Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce apoptosis in multi-drug-resistant cancer cells

PURPOSE: The development of multi-drug resistance (MDR) due to the expression of members of the ATP binding cassette (ABC) transporter family is a major obstacle in cancer treatment. The broad range of substrate specificities associated with these transporters leads to the efflux of many anti-cancer drugs from tumour cells. Therefore, the development of new chemotherapeutic agents that are not substrates of these transporters is important. We have recently demonstrated that some members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds are microtubule-depolymerising agents that potently induce apoptosis in several cancer cell lines and impair growth of mouse breast tumours. The aim of this current study was to establish whether PBOXs were capable of inducing apoptosis in cancer cells expressing either P-glycoprotein or breast cancer resistance protein (BCRP), two of the main ABC transporters associated with MDR.

METHODS: We performed in vitro studies to assess the effects of PBOXs on cell proliferation, cell cycle and apoptosis in human cancer cell lines and their drug-resistant substrains expressing either P-glycoprotein or BCRP. In addition, we performed a preliminary molecular docking study to examine interactions between PBOXs and P-glycoprotein.

RESULTS: We established that three representative PBOXs, PBOX-6, -15 and -16 were capable of inducing apoptosis in drug-resistant HL60-MDR1 cells (expressing P-glycoprotein) and HL60-ABCG2 cells (expressing BCRP) with similar potencies as in parental human promyelocytic leukaemia HL60 cells. Likewise, resistance to PBOX-6 and -16 was not evident in P-glycoprotein-expressing A2780-ADR cells in comparison with parent human ovarian carcinoma A2780 cells. Finally, we deduced by molecular docking that PBOX-6 is not likely to form favourable interactions with the substrate binding site of P-glycoprotein.

CONCLUSION: Our results suggest that pro-apoptotic PBOX compounds may be potential candidates for the treatment of P-glycoprotein- or BCRP-associated MDR cancers.

Radiation to control transgene expression in tumors

Significant evidence has accumulated indicating that certain genes are induced by ionising radiation. An implication of this observation is that their promoter regions include radiation-responsive sequences. These sequences have been isolated in the promoter of several genes including Erg-1, p21/WAF-1, GADD45alpha and t-PA. The mechanism by which radiation induces gene expression remains unclear but involves putative binding sites for selected transcription factors and/or p53. Consensus CC(A/T)6GG sequences have been localized in the Erg-1 promoter and are referred to as serum response elements or CArG elements. The tandem combination of CArG elements has been shown to improve gene expression levels, with a 9-copy motif conferring maximum inducibility. The response of these genes to ionising radiation appears to follow a sigmoid relationship with time and dose. Therapeutic induction of suicide genes and significant cytotoxicity can be achieved at clinically relevant x-rays doses both in vitro and in vivo but was found to be cell-type dependent. Radiation-inducible gene therapy can be potentially enhanced by exploiting hypoxia through the inclusion of hypoxia-response element motifs in the expression cassette, the use of the anaerobic bacteria or the use of neutron irradiation. These results are encouraging and provide significant evidence that gene therapy targeted to the radiation field is a reasonably attractive therapeutic option and could help overcome hypoxic radioresistant tumors.

DNA microarray profiling of genes differentially regulated by the histone deacetylase inhibitors vorinostat and LBH589 in colon cancer cell lines

BACKGROUND: Despite the significant progress made in colon cancer chemotherapy, advanced disease remains largely incurable and novel efficacious chemotherapies are urgently needed. Histone deacetylase inhibitors (HDACi) represent a novel class of agents which have demonstrated promising preclinical activity and are undergoing clinical evaluation in colon cancer. The goal of this study was to identify genes in colon cancer cells that are differentially regulated by two clinically advanced hydroxamic acid HDACi, vorinostat and LBH589 to provide rationale for novel drug combination partners and identify a core set of HDACi-regulated genes.

METHODS: HCT116 and HT29 colon cancer cells were treated with LBH589 or vorinostat and growth inhibition, acetylation status and apoptosis were analyzed in response to treatment using MTS, Western blotting and flow cytometric analyses. In addition, gene expression was analyzed using the Illumina Human-6 V2 BeadChip array and Ingenuity Pathway Analysis.

RESULTS: Treatment with either vorinostat or LBH589 rapidly induced histone acetylation, cell cycle arrest and inhibited the growth of both HCT116 and HT29 cells. Bioinformatic analysis of the microarray profiling revealed significant similarity in the genes altered in expression following treatment with the two HDACi tested within each cell line. However, analysis of genes that were altered in expression in the HCT116 and HT29 cells revealed cell-line-specific responses to HDACi treatment. In addition a core cassette of 11 genes modulated by both vorinostat and LBH589 were identified in both colon cancer cell lines analyzed.

CONCLUSION: This study identified HDACi-induced alterations in critical genes involved in nucleotide metabolism, angiogenesis, mitosis and cell survival which may represent potential intervention points for novel therapeutic combinations in colon cancer. This information will assist in the identification of novel pathways and targets that are modulated by HDACi, providing much-needed information on HDACi mechanism of action and providing rationale for novel drug combination partners. We identified a core signature of 11 genes which were modulated by both vorinostat and LBH589 in a similar manner in both cell lines. These core genes will assist in the development and validation of a common gene set which may represent a molecular signature of HDAC inhibition in colon cancer.

