. In A549 cells, wogonin significantly reduced LDH activity, but exhibited no significant effects on kinase activities or ATP generation. Furthermore, wogonin significantly decreased HIF-1α and MCT-4 protein expression in SGC-7901 cells, but not in A549 cells. The results demonstrated that wogonin inhibited the energy metabolism, cell proliferation and angiogenesis in SGC-7901 and A549 cells by negatively regulating HIF-1α and MCT-4 expression. The differential regulatory roles of wogonin in metabolism-associated enzymes in human gastric cancer and lung adenocarcinoma cells indicated its various antitumor mechanisms. The different metabolic regulatory mechanisms exhibited by wogonin in different tumor tissues should therefore be considered for antitumor therapy.

[Effect of puerarin on hypoxic pulmonary hypertension and accompanying pulmonary fibrosi].

To investigate the effect and regulatory mechanism of puerarin on pulmonary arterial hypertension due to hypoxia and the possible accompanying pulmonary fibrosis, The rat model of hypoxic pulmonary hypertension and the rat model of hypoxia were established. Totally 18 clean-grade SD rats were fed and randomly divided into normal control group, model group and hypoxia+medicine group. Each group received intraperitoneal injection 30 min before modeling every day; hypoxia+medicine group was injected with 20 mg·kg⁻¹ puerarin. Normal control group and model group were injected with the equal volume of 0.9% NaCl solution. Normal control group was cultured under normal conditions in the laboratory, while model group and hypoxia+medicine group were cultured in ahypoxia environment for 21 days to observe rat hypoxic characteristics and make the preliminary judgment about modeling. Afterwards, small animal echocardiography, right cardiac catheterization, HE dyeing and other experiments were used to verify the successful modeling, and puerarin has a therapeutic effect in pulmonary hypertension caused by hypoxia in SD rats. Fluorescence quantitative PCR, Western blot and immunofluorescence method were used to detect the changes caused by hypoxia pulmonary fibrosis-associated protein. It was found that puerarin could be given in anoxia to promote the expressions of CD31, VE-cadherin, inhibit the expressions of α-SMA, vimentin and fibronection, namely the inhibition of vascular wall thickening. Puerarin has the therapeutic effect on the pulmonary hypertension and accompanying pulmonary fibrosis in rats induced by hypoxia.

[Taurine affects expression of ICAM-1, VCAM-1 by p38 pathway in hypoxic endothelial cells].

To investigate the effect of taurine(Tau) on ICAM-1, VCAM-1 by p-p38 pathway in bovine pulmonary artery endothelial cells(PAECs) and explore its mechanism of action. Generation 4-12 cells in primary cultures of PAECs were used in experiments and divided into five groups： control group, hypoxia(hyp) group, inhibitor(SB203580) group, treatment(Tau) group, and treatment+inhibitor(SB+Tau) group. The concentration of Tau：100 mmol•L⁻¹; p38 inhibitor SB203580： 20 µmol•L⁻¹; and the treatment time was 12 h. MTT assay was used to detect the inhibitory effect of different concentrations of Tau on PAECs. Western blot and Real-time PCR method were used to detect the p38 pathway proteins and ICAM-1, VCAM-1 expression levels. Immunofluorescence was used to investigate p38 nuclear displacement situation. The results of MTT showed that the inhibitory effect was gradually increased with increasing concentrations of Tau. Western blot and RT-PCR revealed that the protein and mRNA expression levels of ICAM-1, VCAM-1 were reduced by Tau. Western blot and immunofluorescence showed Tau can inhibit p38 activation. Tau may decrease the expression levels of VCAM-1 and ICAM-1 in endothelial cells induced by hypoxia through MAPK p38 pathway.

Association of interleukin-18 and asthma.

Cytokine-mediated immunity plays a dominant role in the pathogenesis of various immune diseases, including asthma. The recent identification of the family interleukin (IL)-1-related cytokine IL-18 now contributes to our understanding of the fine-tuning of cellular immunity. IL-18 can act as a cofactor for Th2 cell development and IgE production and also plays an important role in the differentiation of Th1 cells. Recent work identified an IL-18 association with the pathogenesis of asthma, wherein increased IL-18 expression was found in the serum of patients. Furthermore, IL-18 polymorphisms with susceptibility to asthma were reported, suggesting that IL-18 may be therapeutically relevant to asthma. In this review, we discuss the role of IL-18 in the pathogenesis of asthma and its therapeutic potential based on current research.

