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BACKGROUND: Immunotherapy with immune checkpoint inhibitors (ICIs) is being explored to improve cholangiocarcinoma (CCA) therapy. However, it remains difficult to predict which ICI will be effective for individual patients. Therefore, the aim of this study is to develop a co-culture method with patient-derived CCA organoids and immune cells, which could represent anti-cancer immunity in vitro. METHODS: CCA organoids were co-cultured with peripheral blood mononuclear cells or T cells. Flow cytometry, time-lapse confocal imaging for apoptosis, and quantification of cytokeratin 19 fragment (CYFRA) release were applied to analyse organoid and immune cell behaviour. CCA organoids were also cultured in immune cell-conditioned media to analyse the effect of soluble factors. RESULTS: The co-culture system demonstrated an effective anti-tumour organoid immune response by a decrease in live organoid cells and an increase in apoptosis and CYFRA release. Interpatient heterogeneity was observed. The cytotoxic effects could be mediated by direct cell-cell contact and by release of soluble factors, although soluble factors only decreased viability in one organoid line. CONCLUSIONS: In this proof-of-concept study, a novel CCA organoid and immune cell co-culture method was established. This can be the first step towards personalised immunotherapy for CCA by predicting which ICIs are most effective for individual patients.
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Neoplasias dos Ductos Biliares , Colangiocarcinoma , Ductos Biliares Intra-Hepáticos/patologia , Humanos , Leucócitos Mononucleares/metabolismo , Organoides , Linfócitos T/patologiaRESUMO
OBJECTIVE: To identify risk factors for a woman to experience pregnancy denial. DESIGN, SETTING AND POPULATION: A French multicentric prospective case-control study with 71 mother-infant dyads having experienced a pregnancy denial versus a control group of 71 dyads. METHODS: Data were collected in the week after delivery using an observational leaflet and two psychiatric scales (MINI and QSSP). MAIN OUTCOME MEASURES: Statistically significant differences between the two groups regarding social, demographic, medical and psychiatric data. RESULTS: Not being in a stable relationship (odds ratio [OR] 17.18, 95% CI 3.37-87.60]; P < 0.0001), not having a high school diploma (OR 1.11, 95% CI 1.04-1.38]; P < 0.0001) and having a psychiatric history (OR 6.33, 95% CI 1.62-24.76; P = 0.0002) were risk factors to experience pregnancy denial, whereas being older was a protective factor (OR 0.86, 95% CI 0.79-0.93; P = 0.0054) (logistic regression, Wald 95% CI). Other risk factors included late declarations of pregnancy history and past pregnancy denials (case n = 7, 9.7% versus 0% in controls; P = 0.01), past pregnancy denials in the family (case n = 13, 18% versus control n = 4, 5.6%; P = 0.03), and use of a contraceptive method (75% for cases versus 7% in control; P < 0.0001), primarily an oral contraceptive (75%). CONCLUSION: Family or personal history of pregnancy denial should be part of the systematic anamnesis during the first visit of a patient of child-bearing age. Further, our study points out that life context (young age, single status, socio-economic precarity, pill-based contraception) could be a trigger for pregnancy denial in certain women. TWEETABLE ABSTRACT: Life context can be a trigger for pregnancy denial.
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Negação em Psicologia , Gravidez não Planejada/psicologia , Adulto , Estudos de Casos e Controles , Anticoncepção/psicologia , Anticoncepção/estatística & dados numéricos , Escolaridade , Feminino , França , Humanos , Modelos Logísticos , Idade Materna , Razão de Chances , Gravidez , Estudos Prospectivos , Fatores de Risco , Adulto JovemRESUMO
The thioester moiety is introduced as a lithium binding unit for the hierarchical formation of titanium(IV) catecholate-based lithium-bridged helicates. In solution, the coordination compounds show a monomer-dimer equilibrium which -in comparison to the oxo esters- is significantly shifted towards the monomers. In addition, the influence of the thioester side chain on the dimerization behavior is investigated and an expansible/compressible molecular switch is synthesized. In the latter case expansion and compression are performed reversibly in methanol, whereas in DMSO spontaneous expansion occurs.