Molecular diversity and transferability of integrons from wastewaters

Na evolução bacteriana, a capacidade de explorar novos ambientes e de responder a diferentes pressões selectivas deve-se principalmente à aquisição de novos genes por transferência horizontal. Integrões são elementos genéticos bacterianos que constituem sistemas naturais de captura e expressão de cassetes de genes, sendo um dos principais mecanismos bacterianos envolvidos na aquisição de resistências a antibióticos. Estudos recentes suportam a hipótese de que os ambientes naturais constituem importantes reservatórios de integrões e cassetes de genes. Uma vez que as águas residuais são descarregadas em receptores naturais, torna-se fundamental conhecer a presença e dispersão de integrões nestes ambientes, assim como a sua associação a outros elementos genéticos móveis e a genes de resistências a antibióticos. Neste trabalho, pretendeu-se avaliar a prevalência e diversidade de integrões em águas residuais de origem animal e doméstica, bem como a sua associação a plasmídeos conjugativos, usando metodologias dependentes e independentes do cultivo de microrganismos em laboratório. Os resultados obtidos sustentam assim a hipótese de que ambientes particularmente ricos em matéria orgânica, como é o caso das águas residuais, constituem ambientes propícios à presença de integrões e à ocorrência de transferência horizontal de genes de resistência a antibióticos, embora a sua prevalência e diversidade seja influenciada pelo tipo de efluente em questão. A presença de integrões em estações de tratamento de águas residuais, e em especial nos efluentes tratados, constitui assim um factor preocupante, uma vez que tal contribui para a sua disseminação e dispersão por outros ecossistemas aquáticos, nomeadamente rios e mares. Os métodos utilizados permitiram também detectar uma elevada diversidade de cassetes de genes associadas a integrões, sendo possível que algumas dessas sequências codifiquem para proteínas que desempenhem um importante papel na adaptação bacteriana às intensas pressões selectivas características deste tipo de ambientes. Assim, é possível concluir que as comunidades bacterianas presentes em águas residuais reúnem diferentes tipos de elementos geneticamente móveis que desempenham um importante papel não só na adaptação bacteriana, mas também na disseminação de determinantes genéticos de resistência para ambientes naturais. Adicionalmente, a presença de potenciais proteínas com possíveis aplicações biotecnológicas reforça a importância das águas residuais como fontes de diversidade funcional. Este trabalho incluiu também a criação e implementação da base de dados INTEGRALL, desenvolvida com o intuito de congregar informação acerca de integrões e de uniformizar a nomenclatura de cassetes de genes.

The role of hypoxia in the regulation of membrane transporters

ATP binding cassette (ABC) and solute carrier (SLC) transporters are responsible for the majority of the transcellular movement of various substrates, including drugs, among epithelial cells. Despite the well characterized regulation of influx (SLC) and efflux (ABC) transporters by endogenous mediators, such as inflammatory cytokines, little is known about how changes in oxygen levels may affect expression of these transporters. In this study we showed that the expression of SLC22A4, SLC22A5, SLC22A1, SLC02B1, SLC10A2, ABCC2 and ABCC3 transporters is upregulated by hypoxia in HT29 colon carcinoma cells, but not in HepG2 hepatocarcinoma cells. Moreover, OCTN1 (SLC22A4), OCT1 (SLC22A1) and OATP-B (SLC02B1) transporter expression is also induced by inflammatory cytokines but in a smaller extent than in hypoxia. Furthermore our experiments indicate that there is no cross talk between HIF-1 and NF-κB pathways in HT-29 cells, but these two pathways act simultaneously activating common genes, such as, some SLC and ABC transporters. Our preliminary results from studies with an in vivo murine model of colitis, suggest that HIF-1is stabilized and OCTN1 is strongly induced during severe inflammation, which can be relevant for a recovery from the inflammatory process. We have also been interested in the distribution of HIF-1α variants among different ethnic groups as well as their contribution for cancer risk. Thus, we have demonstrated that there is an ethnicity-related variation in the frequency of the C1772T (P582S) single nucleotide polymorphism (SNP) in the HIF-1α gene. Furthermore, we performed a case-control study in a breast cancer population and our results suggest that there is no association between this SNP or the rare G1790A (A588T) SNP and the incidence of breast cancer. Taken together, the results obtained in this study contribute to a better knowledge of drug influx and efflux during hypoxia and inflammation as well as to the understanding of the pharmacogenetic variability of the HIF-1.

