[Multi-scenario Simulation of Construction Land Expansion and Its Impact on Ecosystem Carbon Storage in Beijing-Tianjin-Hebei Urban Agglomeration].

It is of great practical significance for regional sustainable development and ecological construction to quantitatively analyze the impact of construction land expansion on terrestrial ecosystem carbon storage and to explore the optimization scheme of simulating construction land expansion to improve future ecosystem carbon storage. Based on the land use and cover change (LUCC) and other geospatial data of the Beijing-Tianjin-Hebei Urban Agglomeration from 2000 to 2020, this study utilized the Integrated Valuation of Ecosystem Services and Tradeoffs (InVEST) model and the patch-generating land-use simulation (PLUS) model to assess and analyze the changes in ecosystem carbon stocks and spatial patterns regionally. In this study, we performed linear regression analysis to investigate the relationship between urban land expansion and changes in ecosystem carbon stocks for varying urban land proportion levels during two distinct time intervals, 2000-2010 and 2010-2020, which was conducted at a spatial resolution of 2 km. Three distinct urban land expansion scenarios were subjected to simulation to forecast the prospective land use pattern by 2030. Subsequently, we quantified the ramifications of these scenarios on ecosystem carbon stocks during the period from 2020 to 2030. The results were as follows:① In the Beijing-Tianjin-Hebei Urban Agglomeration, the ecosystem carbon stocks exhibited notable variations over the study period, with values of 2 088.02, 2 106.78, and 2 121.25 Tg recorded for the years 2000, 2010, and 2020, respectively, resulting in a cumulative carbon sequestration of 33.23 Tg C during the study duration. It is noteworthy that forest carbon storage emerged as the dominant contributor, with an increase from 1 010.17 Tg in 2000 to 1 136.53 Tg in 2020. Throughout the study period, the spatial distribution of carbon stocks displayed relative stability. Regions characterized by lower carbon content were concentrated in the vicinity of the Bohai Rim region and in proximity to cities such as Beijing, Tianjin, and Shijiazhuang, as well as rural settlements. In contrast, grid units with moderate and high carbon stocks were predominantly situated in the western Taihang Mountain and the northern Yanshan Mountain. Additionally, there was a tendency of increasing carbon stocks in the Taihang Mountain and Yanshan Mountain region, whereas those surrounding major urban centers such as Beijing, Tianjin, Shijiazhuang, and Tangshan experienced a notable decline in carbon stocks. Such reductions were most pronounced in regions undergoing urban land expansion during the study period. ② In grid units with an urban land proportion exceeding 10% at each level, a strong correlation was observed between urban land expansion and changes in carbon stocks during both the 2000-2010 and 2010-2020 periods. The changes in urban land proportion adequately explained the variations in carbon stocks. However, the explanatory power of urban land on carbon stocks decreased during the 2010-2020 period, indicating that other factors played a more substantial role in influencing carbon stocks during this time. The regression coefficients for both periods exhibited a fluctuating upward trend. In comparison to that during the 2000-2010 period, the impact of urban land expansion on carbon stocks was relatively smaller during 2010-2020, indicating a weakening influence. ③ In light of three distinct development scenarios, namely natural development (Scenario Ⅰ), a 15% reduction in the rate of urban land expansion (Scenario Ⅱ), and a 30% reduction in the rate of urban land expansion (Scenario Ⅲ), the projected ecosystem carbon stocks for the Beijing-Tianjin-Hebei Urban Agglomeration in the year 2030 were estimated to be 2 129.12, 2 133.55, and 2 139.10 Tg, respectively. These projections indicated an increase of 7.88, 12.30, and 17.85 Tg in comparison to the current carbon stocks. All scenarios demonstrated that the terrestrial ecosystem would play a role of carbon sink, particularly with the greatest carbon sink observed in the scenario with a 30% reduction in urban land expansion. The fit performance between urban land expansion and carbon stock changes during the 2020-2030 period was significantly better than that during the 2000-2010 and 2010-2020 periods, and the regression coefficients showed a fluctuating increase with an increase in urban land proportion. Across grid units with different urban land proportion levels, the regression coefficients exhibited the order of Scenario Ⅰ < Scenario Ⅱ < Scenario Ⅲ. In pursuit of the carbon peaking and carbon neutrality goals, the Beijing-Tianjin-Hebei Urban Agglomeration should prioritize scenarios with reduced rates of urban land expansion, especially in regions with higher urban land proportions.