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BACKGROUND: Ragweed (Ambrosia artemisiifolia) is a strong elicitor of allergic airway inflammation with worldwide increasing prevalence. Various components of ragweed pollen are thought to play a role in the development of allergic responses. The aim of this study was to identify critical factors for allergenicity of ragweed pollen in a physiological model of allergic airway inflammation. METHODS: Aqueous ragweed pollen extract, the low molecular weight fraction or the major allergen Amb a 1 was instilled intranasally on 1-11 consecutive days, and allergic airway inflammation was evaluated by bronchoalveolar lavage, lung histology, serology, gene expression in lung tissue, and measurement of lung function. Pollen-derived adenosine was removed from the extract enzymatically to analyze its role in ragweed-induced allergy. Migration of human neutrophils and eosinophils toward supernatants of ragweed-stimulated bronchial epithelial cells was analyzed. RESULTS: Instillation of ragweed pollen extract, but not of the major allergen or the low molecular weight fraction, induced specific IgG1 , pulmonary infiltration with inflammatory cells, a Th2-associated cytokine signature in pulmonary tissue, and impaired lung function. Adenosine aggravated ragweed-induced allergic lung inflammation. In vitro, human neutrophils and eosinophils migrated toward supernatants of bronchial epithelial cells stimulated with ragweed extract only if adenosine was present. CONCLUSIONS: Pollen-derived adenosine is a critical factor in ragweed-pollen-induced allergic airway inflammation. Future studies aim at therapeutic strategies to control these allergen-independent pathways.
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Adenosina/metabolismo , Antígenos de Plantas/imunologia , Imunização/métodos , Extratos Vegetais/imunologia , Hipersensibilidade Respiratória/fisiopatologia , Administração Intranasal , Animais , Asma/imunologia , Asma/fisiopatologia , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Feminino , Humanos , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Distribuição Aleatória , Medição de Risco , Sensibilidade e Especificidade , Células Th2/imunologia , Células Th2/metabolismoRESUMO
BACKGROUND: B cells play a central role in IgE-mediated allergies. In damaged airway epithelium, they are exposed directly to aeroallergens. We aimed to assess whether direct exposure of B cells to pollen constituents affects allergic sensitization. METHODS: B cells from murine splenocytes and from blood samples of healthy donors were incubated for 8 days under Th2-like conditions with aqueous ragweed pollen extracts (Amb-APE) or its constituents. Secreted total IgM, IgG, and IgE was quantified by ELISA. Additionally, birch, grass, or pine-pollen extracts were tested. The number of viable cells was evaluated by ATP measurements. B-cell proliferation was measured by CFSE staining. IgE class switch was analyzed by quantitation of class switch transcripts. In an OVA/Alum i.p.-sensitization mouse model, Amb-APE was intranasally instilled for 11 consecutive days. RESULTS: Upon Th2 priming of murine B cells, ragweed pollen extract caused a dose-dependent increase in IgE production, while IgG and IgM were not affected. The low-molecular-weight fraction and phytoprostane E1 (PPE1) increased IgE production, while Amb a 1 did not. PPE1 enhanced IgE also in human memory B cells. Under Th1 conditions, Amb-APE did not influence immunoglobulin secretion. The IgE elevation was not ragweed specific. It correlated with proliferation of viable B cells, but not with IgE class switch. In vivo, Amb-APE increased total IgE and showed adjuvant activity in allergic airway inflammation. CONCLUSIONS: Aqueous pollen extracts, the protein-free fraction of Amb-APE, and the pollen-contained substance PPE1 specifically enhance IgE production in Th2-primed B cells. Thus, pollen-derived nonallergenic substances might be responsible for B-cell-dependent aggravation of IgE-mediated allergies.