Development of a Piggybac based direct reprogramming system for derivation of integration free induced pluripotent stem cells

Induced pluripotent stem cells (iPSc) have great potential for applications in regenerative medicine, disease modeling and basic research. Several methods have been developed for their derivation. The original method of Takahashi and Yamanaka involved the use of retroviral vectors which result in insertional mutagenesis, presence in the genome of potential oncogenes and effects of residual transgene expression on differentiation bias of each particular iPSc line. Other methods have been developed, using different viral vectors (adenovirus and Sendai virus), transient plasmid transfection, mRNA transduction, protein transduction and use of small molecules. However, these methods suffer from low efficiencies; can be extremely labor intensive, or both. An additional method makes use of the piggybac transposon, which has the advantage of inserting its payload into the host genome and being perfectly excised upon re-expression of the transposon transposase. Briefly, a policistronic cassette expressing Oct4, Sox2, Klf4 and C-Myc flanked by piggybac terminal repeats is delivered to the cells along with a plasmid transiently expressing piggybac transposase. Once reprogramming occurs, the cells are re-transfected with transposase and subclones free of tranposon integrations screened for. The procedure is therefore very labor intensive, requiring multiple manipulations and successive rounds of cloning and screening. The original method for reprogramming with the the PiggyBac transposon was created by Woltjen et al in 2009 (schematized here) and describes a process with which it is possible to obtain insert-free iPSc. Insert-free iPSc enables the establishment of better cellular models of iPS and adds a new level of security to the use of these cells in regenerative medicine. Due to the fact that it was based on several low efficiency steps, the overall efficiency of the method is very low (<1%). Moreover, the stochastic transfection, integration, excision and the inexistence of an active way of selection leaves this method in need of extensive characterization and screening of the final clones. In this work we aime to develop a non-integrative iPSc derivation system in which integration and excision of the transgenes can be controlled by simple media manipulations, avoiding labor intensive and potentially mutagenic procedures. To reach our goal we developed a two vector system which is simultaneously delivered to original population of fibroblasts. The first vector, Remo I, carries the reprogramming cassette and GFP under the regulation of a constitutive promoter (CAG). The second vector, Eneas, carries the piggybac transposase associated with an estrogen receptor fragment (ERT2), regulated in a TET-OFF fashion, and its equivalent reverse trans-activator associated with a positive-negative selection cassette under a constitutive promoter. We tested its functionality in HEK 293T cells. The protocol is divided in two the following steps: 1) Obtaining acceptable transfection efficiency into human fibroblasts. 2) Testing the functionality of the construct 3) Determining the ideal concentration of DOX for repressing mPB-ERT2 expression 4) Determining the ideal concentration of TM for transposition into the genome 5) Determining the ideal Windows of no DOX/TM pulse for transposition into the genome 6) 3, 4 and 5) for transposition out of the genome 7) Determination of the ideal concentration of GCV for negative selection We successfully demonstrated that ENEAS behaved as expected in terms of DOX regulation of the expression of mPB-ERT2. We also demonstrated that by delivering the plasmid into 293T HEK cells and manipulating the levels of DOX and TM in the medium, we could obtain puromycin resistant lines. The number of puromycin resistant colonies obtained was significantly higher when DOX as absent, suggesting that the colonies resulted from transposition events. Presence of TM added an extra layer of regulation, albeit weaker. Our PCR analysis, while not a clean as would be desired, suggested that transposition was indeed occurring, although a background level of random integration could not be ruled out. Finally, our attempt to determine whether we could use GVC to select clones that had successfully mobilized PB out of the genome was unsuccessful. Unexpectedly, 293T HEK cells that had been transfected with ENEAS and selected for puromycin resistance were insensitive to GCV.

Cholesterol 24S-Hydroxylase overexpression inhibits the liver X receptor (LXR) pathway by activating small guanosine triphosphate-binding proteins (sGTPases) in neuronal cells

The neuronal-specific cholesterol 24S-hydroxylase (CYP46A1) is important for brain cholesterol elimination. Cyp46a1 null mice exhibit severe deficiencies in learning and hippocampal long-term potentiation, suggested to be caused by a decrease in isoprenoid intermediates of the mevalonate pathway. Conversely, transgenic mice overexpressing CYP46A1 show an improved cognitive function. These results raised the question of whether CYP46A1 expression can modulate the activity of proteins that are crucial for neuronal function, namely of isoprenylated small guanosine triphosphate-binding proteins (sGTPases). Our results show that CYP46A1 overexpression in SH-SY5Y neuroblastoma cells and in primary cultures of rat cortical neurons leads to an increase in 3-hydroxy-3-methyl-glutaryl-CoA reductase activity and to an overall increase in membrane levels of RhoA, Rac1, Cdc42 and Rab8. This increase is accompanied by a specific increase in RhoA activation. Interestingly, treatment with lovastatin or a geranylgeranyltransferase-I inhibitor abolished the CYP46A1 effect. The CYP46A1-mediated increase in sGTPases membrane abundance was confirmed in vivo, in membrane fractions obtained from transgenic mice overexpressing this enzyme. Moreover, CYP46A1 overexpression leads to a decrease in the liver X receptor (LXR) transcriptional activity and in the mRNA levels of ATP-binding cassette transporter 1, sub-family A, member 1 and apolipoprotein E. This effect was abolished by inhibition of prenylation or by co-transfection of a RhoA dominant-negative mutant. Our results suggest a novel regulatory axis in neurons; under conditions of membrane cholesterol reduction by increased CYP46A1 expression, neurons increase isoprenoid synthesis and sGTPase prenylation. This leads to a reduction in LXR activity, and consequently to a decrease in the expression of LXR target genes.

