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Sample records for putida alks mrna

  1. Pseudomonas putida AlkA and AlkB proteins comprise different defense systems for the repair of alkylation damage to DNA - in vivo, in vitro, and in silico studies.

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    Damian Mielecki

    Full Text Available Alkylating agents introduce cytotoxic and/or mutagenic lesions to DNA bases leading to induction of adaptive (Ada response, a mechanism protecting cells against deleterious effects of environmental chemicals. In Escherichia coli, the Ada response involves expression of four genes: ada, alkA, alkB, and aidB. In Pseudomonas putida, the organization of Ada regulon is different, raising questions regarding regulation of Ada gene expression. The aim of the presented studies was to analyze the role of AlkA glycosylase and AlkB dioxygenase in protecting P. putida cells against damage to DNA caused by alkylating agents. The results of bioinformatic analysis, of survival and mutagenesis of methyl methanesulfonate (MMS or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG treated P. putida mutants in ada, alkA and alkB genes as well as assay of promoter activity revealed diverse roles of Ada, AlkA and AlkB proteins in protecting cellular DNA against alkylating agents. We found AlkA protein crucial to abolish the cytotoxic but not the mutagenic effects of alkylans since: (i the mutation in the alkA gene was the most deleterious for MMS/MNNG treated P. putida cells, (ii the activity of the alkA promoter was Ada-dependent and the highest among the tested genes. P. putida AlkB (PpAlkB, characterized by optimal conditions for in vitro repair of specific substrates, complementation assay, and M13/MS2 survival test, allowed to establish conservation of enzymatic function of P. putida and E. coli AlkB protein. We found that the organization of P. putida Ada regulon differs from that of E. coli. AlkA protein induced within the Ada response is crucial for protecting P. putida against cytotoxicity, whereas Ada prevents the mutagenic action of alkylating agents. In contrast to E. coli AlkB (EcAlkB, PpAlkB remains beyond the Ada regulon and is expressed constitutively. It probably creates a backup system that protects P. putida strains defective in other DNA repair systems

  2. Pseudomonas putida AlkA and AlkB Proteins Comprise Different Defense Systems for the Repair of Alkylation Damage to DNA – In Vivo, In Vitro, and In Silico Studies

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    Mielecki, Damian; Saumaa, Signe; Wrzesiński, Michał; Maciejewska, Agnieszka M.; Żuchniewicz, Karolina; Sikora, Anna; Piwowarski, Jan; Nieminuszczy, Jadwiga; Kivisaar, Maia; Grzesiuk, Elżbieta

    2013-01-01

    Alkylating agents introduce cytotoxic and/or mutagenic lesions to DNA bases leading to induction of adaptive (Ada) response, a mechanism protecting cells against deleterious effects of environmental chemicals. In Escherichia coli, the Ada response involves expression of four genes: ada, alkA, alkB, and aidB. In Pseudomonas putida, the organization of Ada regulon is different, raising questions regarding regulation of Ada gene expression. The aim of the presented studies was to analyze the role of AlkA glycosylase and AlkB dioxygenase in protecting P. putida cells against damage to DNA caused by alkylating agents. The results of bioinformatic analysis, of survival and mutagenesis of methyl methanesulfonate (MMS) or N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) treated P. putida mutants in ada, alkA and alkB genes as well as assay of promoter activity revealed diverse roles of Ada, AlkA and AlkB proteins in protecting cellular DNA against alkylating agents. We found AlkA protein crucial to abolish the cytotoxic but not the mutagenic effects of alkylans since: (i) the mutation in the alkA gene was the most deleterious for MMS/MNNG treated P. putida cells, (ii) the activity of the alkA promoter was Ada-dependent and the highest among the tested genes. P. putida AlkB (PpAlkB), characterized by optimal conditions for in vitro repair of specific substrates, complementation assay, and M13/MS2 survival test, allowed to establish conservation of enzymatic function of P. putida and E. coli AlkB protein. We found that the organization of P. putida Ada regulon differs from that of E. coli. AlkA protein induced within the Ada response is crucial for protecting P. putida against cytotoxicity, whereas Ada prevents the mutagenic action of alkylating agents. In contrast to E. coli AlkB (EcAlkB), PpAlkB remains beyond the Ada regulon and is expressed constitutively. It probably creates a backup system that protects P. putida strains defective in other DNA repair systems against

  3. High ALK mRNA expression has a negative prognostic significance in rhabdomyosarcoma

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    Bonvini, P; Zin, A; Alaggio, R; Pawel, B; Bisogno, G; Rosolen, A