Synergistic effects and molecular mechanisms of DL-3-n-butylphthalide combined with dual antiplatelet therapy in acute ischemic stroke.

DL-3-n-butylphthalide (NBP) is isolated from the seeds of Apium graveolens L., and has been recently used as a neuroprotective agent for acute ischemic stroke. The present study aimed to determine the efficacy and safety of the combined use of dual antiplatelet therapy (DAPT) and NBP for treating of acute ischemic stroke in rats and to explore the synergistic mechanism of this treatment strategy in rat middle cerebral artery occlusion models. The efficacy of DAPT combined with NBP was evaluated by determining neurological deficits, infarction status, and histological changes. Changes in body weight, blood glucose level, blood count, and serum biochemical parameters were detected to evaluate the safety. To explore the synergistic pharmacological mechanism, the mRNA expression and protein levels of key proteins in the pyroptosis-inflammatory pathway, and the pyroptosis ratio of microglias were examined. Compared with the administration of NBP or DAPT alone, combination of them significantly improved neurological deficits, reduced infarct area, and repaired tissue injury and inflammation after cerebral ischemia. No hepatorenal toxicity was observed. The mRNA expression and protein levels of key proteins in the pyroptosis-inflammation pathway, and the pyroptosis ratio of microglias were significantly downregulated in the combined administration group than in the monotherapy group. We demonstrated that the combined use of NBP and DAPT exhibits better efficacy and high safety and plays a synergistic role by inhibiting the pyroptosis-inflammation pathway in the brain tissues, particularly in microglial cells.

The Synergistic Impact of Crystal Seed and Fluoride Ion in the Synthesis of Silicalite-1 Zeolite in Low-Template Systems.

Silicalite-1 zeolites are widely applied in gas adsorption, catalysis, and separation due to their excellent hydrothermal stability and unique pore structure. However, traditional preparation methods have inherent drawbacks such as high pollution, high cost, etc. Therefore, this work proposed a green and efficient route for preparing Silicalite-1 zeolite by adding NH4F (F/Si = 0.1) and seeds (10 wt%) in a much shorter time (8 h) in a low-template system (TPA+/Si = 0.007). It was found that NH4F is beneficial for inhibiting the formation of SiO2. The S-1 seeds could drastically induce the formation of the zeolite skeleton structure. Noteworthy, the morphology of zeolites was determined by the relative content of NH4F and seeds. The crystal morphology is determined by the higher content of the two substances; however, when the content is similar, the crystal morphology is determined by NH4F. The results showed that simultaneous control of NH4F and seeds can suppress SiO2 formation, can improve the relative crystallinity of products, and can be precisely regulated via the synergistic effect of both in zeolite morphology. This work not only provides new ideas for regulating the morphology of silicate-1 crystals but also offers a new path for industrial large-scale production of low-cost and efficient zeolites.

Effects of Yinzhihuang on Alleviating Cyclosporine A-Induced Cholestatic Liver Injury via Farnesoid X Receptor-Mediated Regulation of Transporters and Enzymes in Vitro and in Vivo.