CD147 stimulates hepatoma cells escaping from immune surveillance of T cells by interaction with Cyclophilin A.

T cells play an important role in tumor immune surveillance. CD147 is a member of immunoglobulin superfamily present on the surface of many tumor cells and mediates malignant cell behaviors. Cyclophilin A (CypA) is an intracellular protein promoting inflammation when released from cells. CypA is a natural ligand for CD147. In this study, CD147 specific short hairpin RNAs (shRNA) were transfected into murine hepatocellular carcinoma Hepa1-6 cells to assess the effects of CD147 on hepatoma cells escaping from immune surveillance of T cells. We found extracellular CypA stimulated cell proliferation through CD147 by activating ERK1/2 signaling pathway. Downregulation of CD147 expression on Hepa1-6 cells significantly suppressed tumor progression in vivo, and decreased cell viability when co-cultured with T cells in vitro. Importantly, knockdown of CD147 on Hepa1-6 cells resulted in significantly increased T cells chemotaxis induced by CypA both in vivo and in vitro. These findings provide novel mechanisms how tumor cells escaping from immune surveillance of T cells. We provide a potential therapy for hepatocellular carcinoma by targeting CD147 or CD147-CypA interactions.

[Effect of puerarin on hypoxia induced proliferation of PASMCs by regulating reactive oxygen].

To discuss the effect of puerarin (Pue) on the proliferation of hypoxia-induced pulmonary artery smooth muscle cells (PASMCs) and discuss whether its mechanism is achieved by regulating reactive oxygen. PASMCs of primarily cultured rats (2-5 generations) were selected in the experiment. MTT, Western blot, FCM and DCFH-DA were used to observe Pue's effect the proliferation of PASMCs. The Western blot was adopted to detect whether ROS participated in Pue's effect in inhibiting PASMC proliferation. The PASMCs were divided into five groups: the normoxia group, the hypoxia group, the hypoxia + Pue group, the hypoxia + Pue + Rotenone group and the hypoxia + Rotenone group, with Rotenone as the ROS blocker. According to the results, under the conditions of normoxia, Pue had no effect on the PASMC proliferation; But, under the conditions of hypoxia, it could inhibit the PASMC proliferation; Under the conditions of normoxia and hypoxia, Pue had no effect on the expression of the tumor necrosis factor-α (TNF-α) among PASMCs, could down-regulate the expression of hypoxia-induced cell cycle protein Cyclin A and proliferative nuclear antigen (PCNA). DCFH-DA proved Pue could reverse ROS rise caused by hypoxia. Both Rotenone and Pue could inhibit the up-regulated expressions of HIF-1α, Cyclin A, PCNA caused by anoxia, with a synergistic effect. The results suggested that Pue could inhibit the hypoxia-induced PASMC proliferation. Its mechanism may be achieved by regulating ROS.

[Effect of puerarin on PI3K/AKT pathway-mediated apoptosis of PASMCs].

To discuss the effect of puerarin (Pue) on the proliferation of hypoxia-induced pulmonary artery smooth muscle cells (PASMCs) and discuss whether the extracellular signal PI3K/AKT pathway was involved in the Pue-induced PASMC apoptosis. With the serum starvation group (SD group) as the control group, the MTT colorimetry method, Annexin V-FITC apoptosis detection kit and Western blot were used to detect Pue's effect on apoptosis of rat PASMCs. The protein immunoblot assay was used to detect whether PI3K/AKT pathway was involved in the inhibition of hypoxia-induced PASMC apoptosis process. The results show that under normoxic conditions, Pue had no effect on PASMC apoptosis; Under hypoxia conditions, Pue can inhibit PASMC apoptosis; Under normoxic and hypoxic conditions, Pue had no effect on TNF-α expression. Pue can reverse hypoxia-induced Bcl-2 (P <0.01), up-regulate it and down-regulated Bax (P <0.01). Under normoxic conditions, Pue had no effect on P-AKT expression. Both LY294002 and Pue can inhibit hypoxia-induced Bcl-2, up-regulation of P-AKT expression and down-regulation of Bax expression. Compared with the hypoxia + Pue group or the hypoxia + LY294002 group, the hypoxia + Pue + LY294002 group showed more significantly changes in Bcl-2, Bax, P-AKT expressions. The results show that, Pue can inhibit the hypoxic-induced PASMC apoptosis, which may be regulated through PI3K/AKT pathway.