    2013-01-01

    Background: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in cancer, but its clinical and functional importance remain controversial. Mutation or amplification of ALK, as well as its expression levels assessed by conventional immunohistochemistry methods, has been linked to prognosis in cancer, although with potential bias because of the semi-quantitative approaches. Herein, we measured ALK mRNA expression in rhabdomyosarcoma (RMS) and determined its clinical impact on patients' stratification and outcome. Methods: Specimens were obtained from RMS patients and cell lines, and ALK expression was analysed by quantitative RT–PCR, western blotting, IHC, and copy number analysis. Results: High ALK mRNA expression was detected in the vast majority of PAX3/7-FOXO1-positive tumours, whereas PAX3/7-FOXO1-negative RMS displayed considerably lower amounts of both mRNA and protein. Notably, ALK mRNA distinguished unfavourable PAX3/7-FOXO1-positive tumours from PAX3/7-FOXO1-negative RMS (Ptumour size (PALK mRNA levels were of prognostic relevance by Cox univariate regression analysis and correlated with increased risk of relapse (P=0.001) and survival (P=0.01), whereas by multivariate analysis elevated ALK mRNA expression resulted a negative prognostic marker when clinical stage was not included. Conclusion: Quantitative assessment of ALK mRNA expression helps to improve risk stratification of RMS patients and identifies tumours with adverse biological characteristics and aggressive behaviour. PMID:24149177

  4. High ALK mRNA expression has a negative prognostic significance in rhabdomyosarcoma

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    Bonvini, P; Zin, A; Alaggio, R; Pawel, B; Bisogno, G; Rosolen, A

    2013-01-01

    Background: Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in cancer, but its clinical and functional importance remain controversial. Mutation or amplification of ALK, as well as its expression levels assessed by conventional immunohistochemistry methods, has been linked to prognosis in cancer, although with potential bias because of the semi-quantitative approaches. Herein, we measured ALK mRNA expression in rhabdomyosarcoma (RMS) and determined its clin...

  5. Characterization and two-dimensional crystallization of membrane component AlkB of the medium-chain alkane hydroxylase system from Pseudomonas putida GPo1.

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    Alonso, Hernan; Roujeinikova, Anna

    2012-11-01

    The alkane hydroxylase system of Pseudomonas putida GPo1 allows it to use alkanes as the sole source of carbon and energy. Bacterial alkane hydroxylases have tremendous potential as biocatalysts for the stereo- and regioselective transformation of a wide range of chemically inert unreactive alkanes into valuable reactive chemical precursors. We have produced and characterized the first 2-dimensional crystals of the integral membrane component of the P. putida alkane hydroxylase system, the nonheme di-iron alkane monooxygenase AlkB. Our analysis reveals for the first time that AlkB reconstituted into a lipid bilayer forms trimers. Addition of detergents that do not disrupt the AlkB oligomeric state (decyl maltose neopentyl glycol [DMNG], lauryl maltose neopentyl glycol [LMNG], and octaethylene glycol monododecyl ether [C(12)E(8)]) preserved its activity at a level close to that of the detergent-free control sample. In contrast, the monomeric form of AlkB produced by purification in n-decyl-β-D-maltopyranoside (DM), n-dodecyl-β-D-maltopyranoside (DDM), octyl glucose neopentyl glycol (OGNG), and n-dodecyl-N,N-dimethylamine-N-oxide (LDAO) was largely inactive. This is the first indication that the physiologically active form of membrane-embedded AlkB may be a multimer. We present for the first time experimental evidence that 1-octyne acts as a mechanism-based inhibitor of AlkB. Therefore, despite the lack of any significant full-length sequence similarity with members of other monooxygenase classes that catalyze the terminal oxidation of alkanes, AlkB is likely to share a similar catalytic mechanism.

  6. Characterization and Two-Dimensional Crystallization of Membrane Component AlkB of the Medium-Chain Alkane Hydroxylase System from Pseudomonas putida GPo1

    OpenAIRE

    Alonso, Hernan; Roujeinikova, Anna

    2012-01-01

    The alkane hydroxylase system of Pseudomonas putida GPo1 allows it to use alkanes as the sole source of carbon and energy. Bacterial alkane hydroxylases have tremendous potential as biocatalysts for the stereo- and regioselective transformation of a wide range of chemically inert unreactive alkanes into valuable reactive chemical precursors. We have produced and characterized the first 2-dimensional crystals of the integral membrane component of the P. putida alkane hydroxylase system, the no...

  7. Regioselective alkane hydroxylation with a mutant AlkB enzyme

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    Koch, Daniel J.; Arnold, Frances H.

    2012-11-13

    AlkB from Pseudomonas putida was engineered using in-vivo directed evolution to hydroxylate small chain alkanes. Mutant AlkB-BMO1 hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. Mutant AlkB-BMO2 similarly hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. These biocatalysts are highly active for small chain alkane substrates and their regioselectivity is retained in whole-cell biotransformations.

  8. The translational repressor Crc controls the Pseudomonas putida benzoate and alkane catabolic pathways using a multi-tier regulation strategy.

