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Contrast acute kidney injury refers to acute renal failure due to the application of contrast agents. Astaxanthin, as an antioxidant, can improve early acute kidney injury in severely burned rats. However, the mechanism of astaxanthin for renal protection is still unclear. In this study, the rat renal tubular epithelial cells (NRK-52E) were treated with iohexol, astaxanthin, astaxanthin plus nicotinamide and nicotinamide. Subsequently, the nuclear morphology was observed by fluorescence staining of DAPI DNA, the apoptosis was detected by flow cytometry, the mitochondrial membrane potential was detected by JC-1 and the SIRT1, P53, Bax, Bcl-2 protein expression level was detected by Western blotting. We found that astaxanthin can reduce nuclear pyknosis and nuclear deep staining, decrease the number of apoptotic cells, up-regulate the expression of proapoptotic proteins P53 and Bax and up-regulate the expression of anti-apoptotic protein Bcl-2 by increasing SIRT1 expression level, thereby exerting protective effects on renal tubular epithelial cells. At the same time, nicotinamide has the opposite effect on the NRK-52E compared with astaxanthin. These results indicated that astaxanthin may provide a new option for the prevention of contrast-induced acute kidney injury., Competing Interests: None.

eIF3a is the largest subunit of eIF3 complex and is a key player in translational control. Recently eIF3a is recognized as a proto-oncogene, which is overexpressed and connected to tumorigenesis of many cancers. However, the mechanistic roles of eIF3a during the tumorigenesis remain largely elusive. Here, we report that depletion of eIF3a significantly reduced HIF1α protein level and cellular glycolysis ability. Mechanistically, we found that eIF3a regulates HIF1α protein synthesis through internal ribosomal entry site (IRES)-dependent translation. Importantly, through analyses of our own sample collection, we found that eIF3a is overexpressed in hepatocellular carcinoma (HCC) tissues, and a high level of eIF3a predicts poor prognosis of HCC patients. TCGA analyses further confirmed that eIF3a is coincident with an elevated activity of HIF1α pathway genes. Collectively, we identify eIF3a as a regulator for glycolysis through HIF1α IRES-dependent translational regulation, which may be a potential therapeutic target for HCC., Competing Interests: None.

Purpose: The present study was designed to further investigate the protective effect of astaxanthin (AST) on contrast-induced acute kidney injury (CI-AKI) in rats and the relationship between SIRT1-p53 pathway and astaxanthin., Methods: 40 adult male Sprague Dawley (SD) rats were randomly divided into five groups (n = 8/group): control (CON), normal rats treated with AST (AST), CM-treated (CM), CM rats treated with isoform of nitric oxide synthase (iNOS) inhibitor (iNOS + CM), and CM rats treated with AST (AST + CM). Serum creatinine (Scr) and blood urea nitrogen (BUN) values were measured at 72 h following the procedure. Hematoxylin and eosin (H-E) staining was used to observe the pathologic changes of kidney. Tunel staining was used to test apoptosis of kidney tubules. Oxidative stress, SIRT1 activity, nitric oxide (NO), and 3-nitrotyrosine (3-NT) content were individually measured with the commercial available kits., Results: Compared with the CON group, Scr and BUN levels significantly increased in the CM group (P < 0.05), and the values in two pre-treatment groups (iNOS + CM and AST + CM) had significantly decreased (P < 0.05). H-E and Tunel staining had shown that renal tubular injury was severe in CM group. The renal injury score and apoptosis index in the two pre-treatment groups also decreased (P < 0.05). The present study showed that in CM group the levels of oxidative stress indicators significantly increased, and the activities of antioxidant stress indicators significantly decreased. These indicators in two pre-treatment groups significantly improved (P < 0.05). In the CM group the expression levels of SITR1 significantly increased, and the ac-p53/p53 significantly increased (P < 0.05). Compared with the CM group, in AST + CM group the expression levels of SIRT1 increased, the expression levels of p53 and ac-p53/p53 decreased (P < 0.05).The levels of NO and 3-NT in CM group significantly increased (P < 0.05). Compared the CM group, the levels in the two pre-treatment groups significantly decreased (P < 0.05)., Conclusions: Astaxanthin has a protective effect on CI-AKI, the mechanism may be related to the SIRT1-p53 pathway. Astaxanthin can reduce the content of NO and 3-NT in renal tissue of CI-AKI, and alleviate the renal injury induced by contrast agents.