The relationship between UGT1A1 gene polymorphism and irinotecan effect on extensive-stage small-cell lung cancer.

AIMS: To analyze the distribution of uridine diphosphate glucuronosyltransferase (UGT)1A1 gene polymorphisms in Chinese patients with extensive-stage small-cell lung cancer (E-SCLC), and to evaluate correlations between the UGT1A1 gene polymorphisms and toxicity, and efficacy of irinotecan (CPT-11) based regimen in the patients with E-SCLC. METHODS: The study analyzed the distribution of UGT1A1*28/*6 gene polymorphisms by polymerase chain reaction amplification and pyrosequencing. The analysis of UGT1A1*28 and UGT1A1*6 gene polymorphisms was performed in 67 patients with E-SCLC admitted to the clinic in the Department of Oncology from June 2011 to January 2013. A total of 67 cases with E-SCLC treated with irinotecan (CPT-11)-based regimen were enrolled to observe the adverse events and efficacy during the chemotherapy, including objective response rate, progression-free survival (PFS) and overall survival (OS). The correlation between UGT1A1 gene polymorphisms and severe adverse events was analyzed. The influences of UGT1A1*6/*28 polymorphisms on objective response rate, PFS, and OS were also analyzed. RESULTS: The distribution of UGT1A1 genotypes among 67 patients was as follows: UGT1A1*28 wild-type (WT) genotype TA6/6 (56, 83.6%), heterozygous mutant genotype TA6/7 (11, 16.4%); UGT1A1*6 WT genotype G/G (45, 67.2%), heterozygous mutant genotype G/A (22, 32.8%); no significant difference of PFS and OS was observed between different genotypes. The incidence of grade 3 and 4 delayed diarrhea and neutropenia in the patients carrying UGT1A1*6 G/A mutation was higher than that in the WT genotype (36.4% vs 6.6% P=0.034; 27.2% vs 4.4% P=0.026, respectively). The incidence of grade 3 and 4 thrombocytopenia in the patients carrying UGT1A1*28 TA6/7 mutation was higher than that in the WT genotype (27.2% vs 1.8% P=0.017). The patients simultaneously carrying UGT1A1*28 TA6/7 and UGT1A1*6 G/A mutations were prone to suffering grade 3 and 4 delayed diarrhea and neutropenia. CONCLUSION: For irinotecan-based regimens in E-SCLC, the UGT1A1*28 and UGT1A1*6 locus mutations can be regarded as predictors for severe adverse events. We also found that neither clinical response nor prognosis was significantly associated with the UGT1A1 gene polymorphisms.

Anti-inflammation Effects of Oxysophoridine on Cerebral Ischemia-Reperfusion Injury in Mice.

Oxysophoridine (OSR) is a bioactive alkaloid extracted from the Sophora alopecuroides Linn. Our aim is to explore the potential anti-inflammation mechanism of OSR in cerebral ischemic injury. Mice were intraperitoneally pretreated with OSR (62.5, 125, and 250 mg/kg) or nimodipine (Nim) (6 mg/kg) for 7 days followed by cerebral ischemia. The inflammatory-related cytokines in cerebral ischemic hemisphere tissue were determined by immunohistochemistry staining, Western blot and enzyme-like immunosorbent assay (ELISA). OSR-treated groups observably suppressed the nuclear factor kappa B (NF-κB), intercellular adhesion molecule-1 (ICAM-1), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). OSR-treated group (250 mg/kg) markedly reduced the inflammatory-related protein prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6), and interleukin-8 (IL-8). Meanwhile, it dramatically increased the interleukin-10 (IL-10). Our study revealed that OSR protected neurons from ischemia-induced injury in mice by downregulating the proinflammatory cytokines and blocking the NF-κB pathway.

Oxymatrine attenuated hypoxic-ischemic brain damage in neonatal rats via improving antioxidant enzyme activities and inhibiting cell death.