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Alérgenos/imunologia , Formação de Anticorpos/imunologia , Linfócitos B/imunologia , Imunoglobulina E/imunologia , Pólen/imunologia , Células Th2/imunologia , Ambrosia/imunologia , Animais , Antígenos de Plantas/imunologia , Linfócitos B/metabolismo , Feminino , Humanos , Imunização , Memória Imunológica , Ativação Linfocitária/imunologia , Camundongos , Ovalbumina/imunologia , Extratos Vegetais/imunologia , Pneumonia/imunologia , Pneumonia/metabolismo , Pneumonia/patologia , Células Th2/metabolismoRESUMO
The extracellular matrix (ECM) is essential for cell support during homeostasis and plays a critical role in cancer. Although research often concentrates on the tumor's cellular aspect, attention is growing for the importance of the cancer-associated ECM. Biochemical and physical ECM signals affect tumor formation, invasion, metastasis, and therapy resistance. Examining the tumor microenvironment uncovers intricate ECM dysregulation and interactions with cancer and stromal cells. Anticancer therapies targeting ECM sensors and remodelers, including integrins and matrix metalloproteinases, and ECM-remodeling cells, have seen limited success. This review explores the ECM's role in cancer and discusses potential therapeutic strategies for cell-ECM interactions.
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Neoplasias , Humanos , Fenômenos Biomecânicos , Neoplasias/patologia , Matriz Extracelular , Integrinas , Microambiente TumoralRESUMO
Bile ducts are essential for bile transport and consist of complex branching tubular networks. Human patient-derived cholangiocyte develops a cystic rather than branching duct morphology. Here, we present a protocol to establish branching morphogenesis in cholangiocyte and cholangiocarcinoma organoids. We describe steps for the initiation, maintenance, and expansion of intrahepatic cholangiocyte organoids branching morphology. This protocol enables the study of organ-specific and mesenchymal-independent branching morphogenesis and provides an improved model to study biliary function and diseases. For complete details on the use and execution of this protocol, please refer to Roos et al. (2022).1.
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Neoplasias dos Ductos Biliares , Colangiocarcinoma , Humanos , Colangiocarcinoma/patologia , Organoides/patologia , Morfogênese , Ductos Biliares Intra-Hepáticos/patologia , Neoplasias dos Ductos Biliares/patologiaRESUMO
Patient-derived tumor organoids have been established as promising tools for in vitro modelling of multiple tumors, including cholangiocarcinoma (CCA). However, organoids are commonly cultured in basement membrane extract (BME) which does not recapitulate the intricacies of the extracellular matrix (ECM). We combined CCA organoids (CCAOs) with native tumor and liver scaffolds, obtained by decellularization, to effectuate a model to study the interaction between epithelial tumor cells and their surrounding ECM. Decellularization resulted in removal of cells while preserving ECM structure and retaining important characteristics of the tissue origin, including stiffness and presence of desmoplasia. The transcriptome of CCAOs in a tumor scaffold much more resembled that of patient-paired CCA tissue in vivo compared to CCAOs cultured in BME or liver scaffolds. This was accompanied by an increase in chemoresistance to clinically-relevant chemotherapeutics. CCAOs in decellularized scaffolds revealed environment-dependent proliferation dynamics, driven by the occurrence of epithelial-mesenchymal transition. Furthermore, CCAOs initiated an environment-specific desmoplastic reaction by increasing production of multiple collagen types. In conclusion, convergence of organoid-based models with native ECM scaffolds will lead to better understanding of the in vivo tumor environment. STATEMENT OF SIGNIFICANCE: The extracellular matrix (ECM) influences various facets of tumor behavior. Understanding the exact role of the ECM in controlling tumor cell fate is pertinent to understand tumor progression and develop novel therapeutics. This is particularly the case for cholangiocarcinoma (CCA), whereby the ECM displays a distinct tumor environment, characterized by desmoplasia. However, current models to study the interaction between epithelial tumor cells and the environment are lacking. We have developed a fully patient-derived model encompassing CCA organoids (CCAOs) and human decellularized tumor and tumor-free liver ECM. The tumor ECM induced recapitulation of various aspects of CCA, including migration dynamics, transcriptome and proteome profiles, and chemoresistance. Lastly, we uncover that epithelial tumor cells contribute to matrix deposition, and that this phenomenon is dependent on the level of desmoplasia already present.