ABCC Subfamily Vacuolar Transporters are Involved in Pb (Lead) Detoxification in Saccharomyces cerevisiae

The present work has as objective to contribute for the elucidation of the mechanism associated with Pb detoxification, using the yeast Saccharomyces cerevisiae as a model organism. The deletion of GTT1 or GTT2 genes, coding for functional glutathione transferases (GST) enzymes in S. cerevisiae, caused an increased susceptibility to high Pb concentrations (500-1000 μmol L(-1)). These results suggest that the formation of glutathione-Pb conjugate (GS-Pb), dependent of GSTs, is important in Pb detoxification. The involvement of ATP-binding cassette (ABC) vacuolar transporters, belonging to class C subfamily (ABCC) in vacuolar compartmentalization of Pb, was evaluated. For this purpose, mutant strains disrupted in YCF1, VMR1, YBT1 or BPT 1 genes were used. All mutants tested, without vacuolar ABCC transporters, presented an increased sensitivity to 500-1000 μmol L(-1) Pb comparative to wild-type strain. Taken together, the obtained results suggest that Pb detoxification, by vacuolar compartmentalization, can occur as a result of the concerted action of GSTs and vacuolar ABCC transporters. Pb is conjugated with glutathione, catalysed by glutathione transferases and followed to the transport of GS-Pb conjugate to the vacuole by ABCC transporters.

Estudo da Toxicidade do Chumbo na Levedura Saccharomyces cerevisiae

O chumbo é um importante poluente ambiental. A levedura Saccharomyces cerevisiae constitui um modelo útil para o estudo dos efeitos tóxicos do chumbo. O conhecimento dos mecanismos de defesa e resistência à presença de metais pesados poderá ser útil em tecnologias de proteção ambiental, nomeadamente no desenvolvimento de novas metodologias para a biorremediação de metais pesados. O presente trabalho teve como objetivo avaliar o impacto do Pb na capacidade proliferativa, na integridade membranar e na produção intracelular de espécies reativas de oxigénio (ROS), na estirpe laboratorial da levedura Saccharomyces cerevisiae BY4741 (estirpe selvagem, WT). Foi também estudado o papel das mitocôndrias, como fonte de ROS induzida por Pb, e o envolvimento da H+-ATPase vacuolar (V-ATPase) e de transportadores vacuolares pertencentes à superfamília ABC (de ATP-binding cassette) na defesa contra a toxicidade do Pb. O estudo cinético do impacto de duas concentrações de Pb na viabilidade das leveduras (avaliado através de um ensaio clonogénico), na integridade da membrana celular (determinada com iodeto de propídio) e na produção intracelular de ROS (o anião superóxido foi detetado com dihidroetídio e o peróxido de hidrogénio com 2’,7’- diclorodihidrofluoresceína), revelou uma perda progressiva da capacidade proliferativa (53 e 17% de células viáveis, após a exposição durante 3h a 250 ou 1000 µmol/l de chumbo, respetivamente), coincidente com a acumulação intracelular de anião superóxido e de peróxido de hidrogénio, na ausência de perda da integridade membranar. A importância das mitocôndrias na produção de ROS, induzida por chumbo, foi levada a cabo usando um mutante deficiente respiratório desprovido de ADN mitocondrial (ƿ0). Quando comparado com a respetiva estirpe parental, o mutante ƿ0 apresentou uma maior resistência ao Pb e uma menor produção de ROS induzida por Pb. A exposição das células da estirpe BY4741 a 250 e 1000 µmol/l de chumbo originou a formação de 49 e 58% de células deficientes respiratórias, respetivamente. A função da V-ATPase, na desintoxicação de chumbo, foi avaliada utilizando mutantes com uma estrutura vacuolar normal mas defetivos em subunidades da VATPase (vma1Δ, vma2Δ, vma3Δ e vph1Δ). Comparativamente às células da estirpe WT, todos os mutantes testados, sem V-ATPase funcional, apresentaram uma maior suscetibilidade ao Pb. O papel dos transportadores vacuolares pertencentes à superfamília ABC, na defesa contra a toxicidade induzida por chumbo, foi levada a cabo utilizando mutantes sem os transportadores Ycf1p ou Vmr1p. Os resultados preliminares mostraram que quando comparadas com as células da estirpe WT, as células das estirpes ycf1Δ ou vmr1Δ não apresentavam uma maior perda da viabilidade. A modificação da morfologia vacuolar, em células expostas a chumbo, foi visualizada utilizando a estirpe Vma2p-GFP. O tratamento das células com Pb originou a fusão dos vacúolos de tamanho médio num único vacúolo de grande dimensão. Em conclusão, os estudos desenvolvidos no presente trabalho, utilizando a estirpe laboratorial BY4741, mostraram que a perda da capacidade proliferativa das leveduras, induzida pelo chumbo, pode ser atribuída à acumulação intracelular do anião superóxido e de peróxido de hidrogénio. As mitocôndrias parecem ser uma das principais fontes de ROS induzido por Pb e, simultaneamente, um dos principais alvos da sua toxicidade. Em S. cerevisiae, o vacúolo desempenha um papel importante na desintoxicação do Pb. A modificação da morfologia vacuolar após exposição ao chumbo poderá ser a consequência da acumulação de Pb no vacúolo. Enquanto os transportadores da superfamília ABC parecem não estar envolvidos na sequestração vacuolar de Pb, é necessária a presença, num estado funcional, da V-ATPase para que ocorra a compartimentação do Pb. Muito provavelmente, a compartimentação do Pb no vacúolo previne a sua acumulação no citosol e o desencadear dos respetivos efeitos tóxicos.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) in Portugal:

Methicillin-resistant Staphylococcus aureus (MRSA), a human pathogen confined to hospitals (HAMRSA) for over 30 years have been emerging worldwide in the last two decades as a leading cause of severe infections in healthy individuals in the community (CA-MRSA). Despite its clinical significance, in the beginning of our studies no information existed on the prevalence, and population structure of CA-MRSA in Portugal. Moreover, it remained to be clarified how CA-MRSA emerged in our country. In particular, it was not known if CA-MRSA emerged locally by acquisition of the staphylococcal cassette chromosome mec (SCCmec) by established methicillin-susceptible S. aureus (MSSA) in the community, if they were imported from abroad or have escaped from the hospital.(...)