Background & Aims: Treatment of liver cancer remains challenging because of a paucity of drugs that target critical dependencies. Sorafenib is a multikinase inhibitor that is approved as the standard therapy for patients with advanced hepatocellular carcinoma, but it only provides limited survival benefit. In this study we aimed to identify potential combination therapies to improve the clinical response to sorafenib., Methods: To investigate the cause of the limited therapeutic effect of sorafenib, we performed a CRISPR-Cas9 based synthetic lethality screen to search for kinases whose knockout synergizes with sorafenib. Synergistic effects of sorafenib and selumetinib on cell apoptosis and phospho-ERK (p-ERK) were analyzed by caspase-3/7 apoptosis assay and western blot, respectively. p-ERK was measured by immunochemical analysis using a tissue microarray containing 78 liver cancer specimens. The in vivo effects of the combination were also measured in two xenograft models., Result: We found that suppression of ERK2 (MAPK1) sensitizes several liver cancer cell lines to sorafenib. Drugs inhibiting the MEK (MEK1/2 [MAP2K1/2]) or ERK (ERK1/2 [MAPK1/3]) kinases reverse unresponsiveness to sorafenib in vitro and in vivo in a subset of liver cancer cell lines characterized by high levels of active p-ERK, through synergistic inhibition of ERK kinase activity., Conclusion: Our data provide a combination strategy for treating liver cancer and suggest that tumors with high basal p-ERK levels, which are seen in approximately 30% of liver cancers, are most likely to benefit from such combinatorial treatment., Lay Summary: Sorafenib is approved as the standard therapy for patients with advanced hepatocellular carcinoma, but only provides limited survival benefit. Herein, we found that inhibition of the kinase ERK2 increases the response to sorafenib in liver cancer. Our data indicate that a combination of sorafenib and a MEK inhibitor is most likely to be effective in tumors with high basal phospho-ERK levels., (Copyright © 2018 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Purpose: The study was processed to investigate the effect of astaxanthin (AST; 3,3-dihydroxybeta, beta-carotene-4,4-dione) on the acute kidney injury induced by iohexol and the relationship with SIRT1/FOXO3a signal pathway., Methods: Thirty male Sprague Dawley rats were randomly divided into five groups as follows: control group (CON; olive oil only), contrast media group, astaxanthin control group (100 mg/kg), low astaxanthin dose group (LAG, 50 mg/kg) and high astaxanthin dose group (HAG, 100 mg/kg). As followed, serum creatinine (SCr), blood urea nitrogen (BUN), the oxidative stress markers and apoptosis-related proteins were detected. Human proximal tubular epithelial cells (HK-2) were cultured in DMEM/F12 medium in vitro and then randomly divided into appropriate experimental groups: normal group (N), dimethyl sulfoxide (DMSO), iohexol group (I), iohexol + (1.0, 10.0 μmol/L) astaxanthin group (I + LAST; I + HAST), iohexol + SIRT1 inhibitors (nicotinamide) group (NA) and iohexol + si-RNA FOXO3a group (si-RNA FOXO3a); when cultured for 24 h, cell proliferation ability was tested by cell counting kit (CCK-8), reactive oxygen species (ROS) were detected by flow cytometry and the expression of SIRT1 and FOXO3a were observed using western blot., Results: At the end of the experiment, the levels of SCr, BUN and malondialdehyde (MDA) were all increased in the CM group. The LAG and HAG reduce superoxide anion (SOD) activity, catalase (CAT) activity, glutathione peroxidase (GPx) activity and glutathione (GSH) content, as well as SCr and BUN level. Moreover, apoptosis-associated proteins, caspase 3 p17, bax and bcl-2 were upregulated. In HK-2 cells, after adding iohexol, proliferation and intracellular ROS levels were significantly increased. Using astaxanthin in advance after the intervention, the result is opposite. SIRTl inhibitors NA can reduce the expression of SIRTl and decrease the expression of FOXO3a protein. Si-RNA FOXO3a reduces the expression of FOXO3a but had no significant effect on the expression of SIRT1., Conclusions: Our study demonstrates that the intervention of astaxanthin could attenuate the oxidative stress and apoptosis in contrast-induced acute kidney injury (CI-AKI), and the SIRT1/FOXO3a pathway participates in the contrast-induced apoptosis of HK-2 cells. Finally, astaxanthin reduces CI-AKI by suppression of apoptosis, which may be through inhibition of SIRT1/FOXO3a pathways.