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Colangiocarcinoma , Neoplasias Epiteliais e Glandulares , Humanos , Matriz Extracelular/química , Colágeno , Organoides , Alicerces Teciduais/química , Engenharia TecidualRESUMO
Tumor initiation and progression are critically dependent on interaction of cancer cells with their cellular and extracellular microenvironment. Alterations in the composition, integrity, and mechanical properties of the extracellular matrix (ECM) dictate tumor processes including cell proliferation, migration, and invasion. Also in primary liver cancer, consisting of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), the dysregulation of the extracellular environment by liver fibrosis and tumor desmoplasia is pertinent. Yet, the exact changes occurring in liver cancer ECM remain uncharacterized and underlying tumor-promoting mechanisms remain largely unknown. Herein, an integrative molecular and mechanical approach is used to extensively characterize the ECM of HCC and CCA tumors by utilizing an optimized decellularization technique. We identified a myriad of proteins in both tumor and adjacent liver tissue, uncovering distinct malignancy-related ECM signatures. The resolution of this approach unveiled additional ECM-related proteins compared to large liver cancer transcriptomic datasets. The differences in ECM protein composition resulted in divergent mechanical properties on a macro- and micro-scale that are tumor-type specific. Furthermore, the decellularized tumor ECM was employed to create a tumor-specific hydrogel that supports patient-derived tumor organoids, which provides a new avenue for personalized medicine applications. Taken together, this study contributes to a better understanding of alterations to composition, stiffness, and collagen alignment of the tumor ECM that occur during liver cancer development.
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Carcinoma Hepatocelular , Neoplasias Hepáticas , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proteômica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Matriz Extracelular/genética , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Microambiente Tumoral/genéticaRESUMO
BACKGROUND: Cutaneous fungal infections are very common, especially in poorer communities and with intercurrent HIV infection. Determining the fungal pathogen in skin-related fungal neglected tropical diseases (NTDs) determines optimal therapy. We undertook a country survey across many African countries to determine the diagnostic capacity for skin fungal diseases. METHODS: A detailed questionnaire was delivered to country contacts to collect data on availability, frequency, and location of testing for key diagnostic procedures and followed up with 2 rounds of validation by video call and by confirmation of individual country data confirmation by email. RESULTS: Of 47 countries with data, seven (15%) and 21 (45%) do not offer skin biopsy in the public or private sector, respectively, but 22 (46%) countries do it regularly, mostly in university hospitals. Direct microscopy is often performed in 20 of 48 (42%) countries in the public sector and not done in 10 (21%). Fungal cultures are often performed in 21 of 48 (44%) countries in the public sector but not done in nine (20%) or 21 (44%) in either public or private facilities. Histopathological examination of tissue is frequently used in 19 of 48 (40%) countries but not in nine (20%) countries in the public sector. The cost of diagnostics to patients was a major limiting factor in usage. CONCLUSION: Major improvements in the availability and use of diagnostic tests for skin, hair, and nail fungal disease are urgently needed across Africa.
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Dermatomicoses , Infecções por HIV , Malária , Humanos , África , Dermatomicoses/diagnóstico , Setor PrivadoRESUMO
Reliable establishment of tumor organoids is paramount to advance applications of organoid technology for personalized medicine. Here, we share our multi-center experience on initiation and tumorigenic confirmation of hepatobiliary cancer organoids. We discuss current concerns, propose potential solutions, and provide future perspectives for improvements in hepatobiliary cancer organoid establishment.