Flippase (FLP) recombinase-mediated marker recycling in the dermatophyte Arthroderma vanbreuseghemii.

Biological processes can be elucidated by investigating complex networks of relevant factors and genes. However, this is not possible in species for which dominant selectable markers for genetic studies are unavailable. To overcome the limitation in selectable markers for the dermatophyte Arthroderma vanbreuseghemii (anamorph: Trichophyton mentagrophytes), we adapted the flippase (FLP) recombinase-recombination target (FRT) site-specific recombination system from the yeast Saccharomyces cerevisiae as a selectable marker recycling system for this fungus. Taking into account practical applicability, we designed FLP/FRT modules carrying two FRT sequences as well as the flp gene adapted to the pathogenic yeast Candida albicans (caflp) or a synthetic codon-optimized flp (avflp) gene with neomycin resistance (nptII) cassette for one-step marker excision. Both flp genes were under control of the Trichophyton rubrum copper-repressible promoter (PCTR4). Molecular analyses of resultant transformants showed that only the avflp-harbouring module was functional in A. vanbreuseghemii. Applying this system, we successfully produced the Ku80 recessive mutant strain devoid of any selectable markers. This strain was subsequently used as the recipient for sequential multiple disruptions of secreted metalloprotease (fungalysin) (MEP) or serine protease (SUB) genes, producing mutant strains with double MEP or triple SUB gene deletions. These results confirmed the feasibility of this system for broad-scale genetic manipulation of dermatophytes, advancing our understanding of functions and networks of individual genes in these fungi.

Directed integration of new insulated lentiviral vectors to heterochromatin towards safer gene transfer to stem cells

The need for better gene transfer systems towards improved risk=benefit balance for patients remains a major challenge in the clinical translation of gene therapy (GT). We have investigated the improvement of integrating vectors safety in combining (i) new short synthetic genetic insulator elements (GIE) and (ii) directing genetic integration to heterochromatin. We have designed SIN-insulated retrovectors with two candidate GIEs and could identify a specific combination of insulator 2 repeats which translates into best functional activity, high titers and boundary effect in both gammaretro (p20) and lentivectors (DCaro4) (see Duros et al, abstract ibid). Since GIEs are believed to shield the transgenic cassette from inhibitory effects and silencing, DCaro4 has been further tested with chimeric HIV-1 derived integrases which comprise C-ter chromodomains targeting heterochromatin through either histone H3 (ML6chimera) or methylatedCpGislands (ML10). With DCaro4 only and both chimeras, a homogeneous expression is evidenced in over 20% of the cells which is sustained over time. With control lentivectors, less than 2% of cells express GFP as compared to background using a control double-mutant in both catalytic and ledgf binding-sites; in addition, a two-times increase of expression can be induced with histone deacetylase inhibitors. Our approach could significantly reduce integration into open chromatin sensitive sites in stem cells at the time of transduction, a feature which might significantly decrease subsequent genotoxicity, according to X-SCIDs patients data.Work performed with the support of EC-DG research within the FP6-Network of Excellence, CLINIGENE: LSHB-CT-2006-018933

今古奇觀Jin gu qi guan.Merveilles de l'antiquité et des temps modernes.