Tryptophan hydroxylase (TPH) was stained in the central nervous system of the Neanthes japonica (Polychaeta, Annelida), using sheep anti-tryptophan hydroxylase antibody by the Streptavidin-Peroxidase immunohistochemical method and Colophony-Paraffin embedded section technique. The immunohistochemistry results revealed that the TPH is distributed in the brain and ventral nerve cord, which is consistent with that of serotonin (5-hydroxytryptamine, 5-HT) that labeled by anti-serotonin antibody. Using the rapid amplification of cDNA ends (RACE) technique, TPH cDNA cloned from Neanthes japonica's central nervous system was 1778bp, which encodes predicted protein of 463 amino acid residues. The co-localization of TPH and 5-HT indicated that the specific TPH was responsible for the central serotonin synthesis in the central nervous system of annelida, TPH and 5-HT not only could be as the novel mutual corroboration marker to detect serotonergic neurons, but also provides the evidences for the evolution of aromatic amino acid hydroxylase genes. Anat Rec, 300:415-424, 2017. © 2016 Wiley Periodicals, Inc., (© 2016 Wiley Periodicals, Inc.)

The aim of this study was to explore the mechanism underlying the antidepression activity of paeoniflorin, the main active ingredient of paeony extract and Shuyu capsules, and determine its effect on the calmodulin/calmodulin-dependent protein kinase II (CaM/CaMKII) signalling pathway and on the possible target, the voltage-gated calcium channel (Ca v ). Rats at the nonacceptance stage were selected for premenstrual syndrome (PMS) depression modelling. Behavioural assays were used for model testing. Rats were given Shuyu capsules, paeony extract, and bupleurum. Western blot analysis was used to assess the expression levels of calcium voltage-gated channel subunit alpha 1 C (CACNA1C), brain-derived neurotrophic factor, and CaM/CaMKII signalling pathway proteins. Intracellular Ca 2+ concentration in CHO cell line was measured using Fluo-4-AM and whole-cell patch clamps. The PMS depression model was successfully established and demonstrated that Shuyu can mitigate depressive behaviour in a rat PMS model. Paeony extract did not affect CACNA1C protein expression in rat hippocampi but did affect Ca v 1.2-mediated CaM/CaMKII signalling pathways. Paeoniflorin significantly inhibited KCl-induced increases in intracellular Ca 2+ concentration and Ca v 1.2 current density. Further, it may function via the CaM/CaMKII pathway and its downstream signalling molecules by regulating Ca v 1.2, thus playing an important role in the treatment and alleviation of affective disorders.

The molecular mechanisms that control metastasis of hepatocellular cancer (HCC) are still poorly understood. It has been determined that microRNA (miRNA) expression has tissue and cell specific, and decreased expression of specific miRNA could induce tumor genesis or metastasis. In this study, we identified that miR-17-5p was expressed lower in high metastatic capability HCC cell lines HCCLM3 and MHCC97H than low metastatic HCC cell line HepG2 by real-time (RT)-PCR. Restoration of miR-17-5p could significantly repress the invasiveness and metastasis of MHCC97H cell line. Furthermore, we validated c-Myc as a downstream and functional target of miR-17-5p using luciferase reporter assay. Immunohistochemical assay revealed that the expression of c-Myc protein levels was significantly increased in cancerous tissues compared with para-tumor tissues. After clinical data analysis, we observed that the higher level of c-Myc was significantly associated with a reduced overall survival (p = 0.0209). Consistent with previous research, we also demonstrated that c-Myc could upregulate the expression of miR-17-5p. Taken together, our data indicated that there is a regulatory feedback loop between miR-17-5p and c-Myc, in which miR-17-5p could suppress some of the distinguishing features, invasion, and metastasis, of oncogenic c-Myc in HCC cells, and meanwhile, miR-17-5p is upregulated by c-Myc role as a transcription factor, although further studies are still needed.