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Neoplasias Gastrointestinais , Organoides , Humanos , Medicina de PrecisãoRESUMO
Advances in biomaterials, particularly in combination with encapsulation strategies, have provided excellent opportunities to increase reproducibility and standardization for cell culture applications. Herein, hybrid microcapsules are produced in a flow-focusing microfluidic droplet generator combined with enzymatic outside-in crosslinking of dextran-tyramine, enriched with human liver extracellular matrix (ECM). The microcapsules provide a physiologically relevant microenvironment for the culture of intrahepatic cholangiocyte organoids (ICO) and patient-derived cholangiocarcinoma organoids (CCAO). Micro-encapsulation allowed for the scalable and size-standardized production of organoids with sustained proliferation for at least 21 days in vitro. Healthy ICO (n = 5) expressed cholangiocyte markers, including KRT7 and KRT19, similar to standard basement membrane extract cultures. The CCAO microcapsules (n = 3) showed retention of stem cell phenotype and expressed LGR5 and PROM1. Furthermore, ITGB1 was upregulated, indicative of increased cell adhesion to ECM in microcapsules. Encapsulated CCAO were amendable to drug screening assays, showing a dose-response response to the clinically relevant anti-cancer drugs gemcitabine and cisplatin. High-throughput drug testing identified both pan-effective drugs as well as patient-specific resistance patterns. The results described herein show the feasibility of this one-step encapsulation approach to create size-standardized organoids for scalable production. The liver extracellular matrix-containing microcapsules can provide a powerful platform to build mini healthy and tumor tissues for potential future transplantation or personalized medicine applications.
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Colangiocarcinoma , Organoides , Humanos , Organoides/metabolismo , Cápsulas , Reprodutibilidade dos Testes , Diferenciação Celular , Fígado/metabolismo , Matriz Extracelular , Colangiocarcinoma/metabolismo , Microambiente TumoralRESUMO
Cholangiocarcinoma (CCA) is a type of liver cancer with an aggressive phenotype and dismal outcome in patients. The metastasis of CCA cancer cells to distant organs, commonly lung and lymph nodes, drastically reduces overall survival. However, mechanistic insight how CCA invades these metastatic sites is still lacking. This is partly because currently available models fail to mimic the complexity of tissue-specific environments for metastatic CCA. To create an in vitro model in which interactions between epithelial tumor cells and their surrounding extracellular matrix (ECM) can be studied in a metastatic setting, we combined patient-derived CCA organoids (CCAOs) (n=3) with decellularized human lung (n=3) and decellularized human lymph node (n=13). Decellularization resulted in removal of cells while preserving ECM structure and retaining important characteristics of the tissue origin. Proteomic analyses showed a tissue-specific ECM protein signature reflecting tissue functioning aspects. The macro and micro-scale mechanical properties, as determined by rheology and micro-indentation, revealed the local heterogeneity of the ECM. When growing CCAOs in decellularized lung and lymph nodes genes related to metastatic processes, including epithelial-to-mesenchymal transition and cancer stem cell plasticity, were significantly influenced by the ECM in an organ-specific manner. Furthermore, CCAOs exhibit significant differences in migration and proliferation dynamics dependent on the original patient tumor and donor of the target organ. In conclusion, CCA metastatic outgrowth is dictated both by the tumor itself as well as by the ECM of the target organ. Convergence of CCAOs with the ECM of its metastatic organs provide a new platform for mechanistic study of cancer metastasis.
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Human cholangiocyte organoids show great promise for regenerative therapies and in vitro modeling of bile duct development and diseases. However, the cystic organoids lack the branching morphology of intrahepatic bile ducts (IHBDs). Here, we report establishing human branching cholangiocyte organoid (BRCO) cultures. BRCOs self-organize into complex tubular structures resembling the IHBD architecture. Single-cell transcriptomics and functional analysis showed high similarity to primary cholangiocytes, and importantly, the branching growth mimics aspects of tubular development and is dependent on JAG1/NOTCH2 signaling. When applied to cholangiocarcinoma tumor organoids, the morphology changes to an in vitro morphology like primary tumors. Moreover, these branching cholangiocarcinoma organoids (BRCCAOs) better match the transcriptomic profile of primary tumors and showed increased chemoresistance to gemcitabine and cisplatin. In conclusion, BRCOs recapitulate a complex process of branching morphogenesis in vitro. This provides an improved model to study tubular formation, bile duct functionality, and associated biliary diseases.