Oxymatrine (OMT), an active constituent of Chinese herb Sophora flavescens Ait, has been proved to possess anti-tumor, anti-oxidant, anti-inflammatory, and anti-apoptotic activities. Previous study has demonstrated that OMT had protective roles on multiple in vitro and in vivo brain injury models including regulation of apoptosis-related proteins caspase-3, Bax and Bcl-2. In this study, we investigated whether this protective effect could apply to neonatal hypoxic-ischemic brain damage. Seven-day-old Sprague-Dawley rats were treated with the left carotid artery ligation followed by exposure to 8% oxygen (balanced with nitrogen) for 2.5 h at 37 °C. In sham group rats, neither ligation nor hypoxia was performed. After two successive days intraperitoneal injection with OMT (30, 60 and 120 mg/kg), Nimodipine (1 mg/kg), and saline, brain infarct volume was estimated, histomorphology changes were performed by hematoxylin-eosin (HE) staining as well as electron microscopy. In addition, the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT), and total antioxidant capacity (T-AOC), as well as production of malondialdehyde (MDA) were assayed in ipsilateral hemisphere homogenates to evaluate the redox status after hypoxic-ischemic. Expression of apoptosis-related proteins Caspase-3, Bax and Bcl-2 in brain were analyzed by western-blot analysis and immunofluorescence. Administration of OMT significantly decreased brain infarct volume and the percentage of injured cells, and ameliorated histopathology and morphological injury as well. Furthermore, OMT obviously increased the activities of SOD, GSH-Px, CAT and T-AOC, and decreased MDA content. Western-blot analysis showed a marked decrease in Caspase-3 expression and increase in the ratio of Bcl-2/Bax after OMT (120 mg/kg) post-treatment as compared with hypoxic-ischemic group. These results suggest that OMT exerts a neuroprotective effect against hypoxic-ischemic brain damage in neonatal rats, which is likely to be mediated through increasing anti-oxidant enzyme activities and inhibiting cell death.

Trefoil factor 3 as a novel biomarker to distinguish between adenocarcinoma and squamous cell carcinoma.

In carcinoma, such as of the lung, the histological subtype is important to select an appropriate therapeutic strategy for patients. However, carcinomas with poor differentiation cannot always be distinguished on the basis of morphology alone nor on clinical findings. Hence, delineation of poorly differentiated adenocarcinoma and squamous cell carcinoma, the 2 most common epithelial-origin carcinomas, is pivotal for selection of optimum therapy. Herein, we explored the potential utility of trefoil factor 3 (TFF3) as a biomarker for primary lung adenocarcinoma and extrapulmonary adenocarcinomas derived from different organs. We observed that 90.9% of lung adenocarcinomas were TFF3-positive, whereas no expression of TFF3 was observed in squamous cell carcinomas. The subtype of lung carcinoma was confirmed by four established biomarkers, cytokeratin 7 and thyroid transcription factor 1 for adenocarcinoma and P63 and cytokeratin 5/6 for squamous cell carcinoma. Furthermore, expression of TFF3 mRNA was observed by quantitative PCR in all of 11 human lung adenocarcinoma cell lines and highly correlated with markers of the adenocarcinomatous lineage. In contrast, little or no expression of TFF3 was observed in 4 lung squamous cell carcinoma cell lines. By use of forced expression, or siRNA-mediated depletion of TFF3, we determined that TFF3 appeared to maintain rather than promote glandular differentiation of lung carcinoma cells. In addition, TFF3 expression was also determined in adenocarcinomas from colorectum, stomach, cervix, esophagus, and larynx. Among all these extrapulmonary carcinomas, 93.7% of adenocarcinomas exhibited TFF3 positivity, whereas only 2.9% of squamous cell carcinomas were TFF3-positive. Totally, 92.9% of both pulmonary and extrapulmonary adenocarcinomas exhibited TFF3 positivity, whereas only 1.5% of squamous cell carcinomas were TFF3-positive. In conclusion, TFF3 is preferentially expressed in adenocarcinoma and may function as an additional biomarker for distinguishing adenocarcinoma from squamous cell carcinoma.

Relationship between the SER treatment period and prognosis of patients with small cell lung cancer.

PURPOSE: To explore the relationship between SER (time between the start of any treatment and the end of radiation therapy) and the survival of patients with limited-stage small cell lung cancer. MATERIALS AND METHODS: Between 2008 and 2013, 135 cases of limited-stage small cell lung cancer (LS-SCLC) treated with consecutively curative chemoradiotherapy were included in this retrospective analysis. In terms of SER, patients were divided into early radiotherapy group (SER<30 days, n=76) and late radiotherapy group (SER≥30 days, n=59) with a cut- off of SER 30 days. Outcomes of the two groups were compared for overall survival. RESULTS: For all analyzable patients, median follow-up time was 23.8 months and median overall survival time was 16.8 months. Although there was no significant differences in distant metastasis free survival between the two groups, patients in early radiotherapy group had a significantly better PFS (p=0.003) and OS (p=0.000). CONCLUSIONS: A short SER may be a good prognostic factor for LD-SCLC patients treated with concurrent chemoradiotherapy.