Objective: To observe the effect of sodium bicarbonate combined with ulinastatin on cholinesterase activity for patients with acute phoxim pesticide poisoning., Methods: A total of 67 eligible patients with acute phoxim pesticide poisoning, Who were admitted to the emeryency department of hospital from March 2011 to February 2014, Acording to different treatments au patients were randomly divided into the conventional treatment group (n=34) and the sodium bicarbonate+ulinastatin group (n=35) . The conventional treatment group were given thorough gastric lavage with water, the sodium bicarbonate + ulinastatin group were given gastric lavage with 2% sodium bicarbonate solution. Both groups were given such treatments as catharsis, administration of oxygen, fluid infusion, diuresis, and antidotes such as atropine and pralidoxime methylchloride. On the basis of comprehensive treatment, people in the sodium bicarbonate+ulinastatin group were given 5% sodium bicarbonate injection and ulinastatin. The clinical effect of the two groups were compared., Results: The serum cholinesterase activity of the sodium bicarbonate+ulinastatin group was significantly higher than the conventional treatment group from the 5th day, and the difference was statistically significant (P<0.05) . The total atropine dosage, total pralidoxime methylchloride dosage and hospitalization days were better than the conventional treatment group, and the differences were statistically significant (P<0.05) . The difference in the time of atropinization between the two groups was not statistically significant (P>0.05) . The results of arterial blood pH, HCO3- of the sodium bicarbonate + ulinastatin group were higher than the conventional treatment group, and the difference of HCO3- at the 10th day was statistically significant (P<0.05) ., Conclusions: Sodium bicarbonate combined with ulinastatin can improve the therapeutic effect and reduce complications in the treatment of acute phoxim pesticide poisoning, and have beneficial effects on the recovery of cholinesterase activity.

Objective: The purpose of the present study was to determine whether type III IFN can modulate the autophagic response in human osteosarcoma cell., Methods: Human osteosarcoma cell were treated with Interferon-λ1. We investigated that Interferon-λ1 could inhibit the invasive ability of osteosarcoma cells by Matrigel invasion assay. Autophagy were assessed by acridine orange staining, MDC staining and Transmission electron microscopy., Results: In this study, we found that Interferon-λ1 could inhibit the invasive ability of osteosarcoma cells. Acridine orange staining and MDC staining showed that Interferon-λ1 triggered the accumulation of acidic vesicular and autolysosomes in osteosarcoma cell. The acridine orange osteosarcoma cell ratios were 3.6 ± 0.5%, 4.5 ± 0.8%, and 12.4 ± 1.7% after treatment with 1, 10, and 100 ng/mL Interferon-λ1 for 48 h. Osteosarcoma cell cells treated with 100 ng/mL Interferon-λ1 for 48 h developed autophagy some-like characteristics, including single or double-membrane vacuoles containing intact and degraded cellular debris., Conclusions: Interferon-λ1 could inhibit the invasive ability of osteosarcoma cells. Autophagy can be induced in a dose-dependent manner by treatment with Interferon-λ1 in osteosarcoma cell.