Yinzhihuang (YZH), a traditional Chinese medicine prescription, was widely used to treat cholestasis. Cholestatic liver injury limited the use of the immunosuppressive drug cyclosporine A (CsA) in preventing organ rejection after solid organ transplantation. Clinical evidences suggested that YZH could enhance bile acids and bilirubin clearance, providing a potential therapeutic strategy against CsA-induced cholestasis. Nevertheless, it remains unclear whether YZH can effectively alleviate CsA-induced cholestatic liver injury, as well as the molecular mechanisms responsible for its hepatoprotective effects. The purpose of the present study was to investigate the hepatoprotective effects of YZH on CsA-induced cholestatic liver injury and explore its molecular mechanisms in vivo and vitro. The results demonstrated that YZH significantly improved the CsA-induced cholestatic liver injury and reduced the level of liver function markers in serum of Sprague-Dawley (SD) rats. Targeted protein and gene analysis indicated that YZH increased bile acids and bilirubin efflux into bile through the regulation of multidrug resistance-associated protein 2 (Mrp2), bile salt export pump (Bsep), sodium taurocholate cotransporting polypeptide (Ntcp) and organic anion transporting polypeptide 2 (Oatp2) transport systems, as well as upstream nuclear receptors farnesoid X receptor (Fxr). Moreover, YZH modulated enzymes involved in bile acids synthesis and bilirubin metabolism including Cyp family 7 subfamily A member 1 (Cyp7a1) and uridine 5'-diphosphate (UDP) glucuronosyltransferase family 1 member A1 (Ugt1a1). Furthermore, the active components geniposidic acid, baicalin and chlorogenic acid exerted regulated metabolic enzymes and transporters in LO2 cells. In conclusion, YZH may prevent CsA-induced cholestasis by regulating the transport systems, metabolic enzymes, and upstream nuclear receptors Fxr to restore bile acid and bilirubin homeostasis. These findings highlight the potential of YZH as a therapeutic intervention for CsA-induced cholestasis and open avenues for further research into its clinical applications.

Metabolic adaptation of the clam Ruditapes philippinarum during air exposure and the positive effects of sodium nitroprusside pretreatment.

The Manila clam (Ruditapes philippinarum), as one of the shellfish living in the intertidal zone, is known for its strong ability to withstand air exposure. Sodium nitroprusside (SNP), a donor of nitric oxide (NO), has been shown to be useful for antioxidant and immune regulation in aquatic animals. In this study, an untargeted metabolomics (LC-MS/MS) technique was employed for the first time in Manila clam to analyze the metabolic and histological impacts after air exposure and the positive effects of SNP pretreatment. During air exposure, a significant increase in taurine, L-glutamate, and several polyunsaturated fatty acids in clams was detected, which indicates that clams may experience inflammatory reactions, oxidative stress, and an increase in blood ammonia content. When clams were exposed to SNP for 6 h, arginine, spermine, L-glutamic acid, and glutathione content were all upregulated, indicating that the SNP exposure induced NO production and improved antioxidant capacity in clams. When the clams were exposed to air after SNP pretreatment, there were no significant differences in the levels of taurine, L-glutamate, or aliphatic acids between the experimental and control groups. Gill tissue was more severely damaged in clams directly exposed to air than in those that experienced air exposure after SNP pretreatment, especially in clams exposed to air for a long time (72 h). Both metabolomics and tissue section structure indicated that SNP pretreatment decreased the stress responses caused by air exposure in R. philippinarum. These findings provided fresh insights and a theoretical foundation for understanding the tolerance to air exposure and physiological functions of SNP (or NO) in R. philippinarum.

Cytotoxicity and apoptosis induced by enrofloxacin in loach fin cells in vitro.

The cytotoxic effect and cell death were studied in loach fin cells in vitro after enrofloxacin (ENR) exposure. The semi-lethal concentration of ENR for loach cells was calculated as 1296.2 ± 3.11 mol/L (about 512.5 mg/L). Loss of cell viability, increase in vacuoles, disappearance of microvilli, and apoptotic bodies were evident in cells exposed to 400, 800, and 1200 µmol/L ENR. Besides, dose-dependent inhibitory effects on SOD, CAT, Na+-K+-ATPase, and Ca2+-ATPase activities were also observed in loach cells exposed to ENR. Quantitative gene expression results showed that ENR induced caspase-3- and caspase-8-mediated apoptosis as well as caspase-activated DNase in loach cells. The findings also indicated a role of JNK pathway in ENR-induced apoptosis in loach cells. Transcriptome sequencing results showed 10,016 differentially expressed genes in ENR vs. control groups, which were all enriched in "Molecular Function" process in GO term. Furthermore, 6763 genes were enriched in 291 KEEG pathways, with most of them belonging to immune and material metabolic pathways. The large number of transcriptome data and pathways determined in this study provide a database foundation for the toxicity analysis of ENR in loach cells, which must be thoroughly examined to further investigate the cytotoxic mechanism of antibiotics in fish cells.

Effect of capsaicin on breast cancer resistance protein (BCRP/Abcg2) and pharmacokinetics of probe substrates in rats.