Contient : I, livre 1三孝廉讓產立高名San xiao lian rang chan li gao ming.Les trois licenciés charitables qui acquièrent de la célébrité ; II, livre 2兩縣令競義婚孤女Liang xian ling jing yi hun gu nü.L'orpheline ; III, livre 3滕大尹鬼斷家私Teng da yin gui duan jia si.Le portrait de famille ; IV, livre 4裴晉公義還原配Pei jin gong yi huan yuan phei.Comment le mandarin Dan bi perdit et retrouva sa fiancée ; V, livre 5杜十娘怒沈百寶箱Du shi niang nu chen bai bao xiang.Du Shi niang, de colère, jette dans l'eau la cassette de bijoux ; VI, livre 6李謫仙醉草嚇蠻書Li zhe xian zui cao he man shu.Le poète Li Tai bai ; VII, livre 7賣油郎獨占花魁Mai you lang du zhan hua khoei.Le vendeur d'huile qui seul possède la reine de beauté ; VIII, livre 8灌園叟（alias 吏）晚逢仙女Guan yuan sou (alias li) wan feng xian nü.Les pivoines ; IX, livre 9轉運漢巧遇洞庭紅Zhuan yun han qiao yu dong ting hong.Le couli des transports, adroitement, reçoit la beauté du Dong ting ; X, livre 10看財奴刁買寃家主Kan cai nu diao mai yuan jia zhu.Richesse mal acquise (Comment le ciel donne et reprend les richesses) ; XI, livre 11吳保安棄家贖友Wu bao an qi jia shu you.Véritable amitié ; XII, livre 12羊角哀舍命全交Yang jue ai she ming quan jiao.Yang Jue ai fait le sacrifice do sa vie par dévouement pour un ami ; XIII, livre 13沈小霞相會出師表Shen xiao xia xiang hui chu shi biao.Shen Xiao xia rencontre et présente le modèle des maîtres ; XIV, livre 14宋金郎團圓破氊笠Song jin lang tuan yuan po zhan li.Les tendres époux ; XV, livre 15盧太學詩酒傲公侯Lu tai xue shi jiu ao gong hou.Lu tai xue, poète et ivre, brave les princes ; XVI, livre 16李汧公窮邸遇俠客Li qian gong qiong di yu xie ke.Li Qian gong, dans sa résidence misérable, traite un hôte magnanime ; XVII, livre 17蘇小妹三難新郎Su xiao mei san nan xin lang.La jeune Su trois fois maltraite un nouveau marié ; XVIII, livre 18劉元普雙生貴子Liu yuan pu shuang sheng gui zi.Liu Yuan pu obtient deux beaux enfants ; XIX, livre 19俞伯牙摔琴謝知音Yu bai ya choai qin xie zhi yin.Le luth brisé ; XX, livre 20莊子休鼓盆成大道Zhuang zi xiu gu phen cheng da dao.La matrone du pays de Song ; XXI, livre 21老門生三世報恩Lao men sheng san shi bao en.Le vieil élève montre sa reconnaissance à la troisième génération ; XXII, livre 22鈍秀才一朝交泰Dun xiu cai yi zhao jiao tai.Le bachelier obtus tout d'un coup exerce son influence formatrice ; XXIII, livre 23蔣興哥重會珍珠衫Jiang xing ge chong hui zheng zhu shan.Le négociant ruiné (La tunique de perles) ; XXIV, livre 24陳御史巧勘金釵鈿Chen yu shi qiao kan jin tchhai tian.Une cause célèbre ; XXV, livre 25徐老僕義憤成家Xu lao bu yi fen cheng jia.Un serviteur méritant ; XXVI, livre 26蔡小姐忍辱報讐Cai xiao jie ren ru bao chou .L'héroïsme de la piété filiale ; XXVII, livre 27錢秀才錯占鳳凰儔.Qian xiu cai tsho zhan feng huang chou .Mariage forcé ; XXVIII, livre 28喬太守亂㸃鴛鴦譜.Qiao tai shou luan dian yuan yang pu.Le préfet Qiao pointe à tort le registre des unions ; XXIX, livre 29懷私怨很僕告主Huai si yuan hen bu gao zhu.Le crime puni ; XXX, livre 30念親恩孝女藏兒Nian qin en xiao nü cang er.La calomnie démasquée ; XXXI, livre 31呂大郎還金完骨肉Lü da lang huan jin wan gu ru.Les trois frères ; XXXII, livre 32金玉奴棒打薄情郎.Jin yu nu bang da bai qing lang.Femme et mari ingrats ; XXXIII, livre 33唐解元玩世出奇.Tang jie yuan wan shi chu qi.Le mariage du licencié Tang (Tang le jie yuan) ; XXXIV, livre 34女秀才移花接木.Nü xiu cai yi hua jie mu.La bachelière du pays de Zhu ; XXXV, livre 35王喬鸞百年長恨.Wang qiao luan bai nian chang hen.Le ressentiment perpétuel de Wang Qiao luan ; XXXVI, livre 36十三郎五歲朝天.Shi san lang wu sui chao tian.Shi san lang pendant cinq ans se tourne vers le ciel ; XXXVII, livre 37崔俊臣巧合芙蓉屏.Cui jun chen qiao he fu yong ping.Paravent révélateur ; XXXVIII, livre 38趙縣君喬送黃柑子.Zhao xian jun qiao song huang gan zi.Chantage ; XXXIX, livre 39誇妙術丹容提金.Khoa miao shu dan yong ti jin.Les alchimistes ; XL, livre 40逞多財（alias 錢多） 白丁橫帶Cheng duo cai (qian duo) bai ding heng dai.Cupidité et dérèglement

今古奇觀Jin gu qi guan.Merveilles de l'antiquité et des temps modernes.