Malic enzyme 1 (ME1) links the glycolytic and citric acid cycles and is important for NADPH production, glutamine metabolism, and lipogenesis. Recently, its deregulation has been implicated in the progression of various cancers. However, the role of ME1 in the progression of hepatocellular carcinoma (HCC) remains unclear. In this study, we utilized short hairpin RNA-mediated gene silencing to investigate the biological effects of ME1 depletion in HCC and determined its prognostic significance in HCC. ME1 expression was examined by real-time (RT)-PCR and Western blot using five HCC cell lines and one normal liver cell line. We used polyethylenimine nanoparticles to deliver a short hairpin RNA to induce cessation of ME1 expression in HCC cells. Changes in NADPH production and reactive oxygen species (ROS) production were studied. Metastatic potentials of HCC cells were evaluated in vitro. Furthermore, we evaluated the protein level of ME1 in para-tumor and cancerous tissues of 65 HCC patients with detailed clinical, pathological, and clinical follow-up data. Patients' survivals were further assessed as well. Upregulated ME1 expression was observed in HCC cell lines. Downregulation of ME1 attenuated NADPH production and stimulated ROS production. Silencing ME1 was noted to inhibit migratory and invasive properties of HCC cells by inducing the E-cadherin expression and decreasing of N-cadherin and vimentin expression in a ROS-dependent pathway. Overexpression of ME1 was observed in a major fraction of HCC samples. Higher level of ME1 in tumors was significantly associated with reduced overall survival (Kaplan-Meier analysis, P = 0.024) and reduced progression-free survival (Kaplan-Meier analysis, P = 0.011). Inhibition of ME1 expression decreases HCC metastasis via suppression of epithelial-mesenchymal transition (EMT) processes in ROS-induced pathways. ME1 overexpression associates with unfavorable prognoses in patients with HCC, suggesting that ME1 is a poor prognostic predictor of hepatocellular carcinoma.

Lysyl oxidase like 4 (LOXL4), a member of the secreted copper-dependent amine oxidases that contribute to the assemble and maintenance of the extracellular matrix (ECM), was found to be up-regulated or down-regulated in different cancer types, suggesting its paradoxical roles in cancer. The specific role of LOXL4 in hepatocellular carcinoma (HCC), however, is still yet to be defined. Twenty-eight pairs of HCC specimens were used for LOXL4 mRNA expression analysis. The mRNA expression in HCC cell lines was examined, and HepG2 was selected for LOXL4 small interfering RNA (siRNA) interference to investigate the biological function of LOXL4, LOXL4 immunohistochemical staining was performed using a tissue microarray containing 298 HCC patients. The prognostic and diagnostic value of LOXL4 was evaluated using Cox regression and Kaplan-Meier analysis. LOXL4 mRNA or protein expression was significantly lower in HCC tissues than peritumoral tissues (LOXL4 mRNA expression, P = 0.018; LOXL4 protein expression, P < 0.001). Low LOXL4 expression was associated with lower overall survival (OS) rates and higher cumulative recurrence rates. Multivariate analysis indicated that LOXL4 was an independent prognostic indicator for OS and time to recurrence (TTR). Our results revealed that LOXL4 was down-regulated in HCC and correlated with aggressive tumors and a worse clinical outcome. LOXL4 may be a potential biomarker to identify the HCC patients with a higher risk of recurrence.

Clusterin (CLU) is a stress-induced chaperone that confers proliferative and survival advantages to cancer cells. However, effects and molecular mechanisms of CLU in hepatocellular carcinoma (HCC) metastasis are still unknown. In this study, HCC tissue array (n = 198) was utilized to investigate correlation between CLU expression and clinicopathological features. Overexpression of CLU in HCC tissues was correlated with shorter overall survival and higher tumor recurrence. In vitro and in vivo assays demonstrated that silencing CLU attenuated the invasion and metastasis of HCC cells, whereas ectopic overexpression of CLU resulted in the forced metastasis of HCC cells. We also revealed that CLU activated Akt signaling through complexing with eukaryotic translation initiation factor 3 subunit I (EIF3I), which in turn promoted matrix metalloproteinase 13 (MMP13) expression and HCC metastasis. Positive correlations between CLU and MMP13, p-Akt, or EIF3I were found in HCC tissues. We further observed that CLU knockdown using the CLU inhibitor OGX-011 significantly suppressed HCC metastasis in two metastatic models through inhibiting EIF3I/Akt/MMP13 signaling. These findings indicate that CLU is an independent predictive factor for prognosis of HCC and it facilitates metastasis through EIF3I/Akt/MMP13 signaling. CLU suppression using OGX-011 may represent a promising therapeutic option for suppressing HCC metastasis.