Breast cancer resistance protein (BCRP/Abcg2 in human, Bcrp/Abcg2 in rat), a member of the ATP-binding cassette (ABC) transporter family, acts as an efflux pump for xenobiotics, with ability to transport various drugs out of cells. Capsaicin may have the potential to modulate the function of Bcrp transport. This study was to evaluate the effects of capsaicin on the pharmacokinetics of sulfasalazine, a Bcrp substrate, in rats and investigate the mechanism of this food-drug interaction.The rats were pre-treated with 5% carboxymethylcellulose sodium (vehicle), capsaicin (3, 8, 25 mg/kg) and cyclosporine A (10 mg/kg) by gastric gavage for 7 days. On day 7, blood, liver and intestine samples were collected after sulfasalazine administered. Liquid chromatography tandem mass spectrometry (LC-MS/MS) was used to study the effects of capsaicin on the pharmacokinetics of sulfasalazine in rats. RT-PCR and western blotting were used to study the mechanism in biomolecules in rats, respectively.Compared with vehicle group, AUC0-∞ of sulfasalazine in rats were increased by 1.5-folds, 1.6-folds and 1.7-folds in 3, 8 and 25 mg/kg/d capsaicin pre-treated groups. At the same time, the CL/F in rats were decreased by 33%, 38% and 42% in the three groups. In addition, we found Bcrp mRNA levels and protein expressions in rat livers and intestines were decreased in 3, 8 and 25 mg/kg/d capsaicin-treated groups.Our study demonstrated that long-term ingestion of capsaicin significantly enhanced the AUC of sulfasalazine involved down-regulate Bcrp gene and protein expression in rat liver and intestine.

Quantitation of six steroid hormones by ultra-performance liquid chromatography-tandem mass spectrometry in plasma and prefrontal cortex samples from rats with chronic unpredictable mild stress-induced depression.

Steroid hormones such as glucocorticoids and their metabolites are closely related to mental diseases and neuroendocrine diseases. Quantitative analysis of these substances will help in understanding their roles in related research fields. In this study, an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed to detect the concentration of corticosterone (CORT) and its metabolites, progesterone (PROG) and testosterone in rat plasma and prefrontal cortex (PFC), and was applied to investigate the changes in hormones in rats with depression induced by chronic unpredictable mild stress (CUMS). The method was shown to be linear in the quantitation range for all analytes. Intra- and inter-day accuracy and precision were between 80% and 120%. Furthermore, we found that the level of CORT in plasma and PFC increased, whereas that of 11-dehydrocorticosterone (11-DHCORT) as well as the ratio of 11-DHCORT and CORT declined in rats with CUMS-induced depression. The trends of these changes in central PFC and peripheral plasma were consistent. In conclusion, this study successfully established an UPLC-MS/MS method for simultaneous measurement of CORT and its metabolites, PROG and testosterone in rat plasma and PFC, and applied it to rats with depression. The method could be further applied to the research of depression and diseases related to these steroid hormones.

Identification of shell-color-related microRNAs in the Manila clam Ruditapes philippinarum using high-throughput sequencing of small RNA transcriptomes.

Shell-color polymorphism is a common phenomenon in several mollusk species and has been associated with thermal capacity, developmental stability, shell strength, and immunity. Shell-color polymorphism has been related to the differential expression of genes in several signal transduction pathways; however, the functions of micro-RNAs (miRNAs) in shell-color formation remain unclear. In the present study, we compared high-quality, small-RNA transcriptomes in three strains of the Manila clam Ruditapes philippinarum with specific shell-color patterns, artificially selected for six generations. Totals of 114 known and 208 novel miRNAs were identified by high-throughput sequencing, of which nine known and one novel miRNA were verified by stem-loop quantitative real time-polymerase chain reaction. Predicted miRNA targets were subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. miR-137 and miR-216b and the Hedgehog signaling pathway and Wnt signaling pathway were identified as being potentially involved in pigment formation and regulation in R. philippinarum. These results may help to clarify the role of miRNAs in shell coloration and shed light on the mechanisms regulating color formation in bivalve shells.