Contient : I, livre 1三孝廉讓產立高名San xiao lian rang chan li gao ming.Les trois licenciés charitables qui acquièrent de la célébrité ; II, livre 2兩縣令競義婚孤女Liang xian ling jing yi hun gu nü.L'orpheline ; III, livre 3滕大尹鬼斷家私Teng da yin gui duan jia si.Le portrait de famille ; IV, livre 4裴晉公義還原配Pei jin gong yi huan yuan phei.Comment le mandarin Dan bi perdit et retrouva sa fiancée ; V, livre 5杜十娘怒沈百寶箱Du shi niang nu chen bai bao xiang.Du Shi niang, de colère, jette dans l'eau la cassette de bijoux ; VI, livre 6李謫仙醉草嚇蠻書Li zhe xian zui cao he man shu.Le poète Li Tai bai ; VII, livre 7賣油郎獨占花魁Mai you lang du zhan hua khoei.Le vendeur d'huile qui seul possède la reine de beauté ; VIII, livre 8灌園叟（alias 吏）晚逢仙女Guan yuan sou (alias li) wan feng xian nü.Les pivoines ; IX, livre 9轉運漢巧遇洞庭紅Zhuan yun han qiao yu dong ting hong.Le couli des transports, adroitement, reçoit la beauté du Dong ting ; X, livre 10看財奴刁買寃家主Kan cai nu diao mai yuan jia zhu.Richesse mal acquise (Comment le ciel donne et reprend les richesses) ; XI, livre 11吳保安棄家贖友Wu bao an qi jia shu you.Véritable amitié ; XII, livre 12羊角哀舍命全交Yang jue ai she ming quan jiao.Yang Jue ai fait le sacrifice do sa vie par dévouement pour un ami ; XIII, livre 13沈小霞相會出師表Shen xiao xia xiang hui chu shi biao.Shen Xiao xia rencontre et présente le modèle des maîtres ; XIV, livre 14宋金郎團圓破氊笠Song jin lang tuan yuan po zhan li.Les tendres époux ; XV, livre 15盧太學詩酒傲公侯Lu tai xue shi jiu ao gong hou.Lu tai xue, poète et ivre, brave les princes ; XVI, livre 16李汧公窮邸遇俠客Li qian gong qiong di yu xie ke.Li Qian gong, dans sa résidence misérable, traite un hôte magnanime ; XVII, livre 17蘇小妹三難新郎Su xiao mei san nan xin lang.La jeune Su trois fois maltraite un nouveau marié ; XVIII, livre 18劉元普雙生貴子Liu yuan pu shuang sheng gui zi.Liu Yuan pu obtient deux beaux enfants ; XIX, livre 19俞伯牙摔琴謝知音Yu bai ya choai qin xie zhi yin.Le luth brisé ; XX, livre 20莊子休鼓盆成大道Zhuang zi xiu gu phen cheng da dao.La matrone du pays de Song ; XXI, livre 21老門生三世報恩Lao men sheng san shi bao en.Le vieil élève montre sa reconnaissance à la troisième génération ; XXII, livre 22鈍秀才一朝交泰Dun xiu cai yi zhao jiao tai.Le bachelier obtus tout d'un coup exerce son influence formatrice ; XXIII, livre 23蔣興哥重會珍珠衫Jiang xing ge chong hui zheng zhu shan.Le négociant ruiné (La tunique de perles) ; XXIV, livre 24陳御史巧勘金釵鈿Chen yu shi qiao kan jin tchhai tian.Une cause célèbre ; XXV, livre 25徐老僕義憤成家Xu lao bu yi fen cheng jia.Un serviteur méritant ; XXVI, livre 26蔡小姐忍辱報讐Cai xiao jie ren ru bao chou .L'héroïsme de la piété filiale ; XXVII, livre 27錢秀才錯占鳳凰儔.Qian xiu cai tsho zhan feng huang chou .Mariage forcé ; XXVIII, livre 28喬太守亂㸃鴛鴦譜.Qiao tai shou luan dian yuan yang pu.Le préfet Qiao pointe à tort le registre des unions ; XXIX, livre 29懷私怨很僕告主Huai si yuan hen bu gao zhu.Le crime puni ; XXX, livre 30念親恩孝女藏兒Nian qin en xiao nü cang er.La calomnie démasquée ; XXXI, livre 31呂大郎還金完骨肉Lü da lang huan jin wan gu ru.Les trois frères ; XXXII, livre 32金玉奴棒打薄情郎.Jin yu nu bang da bai qing lang.Femme et mari ingrats ; XXXIII, livre 33唐解元玩世出奇.Tang jie yuan wan shi chu qi.Le mariage du licencié Tang (Tang le jie yuan) ; XXXIV, livre 34女秀才移花接木.Nü xiu cai yi hua jie mu.La bachelière du pays de Zhu ; XXXV, livre 35王喬鸞百年長恨.Wang qiao luan bai nian chang hen.Le ressentiment perpétuel de Wang Qiao luan ; XXXVI, livre 36十三郎五歲朝天.Shi san lang wu sui chao tian.Shi san lang pendant cinq ans se tourne vers le ciel ; XXXVII, livre 37崔俊臣巧合芙蓉屏.Cui jun chen qiao he fu yong ping.Paravent révélateur ; XXXVIII, livre 38趙縣君喬送黃柑子.Zhao xian jun qiao song huang gan zi.Chantage ; XXXIX, livre 39誇妙術丹容提金.Khoa miao shu dan yong ti jin.Les alchimistes ; XL, livre 40逞多財（alias 錢多） 白丁橫帶Cheng duo cai (qian duo) bai ding heng dai.Cupidité et dérèglement

今古奇觀Jin gu qi guan.Merveilles de l'antiquité et des temps modernes.