Purpose: Previously, we have established a tissue-like HCC spheroid which better mirrors the biological features of tumorigenesis and metastasis. This study was to find out metastasis-associated genes between two 3D HCC spheroids with different metastasis potential using comparative PCR arrays., Materials and Methods: Two HCC spheroids derived from high-metastatic MHCC97H cells and low-metastatic Hep3B cells were formed respectively in a rotating wall vessel bioreactor after 3D culture for 15 days. The candidate metastasis-associated genes related to cell adhesion, matrix secretion and invasion in HCC spheroids were screened by RT² profiler PCR arrays. The expression patterns of several differentially-expressed genes were further confirmed by real-time RT-PCR., Results: Total of 123 differential expression genes (fold-change>2) were found between two HCC spheroids, including 70 up-regulated genes (VCAM-1, IL-1β, CD44, tenascin C, SPP1, fibronectin, MMP-2, MMP-7, etc) and 53 down-regulated genes (E-cadherin, CTNND2, etc) in the high-metastatic spheroid. Function classification showed that the number of up-regulated genes related to adhesion molecules mediating cell-matrix interactions and matrix secretion was significantly higher in high-metastatic spheroid than that in low-metastatic spheroid. In contrast, the expressions of adhesion molecules maintaining homotypic tumor cell adhesion were decreased in metastatic spheroid as compared with that in low-metastatic spheroid. In addition, the expression pattern of seven selected genes associated with tumor metastasis measured by real-time RT-PCR were consistent with results of PCR arrays., Conclusions: Obvious differences between two HCC spheroids in gene expression patterns of adhesion molecules, matrix secretion, invasion and other molecules may determine the different metastatic characteristics and malignant phenotype of HCC spheroid.

Interferon (IFN)-α is generated in response to viral infections and is used clinically in the therapy of a variety of viral infections and cancers. The present study investigated whether IFN-α could inhibit the invasive ability of osteosarcoma cells using a Matrigel invasion assay. In addition, the osteosarcoma cells were treated with cisplatin and/or IFN-α. Apoptosis and autophagy were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, Hoechst 33258 staining, flow cytometry assay, acridine orange staining, green fluorescent protein-LC3 dot assay and transmission electron microscopy. Further analysis revealed that the efficacy of cisplatin was enhanced by the addition of the cytokine, IFN-α. These results indicate that the combination therapy of chemotherapeutics and IFN-α is a new approach for osteosarcoma, which requires validation by experiments in vivo.

We present adaptive neural control design for a class of perturbed nonlinear MIMO time-varying delay systems in a block-triangular form. Based on a neural controller, it is obtained by constructing a quadratic-type Lyapunov-Krasovskii functional, which efficiently avoids the controller singularity. The proposed control guarantees that all closed-loop signals remain bounded, while the output tracking error dynamics converge to a neighborhood of the desired trajectories. The simulation results demonstrate the effectiveness of the proposed control scheme.

To evaluate the reagent 2-methoxy-4,5-dihydro-1H-imidazole used for isotopic labeling in quantitative proteomics, we synthesized 2-methoxy-4,5-dihydro-1H-imidazole and its tetradeuterated analog in three steps. Prior to tryptic cleavage, bovine serum albumin (BSA) was reduced and alkylated. Tryptic peptides were derivatized with an equal volume of either DO or D4 and D4-derivatized peptides were mixed with at variable ratio (from 10:1 to 1:5) prior to MS and MS/MS analysis. We used matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and Electro spray ionization-mass spectrometry (ESI-MS) to evaluate the quantitative capability of labeling. The specificity of the reagent is excellent: only lysine side chains were modified among tryptic peptides. MALDI and ESI ionization modes not only could achieve the quantification of differentially expressed proteins but also facilitate the de novo sequencing. This side-chain modification can be used for quantitative analysis with proteomic strategies involving liquid chromatography. Reverse phase liquid chromatography (RPLC) kept a good resolution, and the introduction of D atoms did not introduce a variation of retention time between heavy and light peptides in RPLC.