Oxysophoridine protects against focal cerebral ischemic injury by inhibiting oxidative stress and apoptosis in mice.

Our previous studies have demonstrated that oxysophoridine (OSR) has protective effects on cerebral neurons damage in vitro induced by oxygen and glucose deprivation. In this study, we further investigated whether OSR could reduce ischemic cerebral injury in vivo and its possible mechanism. Male Institute of cancer research mice were intraperitoneally injected with OSR (62.5, 125 and 250 mg/kg) for seven successive days, then subjected to brain ischemia induced by the model of middle cerebral artery occlusion. After reperfusion, neurological scores and infarct volume were estimated. Morphological examination of tissues was performed. Apoptotic neurons were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining. Oxidative stress levels were assessed by measurement of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels. The expression of various apoptotic markers as Caspase-3, Bax and Bcl-2 were investigated by immunohistochemistry and Western-blot analysis. OSR pretreatment groups significantly reduced infract volume and neurological deficit scores. OSR decreased the percentage of apoptotic neurons, relieved neuronal morphological damage. Moreover, OSR markedly decreased MDA content, and increased SOD, GSH-Px activities. Administration of OSR (250 mg/kg) significantly suppressed overexpression of Caspase-3 and Bax, and increased Bcl-2 expression. These findings indicate that OSR has a protective effect on focal cerebral ischemic injury through antioxidant and anti-apoptotic mechanisms.

Effect of stachydrine on endoplasmic reticulum stress-induced apoptosis in rat kidney after unilateral ureteral obstruction.

Our study aimed at determining the effect of stachydrine on the PERK, CHOP, and caspase-3 in rat kidney with RIF. Rats were randomly divided into control group, model group, enalapril group, high stachydrine group, medium stachydrine group, and low stachydrine group. RIF models of five groups were developed by unilateral ureteral obstruction except the control group. The rats were sacrificed 12 days after surgery and blood samples were collected. Serum creatinine (Scr) and blood urea nitrogen (BUN) levels were detected. Renal tubular damage index was determined by HE staining. The area percentage of RIF was determined by the Masson method. Expressions of PERK, CHOP, and caspase-3 in kidney were determined by immunohistochemistry. Tubulointerstitial injury index, RIF, serum Scr, BUN level, and expressions of PERK, CHOP, and caspase-3 were different between the model and treatment groups (P < 0.05; P < 0.01). The expressions of PERK, CHOP, and caspase-3 in nephridial tissue were reduced (P < 0.05), tubulointerstitial injury and RIF were reduced (P < 0.05), and Scr and BUN were lower (P < 0.05) in the high stachydrine group than those in the enalapril group. The expressions of PERK, CHOP, and caspase-3 were reduced in the endoplasmic reticulum stress-related apoptosis pathway after stachydrine treatment. Consequently, apoptosis was prevented, and RIF was inhibited.

[Preliminary study on the difference in immune protection between Echinococcus granulosus recombinant ferritin and recombinant mMDH].

OBJECTIVE: To evaluate the immunoprotective activity of the Egrecombinant ferritin and Egrecombinant mMDH proteins in mice. METHODS: Thirty ICR mice were divided into 3 groups and immunized by injection of adjuvant-emulsified rEgferritin, rEgmMDH and PBS, respectively, in multiple spots at back, for 3 times with an interval of 2 wk. Two weeks after the last immunization, the mice of the 3 groups were infected intraperitoneally with 0.1 ml suspension containing about 1 500 Echinococcus granulosus (Eg) protoscoleces. The mice were sacrificed 22 wk after infection and the Eg cysts were collected and measured. Spleens were taken for detecting CD4+ T cells and CD8+ T cells and ratio calculated. RESULTS: Eg cysts were found in 30% (3/10) of the mice in the rEgferritin group with 5 cysts altogether; cysts were received in all the mice in the rEgmMDH group with 118 cysts totally; and cysts were found in 7 of 9 mice in the PBS control with 35 cysts totally. The mice in the rEgferritin group showed an 84.7% protection but revealed no protection in the rEgmMDH group. The CD4+ T cells were significantly higher in the rEgferritin group than the control, but no statistical difference was found in CD8+ T cells and CD4+/CD8+ ratio between the 2 groups. There was no considerable change in the T cells and ratio in the rEgmMDH group compared to the control. CONCLUSION: The Egrecombinant ferritin can inhibit the growth of Eg while the Egrecombinant mMDH seems promoting its growth in mice.