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Colangiocarcinoma , Organoides , Ductos Biliares , Células Epiteliais , Humanos , TranscriptomaRESUMO
Chemical templates for the patterned immobilization of gold nanoparticles were fabricated by soft UV nanoimprint lithography. The template structures were fabricated by means of the consecutively performed process steps of nanoimprint lithography, reactive ion etching, chemical functionalization with amino groups, and lift-off of imprint resist. These chemical templates were used for the defined assembly of 20 nm diameter citrate stabilized gold nanoparticles from aqueous solution. By reducing the ionic strength of the solution, one- and zero-dimensional particle assemblies were generated on sub-100-nm template structures. By this means, the pattern resolution predefined by the lithography process could be easily enhanced by dilution of the nanoparticle solution.
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BACKGROUND: Evidence is accumulating that the pollen exsudate contains an array of non-allergenic, pro-inflammatory and immunomodulatory substances acting on the innate and adaptive immune system. In this context, pollen-associated E(1)-phytoprostanes (PPE(1)) were shown to licence human monocyte-derived dendritic cells for T-helper type 2 (Th2) polarization of naïve T cells. OBJECTIVE: This study aims at analysing the impact of pollen-associated lipid mediators on cytokine secretion and maturation of 6-sulfo LacNAc(+) dendritic cells (slanDCs), the most abundant native dendritic cell (DC) in human peripheral blood, and further dissecting the biologically active substance(s) within aqueous pollen extracts. RESULTS: Aqueous birch pollen extracts dose-dependently inhibited the lipopolysaccharide (LPS)-induced IL-12 p70 production, while the levels of IL-6 remained unaffected. PPE(1) inhibited secretion of both IL-12 p70 and IL-6. Aqueous pollen extracts, but not PPE(1) or F(1)-phytoprostanes significantly reduced the LPS-induced surface expression of the maturation markers CD80, CD83, CD40 and CCR-7, an effect that was independent of proteins and that was still present in a 3 kDa cut-off fraction of the pollen extract. These effects were observed irrespective of the atopy status of the donors. Finally, slanDCs exposed to aqueous pollen extracts were impaired in eliciting an IFN-gamma response in naïve CD4(+) T cells. CONCLUSION: Our data show that slanDCs, a subset of human blood DCs with constitutively high potency to induce Th1 responses, are susceptible to the Th2 polarizing effect of low molecular weight, non-protein factors derived from pollen.
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Amino Açúcares/imunologia , Células Dendríticas/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Extratos Vegetais/farmacologia , Pólen/química , Pólen/imunologia , Células Th1/imunologia , Adulto , Idoso , Células Dendríticas/imunologia , Relação Dose-Resposta a Droga , Humanos , Fatores Imunológicos/imunologia , Interleucina-12/biossíntese , Interleucina-12/imunologia , Lipopolissacarídeos/antagonistas & inibidores , Lipopolissacarídeos/farmacologia , Pessoa de Meia-Idade , Peso Molecular , Extratos Vegetais/imunologia , Células Th1/efeitos dos fármacos , Adulto JovemRESUMO
Primary liver cancer, consisting predominantly of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), remains one of the most lethal malignancies worldwide. This high malignancy is related to the complex and dynamic interactions between tumour cells, stromal cells and the extracellular environment. Novel in vitro models that can recapitulate the tumour are essential in increasing our understanding of liver cancer. Herein, primary liver cancer-derived organoids have opened up new avenues due to their patient-specificity, self-organizing ability and potential recapitulation of many of the tumour properties. Organoids are solely of epithelial origin, but incorporation into co-culture models can enable the investigation of the cellular component of the tumour microenvironment. However, the extracellular component also plays a vital role in cancer progression and representation is lacking within current in vitro models. In this review, organoid technology is discussed in the context of liver cancer models through comparisons to other cell culture systems. In addition, the role of the tumour extracellular environment in primary liver cancer will be highlighted with an emphasis on its importance in in vitro modelling. Converging novel organoid-based models with models incorporating the native tumour microenvironment could lead to experimental models that can better recapitulate liver tumours in vivo.