In the title compound, C(18)H(15)NO(4), the benzene rings form quite different dihedral angles [16.07 (1) and 59.50 (1)°] with the central pyrrole ring, indicating a twisted mol-ecule. Conjugation is indicated between the five- and six-membered rings by the lengths of the C-C bonds which link them [1.462 (3) and 1.477 (3) Å]. The most prominent feature of the crystal packing is the formation of inversion dimers via eight-membered {⋯HNCO}(2) synthons.

In the title mol-ecule, C(22)H(13)Br(2)NO(2), the three benzene rings are arranged in a propeller-like fashion around the central malemide ring, making dihedral angles of 48.2 (4), 30.2 (4) and 34.8 (4)° with the malemide ring. The C-C single-bond lengths connecting benzene groups and maleimide are significantly shorter [C-C = 1.468 (9) and 1.478 (9) Å] than a typical Csp(3)-Csp(3) single bond, indicating partial conjugation between the benzene and the mal-eimide. A weak non-classical C-H⋯O hydrogen bond helps to stabilize the crystal structure.

Objective: To prepare the infected Oncomelania hupensis by artificial method for the research on the activity, vaccine, and genetic variation of Schistosoma Japonicum (S. Japonicum)., Methods: The mature eggs of S. Japonicum were collected by Nylon silk method and the miracidia were incubated under appropriate conditions. Negative snails were infected with miracidia in different proportion by means of individual or collective infection to seek the best method and proportion of infection between miracidia and snails. Infected snails were divided into 12 groups in total. I-VI groups were for individual infection and VII-XII groups were for collective infection. There were 200 snails in each group. The infection ratios between snails and miracidia in Group I-VI or VII-XII were 1:0,1:5,1:10,1:15,1:20,1:25, respectively. The infected snails were screened, numbered, and reared singly. The amount of cercariae was calculated once every 10 days until the infected snails died. Then cercariae shedding quantity, infection quantity, and mortality of infected snails in every group were compared to find the best infection method and the best infection proportion between miracidia and snails. The cercariae were collected from the first generation of infected snails and were used to infect experimental animals. The mature eggs of S. Japonicum were saved from the infected experimental animals and incubated to get miracidia. The snails were artificially infected by miracidium to get the second generation of infected snails. The developmental rates of adult worms, the egg density in fecal and liver were compared between artificially and naturally infected snails. Results In individual infection Group I-VI,the average infection value of snails were 0 ± 0,22.7 ± 4.2,31.7 ± 4.5,53.0 ± 5.3,39.3 ± 5.9,32.7 ± 4.7,the average fatality of snails were 21.7 ± 3.1,25.0 ± 3.6,31.3 ± 4.9,44.7 ± 6.5,78.3 ± 9.5,89.7 ± 13.6, and the average value of cercariae shedding from infected snails were 0.0 ± 0.0,308.0 ± 96.6,428.1 ± 146.2,527.0 ± 171.1,571.4 ± 148.9,602.9 ± 356.3, respectively. In collective infection Group VII-XII,the average infection value of snails were 0 ± 0,12.3 ± 2.5,18.7 ± 4.7,28.3 ± 4.2,33.3 ± 4.7,29.3 ± 5.5,and the average fatality of snails were 22.7 ± 3.8,23.7 ± 4.5,28.3 ± 5.5,47.0 ± 9.5,75.7 ± 8.5,86.3 ± 12.2, and the average value of cercariae shedding from infected snails were 0 ± 0,244.5 ± 57.3,292.3 ± 74.8,347.1 ± 100.8,477.2 ± 142.1,447.3 ± 161.4, respectively. The second generation of artificially infected snails was obtained successfully. The average infection rate and fatality rate for the second generation of artificially infected snails were 24.65% and 24.50%, both of which were not obviously different from that of the first generation of artificially infected snails (P>0.05). In the animal experiment, the worm growth rate for the naturally infected snails, the first or second generation of artificially infected snails were 68.50%,73.50% or 71.00%. There was no obvious difference among them (P>0.05). The fecal (or liver) eggs per gram for the naturally infected snails, the first or the second generation of artificially infected snails were 1 503 ± 269,1 683 ± 233, or 1 541 ± 117 (or 6 641 ± 1 819,6 272 ± 1 419, or 7 263 ± 1 643). There was no significant difference among the 3 groups (P>0.05)., Conclusion: Infected snails can be obtained through the artificial method by using S. Japonicum miracidia to infect snails. Individual infection has the advantage over collective infection. The optimal proportion of infection between snails and miracidia is 1:15. There was no significant difference between the first and the second generation of artificially infected snails in the average of cercariae shedding, infection, and fatality average of snails. There was no significant difference between artificially and naturally infected snails in the developmental rate of adult worms, fecal and liver eggs per gram.