Effects of cyclosporin A on the pharmacokinetics and toxicities of irinotecan mediated by UGT1A1 in vitro and in vivo.

Irinotecan (CPT-11) is a broad spectrum agent for the treatment of solid tumor malignancies, despite severe diarrhea is limiting its widespread usage. The local effects of SN-38 in the small intestine were considered to be responsible for the irinotecan-induced delayed diarrhea. It was proposed that cyclosporin A (CsA) inhibiting biliary excretion could attenuate this side effect, but in fact, it could not improve the therapeutic index of irinotecan. At present, most studies focused on the inhibition of bile excretion by cyclosporin A through the transporters MRP2 and MDR1 and its effect the irinotecan treatment in vivo. However, UDP glucuronyltransferase-1 polypeptide A1 (UGT1A1) was related to a significantly altered disposition of irinotecan and its metabolites, and was therefore associated with irinotecan-induced toxicity. This study focused on UGT1A1-mediated conversion of SN-38 to SN-38G, and systematically investigated the CsA-irinotecan interactions in vitro and in vivo. After treatment with 10 mg·kg-1 CsA for 7 days, the bile excretion of irinotecan and its metabolites decreased and AUC0-∞ increased significantly. The AUC0-∞ (SN-38G)/AUC0-∞ (SN-38) was significantly reduced when compared with that in vehicle-treated rats. In the liver microsome incubation system, the IC50 of CsA for UGT1A1 enzyme was 9.4 µM. Furthermore, the UGT1A1 mRNA and protein expression levels were significantly reduced. The present study indicated that CsA treatment could enhance the systemic exposure and toxicity of SN-38 by inhibiting the UGT1A1 enzyme. The inhibition of UGT1A1 enzyme might be a critical factor in the failure of CsA improving irinotecan's treatment index.

Development of an underivatized LC-MS/MS method for quantitation of 14 neurotransmitters in rat hippocampus, plasma and urine: Application to CUMS induced depression rats.

Sensitive and comprehensive measurement of systemic metabolites of tryptophan, phenylalanine and glutamate metabolism in biological samples is effective for understanding the pathogenesis of depression and other neurological diseases. Therefore, this study developed an underivatized liquid chromatography tandem mass spectrometry (LC-MS/MS) method for simultaneous monitoring the 3 components of glutamate metabolism in rat hippocampus and 11 components of tryptophan and phenylalanine metabolism in rat hippocampus, plasma and urine, and applied it to investigate their changes in rats induced by chronic unpredictable mild stress (CUMS). The investigated analytes are as follows: tryptophan, serotonin, 5-hydroxyindoleacetic acid, kynurenine, kynurenic acid, xanthurenic acid, 3-hydroxyanthranilic acid, quinolinic acid, phenylalanine, tyrosine, tyramine, glutamate, glutamine and gamma-aminobutyric acid. The method was verified to be sensitive and effective with satisfactory linearity, accuracies in the range of 78.2%-120.4%, and precisions less than 17.8% for all identified analytes. A series of significant changes in CUMS-induced rats can be detected: tryptophan, serotonin and tyrosine levels decreased and quinolinic acid increased in both hippocampus and plasma. In addition, the kynurenine/tryptophan ratios increased in hippocampus and plasma, the kynurenic acid/quinolinic acid ratios of plasma and urine were significantly reduced. These findings demonstrated that the CUMS procedure could lead to the central and peripheral imbalances of tryptophan and phenylalanine metabolism. In conclusion, a LC-MS/MS method for simultaneous measurement of several neurotransmitters in rat hippocampus, plasma and urine was developed and successfully applied to investigation of the central and peripheral changes in CUMS-induced rats. The method would be expected to provide applicability to the study of the mechanisms of depression and other related diseases associated with these neurotransmitters.

Toxic effects of copper sulfate on diploid and triploid fin cell lines in Misgurnus anguillicaudatus.