Contient : I, livre 1三孝廉讓產立高名San xiao lian rang chan li gao ming.Les trois licenciés charitables qui acquièrent de la célébrité ; II, livre 2兩縣令競義婚孤女Liang xian ling jing yi hun gu nü.L'orpheline ; III, livre 3滕大尹鬼斷家私Teng da yin gui duan jia si.Le portrait de famille ; IV, livre 4裴晉公義還原配Pei jin gong yi huan yuan phei.Comment le mandarin Dan bi perdit et retrouva sa fiancée ; V, livre 5杜十娘怒沈百寶箱Du shi niang nu chen bai bao xiang.Du Shi niang, de colère, jette dans l'eau la cassette de bijoux ; VI, livre 6李謫仙醉草嚇蠻書Li zhe xian zui cao he man shu.Le poète Li Tai bai ; VII, livre 7賣油郎獨占花魁Mai you lang du zhan hua khoei.Le vendeur d'huile qui seul possède la reine de beauté ; VIII, livre 8灌園叟（alias 吏）晚逢仙女Guan yuan sou (alias li) wan feng xian nü.Les pivoines ; IX, livre 9轉運漢巧遇洞庭紅Zhuan yun han qiao yu dong ting hong.Le couli des transports, adroitement, reçoit la beauté du Dong ting ; X, livre 10看財奴刁買寃家主Kan cai nu diao mai yuan jia zhu.Richesse mal acquise (Comment le ciel donne et reprend les richesses) ; XI, livre 11吳保安棄家贖友Wu bao an qi jia shu you.Véritable amitié ; XII, livre 12羊角哀舍命全交Yang jue ai she ming quan jiao.Yang Jue ai fait le sacrifice do sa vie par dévouement pour un ami ; XIII, livre 13沈小霞相會出師表Shen xiao xia xiang hui chu shi biao.Shen Xiao xia rencontre et présente le modèle des maîtres ; XIV, livre 14宋金郎團圓破氊笠Song jin lang tuan yuan po zhan li.Les tendres époux ; XV, livre 15盧太學詩酒傲公侯Lu tai xue shi jiu ao gong hou.Lu tai xue, poète et ivre, brave les princes ; XVI, livre 16李汧公窮邸遇俠客Li qian gong qiong di yu xie ke.Li Qian gong, dans sa résidence misérable, traite un hôte magnanime ; XVII, livre 17蘇小妹三難新郎Su xiao mei san nan xin lang.La jeune Su trois fois maltraite un nouveau marié ; XVIII, livre 18劉元普雙生貴子Liu yuan pu shuang sheng gui zi.Liu Yuan pu obtient deux beaux enfants ; XIX, livre 19俞伯牙摔琴謝知音Yu bai ya choai qin xie zhi yin.Le luth brisé ; XX, livre 20莊子休鼓盆成大道Zhuang zi xiu gu phen cheng da dao.La matrone du pays de Song ; XXI, livre 21老門生三世報恩Lao men sheng san shi bao en.Le vieil élève montre sa reconnaissance à la troisième génération ; XXII, livre 22鈍秀才一朝交泰Dun xiu cai yi zhao jiao tai.Le bachelier obtus tout d'un coup exerce son influence formatrice ; XXIII, livre 23蔣興哥重會珍珠衫Jiang xing ge chong hui zheng zhu shan.Le négociant ruiné (La tunique de perles) ; XXIV, livre 24陳御史巧勘金釵鈿Chen yu shi qiao kan jin tchhai tian.Une cause célèbre ; XXV, livre 25徐老僕義憤成家Xu lao bu yi fen cheng jia.Un serviteur méritant ; XXVI, livre 26蔡小姐忍辱報讐Cai xiao jie ren ru bao chou .L'héroïsme de la piété filiale ; XXVII, livre 27錢秀才錯占鳳凰儔.Qian xiu cai tsho zhan feng huang chou .Mariage forcé ; XXVIII, livre 28喬太守亂㸃鴛鴦譜.Qiao tai shou luan dian yuan yang pu.Le préfet Qiao pointe à tort le registre des unions ; XXIX, livre 29懷私怨很僕告主Huai si yuan hen bu gao zhu.Le crime puni ; XXX, livre 30念親恩孝女藏兒Nian qin en xiao nü cang er.La calomnie démasquée ; XXXI, livre 31呂大郎還金完骨肉Lü da lang huan jin wan gu ru.Les trois frères ; XXXII, livre 32金玉奴棒打薄情郎.Jin yu nu bang da bai qing lang.Femme et mari ingrats ; XXXIII, livre 33唐解元玩世出奇.Tang jie yuan wan shi chu qi.Le mariage du licencié Tang (Tang le jie yuan) ; XXXIV, livre 34女秀才移花接木.Nü xiu cai yi hua jie mu.La bachelière du pays de Zhu ; XXXV, livre 35王喬鸞百年長恨.Wang qiao luan bai nian chang hen.Le ressentiment perpétuel de Wang Qiao luan ; XXXVI, livre 36十三郎五歲朝天.Shi san lang wu sui chao tian.Shi san lang pendant cinq ans se tourne vers le ciel ; XXXVII, livre 37崔俊臣巧合芙蓉屏.Cui jun chen qiao he fu yong ping.Paravent révélateur ; XXXVIII, livre 38趙縣君喬送黃柑子.Zhao xian jun qiao song huang gan zi.Chantage ; XXXIX, livre 39誇妙術丹容提金.Khoa miao shu dan yong ti jin.Les alchimistes ; XL, livre 40逞多財（alias 錢多） 白丁橫帶Cheng duo cai (qian duo) bai ding heng dai.Cupidité et dérèglement