Objective: To develop a rapid and simple immunoassay to detect antibodies in the sera of patients infect Schistosoma japonicum (S. japonicum)., Methods: Soluble egg antigen (SEA) of S. japonicum conjugated with colloidal carbon in advance was used to react with the antibodies in the sera of patients with schistosomiasis. Then the carbon-antigen-antibody complex would be captured by SEA which had been absorbed on the nitrocellulose membrane and a gray band was shown., Results: A total of 137 sera samples from S. japonicum epidemic area were tested, and the consistency, sensitivity, and specificity of colloidal carbon dipstick assay were 98.54%, 98.99%, and 97.37%, respectively, compared with the IHA method. The gray scale of bands on the dipstick was curvilinear to serum titer which revealed that the assay could be used semi-quantitatively in serum analysis., Conclusion: Colloidal carbon dipstick assay is not only rapid and simple, but also sensitive and specific for the detection of serum antibodies of schistosomiasis japonica. It will be a practical immunological assay for the diagnosis of schistosomiasis in the field testing.

Objective: To establish a hepatocellular carcinoma (HCC) cell line from lung metastatic lesions of human HCC in nude mice so as to provide suitable model for the study of lung-metastasis-related molecular mechanisms., Methods: HCC clone cells MHCC97-H were inoculated subcutaneously into nude mice. The pulmonary metastatic lesions were harvested from the moribund animals and then re-implanted into the nude mice for the second round of selection. The same procedure was repeated twice. New cell line from the third round of lung metastasis was thus established and the following parameters were studied: morphology, in vitro growth curve, plate efficiency, migration, karyotype, flow cytometry, immunocytochemistry for AFP, albumin and cytokeratin 8 (CK8), flourescent PCR for HBV DNA, tumorigenicity and spontaneous metastasis via subcutaneous injection and orthotopic implantation of tumor tissue., Results: The cell line established from nude mouse lung metastasis was designated as HCCLM3, consisting of polygonal epithelial cells with hypotriploid karyotype, its main range of chromosome being 55 - 58. The cell population doubling time was 34.9 hours, plate efficiency 32.4 +/- 3.2%, and cell migration rate 20 +/- 2 microm/h. The cells were positive for AFP, albumin, CK8, and P16 and negative for P53, nm23 and HBsAg. Fluorescent PCR showed HBV DNA integration into cellular genome. When 5 x 10(6) cells were injected subcutaneously into nude mice, the tumorigenicity was 100% with a latency period of 11 +/- 1 d. Five weeks after subcutaneous injection, the pulmonary metastatic rate was 100%, the median number of lung metastases being 121 per mouse. After orthotopic implantation of tumor tissue into nude mouse liver for 35 d, wide spread regional and distant metastases occurred, the metastatic rate was 100% for abdominal wall, 80% for organs in abdominal cavity, 100% for liver, 70% for diaphragm, and 100% for lung. The median number of lung metastatic lesions was 268 per mouse., Conclusion: A new HCC cell line has been established, which is characterized by high pulmonary metastasis via both subcutaneous and orthotopic inoculation. It provides a new model for the study of liver cancer metastasis.