The effects of different concentrations of copper sulfate on diploid and triploid fin cell lines (named DIMF and TRMF, respectively) in Misgurnus anguillicaudatus were studied. The LC50 of copper sulfate estimated by an MTT assay was 268.39 in DIMF cells, and 311.54 µmol/L in TRMF cells, respectively. Activity of superoxide dismutase (SOD) in DIMF cells gradually increased as the concentration of copper sulfate increased (up to 200 µmol/L), and then gradually decreased. SOD activity in triploid loach fin cells, as well as glutathione peroxidase (GSH-Px) and glutathione-S-transferase (GST) activity in both diploid and triploid cells, decreased as the concentration of copper sulfate increased, which suggested that excessive copper exposure at the concentrations tested in this study was detrimental to anti-oxidative capability. In general, SOD, GST and GSH-Px activity was higher in triploid fin cells than in diploid cells. DNA breaks were observed by comet assays after 24 h exposure to 400 and 800 µM copper; DNA percent in the comet's tail was lower in TRMF than in DIMF. Ultrastructurally, there were no significant differences in the organelles of both cells, although a higher number of vesicles were observed in TRMF cells after copper exposure. Pathological changes induced by copper sulfate were similar in DIMF and TRMF cells, and were indicative of cell necrosis. Results above suggested that excessive copper sulfate exposure would lead to antioxidant enzymes activity reduction, along with antioxidant defenses disruption and superoxide radicals increasing, and then to DNA damage, ultrastructural changes and necrosis features in DIMF and TRMF M. anguillicaudatus fin cells. Triploid cell lines had higher resistance to copper than their diploid counterparts especially at higher concentrations of copper due to larger cells and higher intracellular content of detoxification enzymes to resist the toxicity of heavy metals.

IDO and TDO as a potential therapeutic target in different types of depression.

Depression is highly prevalent worldwide and a leading cause of disabilty. However, the medications currently available to treat depression fail to adequately relieve depressive symptoms for a large number of patients. Research into the aberrant overactivation of the kynurenine pathway and the production of various active metabolites has brought new insight into the progression of depression. IDO and TDO are the first and rate-limiting enzymes in the kynurenine pathway and regulate the production of active metabolites. There is substantial evidence that TDO and IDO enzyme are activated during depression, and therefore, IDO and TDO inhibitors have been identified as ideal therapeutic targets for depressive disorder. Hence, this review will focus on the kynurenine branch of tryptophan metabolism and describe the role of IDO and TDO in the pathology of depression. In addition, this review will compare the relative imbalance between KYNA and neurotoxic kynurenine metabolites in different psychiatric disorders. Finally, this review is also directed toward assessing whether IDO and TDO are potential therapeutic target in depression associated with other diseases such as diabetes and/or cancer, as well as the development of potent IDO and TDO inhibitors.

Effects of aprepitant on the pharmacokinetics of imatinib and its main metabolite in rats.

Aprepitant (APT), an antiemetic drug belonging to the class of substance P antagonists is efficiently used in both acute and delayed chemotherapy-induced nausea and vomiting. Nausea and vomiting induced by imatinib (IMA) as a chemotherapeutic drug could be reduced by APT. This study investigated the effect of APT on the pharmacokinetics of IMA and its major metabolite N-desmethyl imatinib (N-D IMA) in rats and the mechanism of this drug-drug interaction. The results indicated that after 3 days of pretreatment with APT (10 mg/kg), the blood concentration of IMA was decreased in both of oral and intravenous routes of IMA administration compared to vehicle treated rats, whereas the blood concentration of N-D IMA was not significantly changed. The total clearance (CL/F) of oral and intravenous given IMA was increased by 1.41 and 1.32-fold, and the bioavailability was greatly decreased about 30.43% and 24.40% respectively. At this time, the P-gp and the hepatic CYP3A1 were increased at both the mRNA and protein levels. These results demonstrated that ingestion of APT will decrease the bioavailability of IMA to a significant extent in rats and the drug-drug interaction between APT and IMA appears to be due to modulation of P-gp and CYP3A1.

Toxic effects of glyphosate on diploid and triploid fin cell lines from Misgurnus anguillicaudatus.