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We report the first mutational study of thymidine kinase 1 (TK1) performed in human solid tumors. We sequenced cDNAs representing the complete coding region of TK1 in human breast (n=22) and colorectal (n=26) cancer. Codon 106 near the ATP binding site constantly differed (ATG --> GTG; Met --> Val) from the one deposited by Bradshaw and Deininger in the Genbank database (Accession number NM_003258). Silent polymorphisms at codon 11 (CCC --> CCT; Pro --> Pro) and codon 75 (GCG --> GCA; Ala --> Ala) were frequently detected in tumors as well as in normal tissues. In breast cancer the two polymorphisms were observed in 63.6% of the samples analyzed. No significant association could be found between polymorphisms and TK activity. In colorectal cancer the incidence of the two changes was 73.1% and 69.2%, respectively. Interestingly, one colon cancer with high cytosolic TK activity displayed two missense mutations located in and near the putative phosphorylation site by tyrosine kinase (s) (TAT --> CAT; Tyr --> His) and by cAMP-, cGMP-dependent protein kinase (TAC --> TGC; Tyr --> Cys), respectively; adjacent normal mucosa showed no mutation. This may open new avenues that imply TK1 activity in tumor cell proliferation.
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Neoplasias da Mama/genética , Neoplasias Colorretais/genética , Análise Mutacional de DNA , Timidina Quinase/genética , Trifosfato de Adenosina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Sítios de Ligação , Linhagem Celular Tumoral , Códon , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Citosol/metabolismo , DNA Complementar/metabolismo , Bases de Dados Genéticas , Humanos , Pessoa de Meia-Idade , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Mutação de Sentido Incorreto , Polimorfismo GenéticoRESUMO
A prototype of an integrated closed system for fish-plankton aquaculture was developed in Iquitos (Peruvian Amazonia) in order to cultivate the Tiger Catfish, Pseudoplatystoma punctifer (Castelnau, 1855). This freshwater recirculating system consisted of two linked sewage tanks with an intensive rearing unit (a cage) for P. punctifer placed in the first, and with a fish-plankton trophic chain replacing the filters commonly used in clear water closed systems. Detritivorous and zooplanktivorous fishes (Loricariidae and Cichlidae), maintained without external feeding in the sewage volume, mineralized organic matter and permitted the stabilization of the phytoplankton biomass. Water exchange and organic waste discharge were not necessary. In this paper we describe the processes undertaken to equilibrate this ecosystem: first the elimination of an un-adapted spiny alga, Golenkinia sp., whose proliferation was favored by the presence of a small rotifer, Trichocerca sp., and second the control of this rotifer proliferation via the introduction of two cichlid species, Acaronia nassa Heckel, 1840 and Satanoperca jurupari Heckel, 1840, in the sewage part. This favored some development of the green algae Nannochloris sp. and Chlorella sp. At that time we took the opportunity to begin a 3-month rearing test of P. punctifer. The mean specific growth rate and feed conversion ratio (FCR) of P. punctifer were 1.43 and 1.27, respectively, and the global FCR, including fish in the sewage part, was 1.08. This system has proven to be suitable for growing P. punctifer juveniles out to adult, and provides several practical advantages compared with traditional recirculating clear water systems, which use a combination of mechanical and biological filters and require periodic waste removal, leading to water and organic matter losses.