[Development of a loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus].

OBJECTIVE: This study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for detection of Vibrio parahaemolyticus (V. parahaemolyticus). METHODS: The specificity of this assay was evaluated by using a panel of 33 strains of V. parahaemolyticus and 22 strains of other species bacteria. The sensitivity was determined by using serial dilutions of V. parahaemolyticus (ATCC 17802) chromosomal DNA (5×10(0) - 5×10(5) copies/µl). The samples were also tested by using qualification PCR assay and Taqman real-time PCR assay in parallel for comparison with LAMP. RESULTS: Both sensitivity and specificity of LAMP assay, PCR assay and Taqman real-time PCR assay were 100% (22/22, 33/33, respectively). The detection limits of above three methods assay were 5×10(1) copies/µl, 5×10(3) copies/µl and 5×10(2) copies/µl, respectively. The reaction period of time needed of the above three assays was 22 min, 3 h, 50 min, respectively. CONCLUSION: Compared to qualification PCR assay and Taqman real-time PCR assay, the established LAMP assay was better in low detection limit and less reaction time, which made it an ideal method for quick detection of V. parahaemolyticus.

ORF3 of duck circovirus: a novel protein with apoptotic activity.

Duck circovirus (DuCV) is classified in the genus Circovirus of the Circoviridae family. Two major open reading frames (ORFs), encoding the replicase (ORF1/rep) and the capsid protein (ORF2/cap), have been recognized for DuCV. Sequence analysis show that another major conserved ORF (named ORF3) is located in the complementary strand of ORF1/rep of DuCV, and its function remains to be investigated. In this study, the ORF3 of DuCV was expressed in recombinant baculovirus-infected Sf9 cells. By IFA and Western blot analysis, the ORF3 protein was positive for the sera from ducks infected with DuCV. The percentages of apoptotic cells of the Sf9 cells infected with the recombinant baculovirus encoding ORF3 of DuCV were significantly higher than (P<0.05) that of the Sf9 cells infected with wild-type baculovirus at 24, 48 and 72 h postinfection. Based on our knowledge, we deduced that the ORF3 protein of DuCV might play an important role in viral pathogenesis via its apoptotic activity.

Effects of small interfering RNA targeting heparanase-1 combined with heparin on invasiveness of mouse hepatocellular carcinoma cell lines.

BACKGROUND AND OBJECTIVE: Heparanase-1 (HPA-1) can promote angiogenesis and metastasis of malignant tumors and plays an important role in the genesis and development of tumors. This study was to explore the effects of specific small interfering RNA (siRNA) targeting HPA-1 combined with heparin on invasiveness of mouse hepatocellular carcinoma cells. METHODS: The expression of HPA-1 in Hca-F, Hca-P, and Hepa1-6 cells, which have high, low, and no metastatic potential, respectively, was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and enzyme-linked immunosorbent assay (ELISA). After transfection with two specific siRNAs targeting HPA-1, siRNA-1 and siRNA-2, and treatment with heparin, invasiveness of Hca-F cells was observed by Matrigel invasion assay. RESULTS: HPA-1 was negative in Hepa1-6 cells while positive in both Hca-F and Hca-P cells. The expression levels of both HPA-1 mRNA and protein were obviously higher in Hca-F cells than in Hca-P cells. HPA-1 proteins could be secreted into culture supernatant of Hca-F and Hca-P cells, and the amount of secreted HPA-1 detected by Western blot analysis was larger in Hca-F cells than in Hca-P cells (1.34 ± 0.02 vs. 0.60 ± 0.01, P < 0.001), which was consistent with the results of ELISA. Both siRNA-1 and siRNA-2 downregulated the expression of HPA-1 and the siRNA-2 did more efficiently. The number of invasive Hca-F cells treated with siRNA-2 or heparin alone was larger than that of Hca-F cells treated with combination of them (9 ± 1 vs. 4 ± 1, P = 0.013; 15 ± 2 vs. 4 ± 1, P = 0.008), but smaller than that of untreated Hca-F cells (9 ± 1 vs. 22 ± 2, P = 0.006; 15 ± 2 vs. 22 ± 2, P = 0.026). CONCLUSION: The combined application of specific siRNA targeting HPA-1 and heparin is more effective in inhibiting the invasiveness of mouse hepatoma cells.