We examined the toxic effects of glyphosate on diploid (DIMF) and triploid (TRMF) fin cell lines from the Oriental Weather Loach Misgurnus anguillicaudatus. The LC50 values of glyphosate estimated by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay were 315.34 and 371.77 mg/L for DIMF and TRMF, respectively. Superoxide dismutase (SOD) and glutathione-S-transferase (GST) activities in DIMF and TRMF cells gradually increased and then decreased with increasing glyphosate concentrations, reaching a maximum at 240 mg/L glyphosate. In contrast, acetylcholinesterase (AChE) activities in DIMF and TRMF decreased with increasing concentrations of glyphosate in a concentration-dependent manner. SOD and AChE activities were generally significantly higher in TRMF compared with DIMF cells (P < 0.05). The rates of micronucleus and abnormal nuclei were significantly higher in DIMF and TRMF groups treated with 80-560 mg/L glyphosate compared with the control groups (P < 0.01). The highest micronuclei rates in both DIMF and TRMF cells (both 4.30‰) occurred at 400 mg/L glyphosate. There were no differences in the rates of micronuclei and abnormal nuclei between DIMF and TRMF cells at any glyphosate concentration. Cell damage, including chromatin condensation, nucleus distortion, and broken and reduced endoplasmic reticulum, mitochondria, and ribosomes, were found in both cells treated with the LC50 concentration of glyphosate. Moreover, vacuolization and apoptotic bodies occurred in glyphosate-exposed DIMF and TRMF cells, indicating apoptosis. These results indicate that glyphosate in the range of tested concentrations represent a potential risk to loach through inhibiting proliferation of diploid and triploid cell lines and induces micronuclei and apoptosis.

Analysis of Different Ploidy and Parent-Offspring Genomic DNA Methylation in the Loach Misgurnus anguillicaudatus.

In this study, we selected natural polyploidy loach (diploid, triploid and tetraploid) and hybrid F1 generation obverse cross (4 × 2) and inverse cross (2 × 4) by diploids and tetraploids as the research model. The MSAP (methylation-sensitive amplified polymorphism) reaction system was established by our laboratory to explore methylation levels and pattern diversification features at the whole genome level of the polyploidy loach. The results showed that the total methylation and full methylation rates decreased on increased ploidy individuals; moreover, the hemimethylation rate showed no consistent pattern. Compared with diploid loach, the methylation patterns of tetraploid sites changed 68.17%, and the methylation patterns of triploid sites changed 73.05%. The proportion of hypermethylation genes is significantly higher than the proportion of demethylation genes. The methylation level of reciprocal cross F1 generation is lower than the male diploid and higher than the female tetraploid. The hemimethylation and total methylation rate of the cross hybrid F1 generation is significantly higher than the orthogonal F1 generation (p < 0.01). After readjusting, the methylation pattern of genome DNA of reciprocal hybrids changed 69.59% and 72.83%, respectively.

Identification of functional genes involved in Cd(2+) response of Chinese surf clam (Mactra chinensis) through transcriptome sequencing.

The Chinese surf clam Mactra chinensis is an economically important bivalve species in the coastal waters of Liaoning and Shandong Province, China. In this study, we carried out transcriptome sequencing to develop molecular resources for M. chinensis and conducted an acute test of Cd(2+) stimulation through quantitative real-time PCR (qRT-PCR) to analyze the relative expression of six functional genes. A total of 100,839 transcripts and 56,712 unigenes were obtained from 39.9 million filtered reads and 21,305 unigenes were annotated by hitting against NCBI database. According to the results of qRT-PCR, heat shock protein 22 (Hsp22) and cytochrome P450 (CYP450(2C31)) were inhibited in the low concentration, and induced in the high concentration of Cd(2+); thioredoxin peroxidase (TPx-A) was at normal level in low concentration, but induced in high concentration of Cd(2+); glutathione peroxidase A (GPA), glutathione peroxidase 1 (GPA1) and Mn superoxide dismutase gene (MnSOD) were down-regulated when exposed to any treatment groups. Expression levels of the six functional genes following Cd(2+) exposure indicated that these genes were linked to environmental stress. Moreover, the present work enriched the molecule genetic data of M. chinensis.

Establishment and characterization of fin cell lines from diploid, triploid, and tetraploid oriental weatherfish (Misgurnus anguillicaudatus).

Continuous fin cell lines from diploid, triploid, and tetraploid oriental weatherfish, Misgurnus anguillicaudatus, were established and characterized. The cell lines, designated DIMF, TRMF, and TEMF, respectively, were subcultured more than 80 times since initiation in October 2012 and were preserved at the China Center for Type Culture Collection as sample numbers C2013109, C2013110, C2013111, respectively. The cell lines consist predominantly of fibroblast-like cells. At the 50th passage, the population doubling times were 48.43 h (DIMF), 36.01 h (TRMF), and 41.45 h (TEMF). Cell survival rate of these three kinds of cells was 80.88 ± 1.38, 84.48 ± 1.13, and 81.57 ± 1.28 %, respectively, when recovered after storage in liquid nitrogen for 60 days at the 40th passage. The chromosome numbers measured from 100 metaphase plates at the 50th passage were 2n = 50 (68 %), 3n = 75 (59 %), and 4n = 100 (54 %) for DIMF, TRMF, and TEMF cells, respectively. At the 60th passage, the chromosome numbers for DIMF and TRMF cells were still 50 (52 %) and 75 (70 %), but the chromosome number for TEMF cells ranged from 88 to 100; a chromosome number of 96 accounted for 26 % of the cells, and the karyotype analysis showed 4n = 96, 16 m + 8sm + 72t, NF = 120; thus, compared with cells at the 50th passage, a group of metacentric chromosomes was missing. Microsatellite marker analysis was conducted using DIMF, TRMF, and TEMF cells and muscle tissue of oriental weatherfish, which confirmed that the three cell lines established in this study were from oriental weatherfish. The cell lines were exposed to two fish viruses to determine their susceptibility to infection; they were susceptible to spring viremia of carp virus but not to piscine nodavirus. Establishment of fin cell lines from different ploidy oriental weatherfish increases the existing number of fish cell lines available for research, and it provides a model for investigating the mechanisms of growth and genetics in polyploid fish.

Identification and characterization of a novel phage-type like lysozyme from Manila clam, Ruditapes philippinarum.

A novel lysozyme gene (RpLysPh) with high similarity to the bacteriophage lysozymes was identified in Manila clam, Ruditapes philippinarum. The full length cDNA of RpLysPh is 828bp and contains a 462bp open reading frame (ORF) that codes for a 154 amino acid protein. Multiple sequence alignment analysis revealed that the three residues essential for catalytic activity in phage-type lysozyme (Glu(20), Asp(29), and Thr(35)) are conserved in RpLysPh. The comparison of the 3D models of RpLysPh and Coxiella burnetii lysozyme also suggested that the active sites involved in the binding of substrate have similar conformations. Phylogenetic analysis suggested that RpLysPh shares a similar origin with the bacterial phage-type lysozyme group. The highest level of expression of RpLysPh was observed in hemocytes, followed by mantle. Induction of RpLysPh expression was observed in gills in response to lipopolysaccharide (LPS), peptidoglycan (PGN), polyinosinic-polycytidylic acid (Poly(I:C)), and whole glucan particles (WGP) challenge. The recombinant protein of RpLysPh showed antibacterial activity against both Gram-positive and Gram-negative bacteria.

Molecular cloning and expression analysis of inhibitor of growth protein 3 (ING3) in the Manila clam, Ruditapes philippinarum.

Inhibitor of growth protein 3 (ING3), a new member of ING family, is involved in the regulation of various processes. In this study, a full-length cDNA of ING3 (named as RpING3) was cloned from the gill of Ruditapes philippinarum by rapid amplification of cDNA ends method for the first time. The cDNA obtained was 1442 bp exclusive of poly (A) residues with a 1248 bp open reading frame encoding 415 amino acids. The RpING3 protein has a calculated molecular weight of 46.59 kDa and isoelectric point of 6.62. Two conserved motif and some functional sites were found. Tissue distribution analysis of the RpING3 mRNA revealed that the RpING3 expression level was much higher in gill and digestive gland while lower in mantle, foot and adductor muscle. The temporal expression of RpING3 in digestive gland after lead exposure was recorded by quantitative real-time PCR. The result showed that RpING3 was rapidly up-regulated at 6 h post-exposure and reached tenfold of the control group. These results suggest that RpING3 dependent signaling pathway is present in Manila clam and RpING3 may play important roles in protecting cells from heavy metal damage in R. philippinarum.