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    Hernández-Arranz, Sofía; Moreno, Renata; Rojo, Fernando

    2013-01-01

    Metabolically versatile bacteria usually perceive aromatic compounds and hydrocarbons as non-preferred carbon sources, and their assimilation is inhibited if more preferable substrates are available. This is achieved via catabolite repression. In Pseudomonas putida, the expression of the genes allowing the assimilation of benzoate and n-alkanes is strongly inhibited by catabolite repression, a process controlled by the translational repressor Crc. Crc binds to and inhibits the translation of benR and alkS mRNAs, which encode the transcriptional activators that induce the expression of the benzoate and alkane degradation genes respectively. However, sequences similar to those recognized by Crc in benR and alkS mRNAs exist as well in the translation initiation regions of the mRNA of several structural genes of the benzoate and alkane pathways, which suggests that Crc may also regulate their translation. The present results show that some of these sites are functional, and that Crc inhibits the induction of both pathways by limiting not only the translation of their transcriptional activators, but also that of genes coding for the first enzyme in each pathway. Crc may also inhibit the translation of a gene involved in benzoate uptake. This multi-tier approach probably ensures the rapid regulation of pathway genes, minimizing the assimilation of non-preferred substrates when better options are available. A survey of possible Crc sites in the mRNAs of genes associated with other catabolic pathways suggested that targeting substrate uptake, pathway induction and/or pathway enzymes may be a common strategy to control the assimilation of non-preferred compounds. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  9. The orphan receptor ALK7 and the Activin receptor ALK4 mediate signaling by Nodal proteins during vertebrate development

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    Reissmann, Eva; Jörnvall, Henrik; Blokzijl, Andries; Andersson, Olov; Chang, Chenbei; Minchiotti, Gabriella; Persico, M. Graziella; Ibáñez, Carlos F.; Brivanlou, Ali H.

    2001-01-01

    Nodal proteins have crucial roles in mesendoderm formation and left–right patterning during vertebrate development. The molecular mechanisms of signal transduction by Nodal and related ligands, however, are not fully understood. In this paper, we present biochemical and functional evidence that the orphan type I serine/threonine kinase receptor ALK7 acts as a receptor for mouse Nodal and Xenopus Nodal-related 1 (Xnr1). Receptor reconstitution experiments indicate that ALK7 collaborates with ActRIIB to confer responsiveness to Xnr1 and Nodal. Both receptors can independently bind Xnr1. In addition, Cripto, an extracellular protein genetically implicated in Nodal signaling, can independently interact with both Xnr1 and ALK7, and its expression greatly enhances the ability of ALK7 and ActRIIB to respond to Nodal ligands. The Activin receptor ALK4 is also able to mediate Nodal signaling but only in the presence of Cripto, with which it can also interact directly. A constitutively activated form of ALK7 mimics the mesendoderm-inducing activity of Xnr1 in Xenopus embryos, whereas a dominant-negative ALK7 specifically blocks the activities of Nodal and Xnr1 but has little effect on other related ligands. In contrast, a dominant-negative ALK4 blocks all mesoderm-inducing ligands tested, including Nodal, Xnr1, Xnr2, Xnr4, and Activin. In agreement with a role in Nodal signaling, ALK7 mRNA is localized to the ectodermal and organizer regions of Xenopus gastrula embryos and is expressed during early stages of mouse embryonic development. Therefore, our results indicate that both ALK4 and ALK7 can mediate signal transduction by Nodal proteins, although ALK7 appears to be a receptor more specifically dedicated to Nodal signaling. PMID:11485994

  10. Fusion of EML4 and ALK is associated with development of lung adenocarcinomas lacking EGFR and KRAS mutations and is correlated with ALK expression

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    Zhang Xuchao

    2010-07-01

    Full Text Available Abstract Background The anaplastic lymphoma kinase (ALK gene is frequently involved in translocations that lead to gene fusions in a variety of human malignancies, including lymphoma and lung cancer. Fusion partners of ALK include NPM, EML4, TPM3, ATIC, TFG, CARS, and CLTC. Characterization of ALK fusion patterns and their resulting clinicopathological profiles could be of great benefit in better understanding the biology of lung cancer. Results RACE-coupled PCR sequencing was used to assess ALK fusions in a cohort of 103 non-small cell lung carcinoma (NSCLC patients. Within this cohort, the EML4-ALK fusion gene was identified in 12 tumors (11.6%. Further analysis revealed that EML4-ALK was present at a frequency of 16.13% (10/62 in patients with adenocarcinomas, 19.23% (10/52 in never-smokers, and 42.80% (9/21 in patients with adenocarcinomas lacking EGFR and KRAS mutations. The EML4-ALK fusion was associated with non-smokers (P = 0.03, younger age of onset (P = 0.03, and adenocarcinomas without EGFR/KRAS mutations (P = 0.04. A trend towards improved survival was observed for patients with the EML4-ALK fusion, although it was not statistically significant (P = 0.20. Concurrent deletion in EGFR exon 19 and fusion of EML4-ALK was identified for the first time in a Chinese female patient with an adenocarcinoma. Analysis of ALK expression revealed that ALK mRNA levels were higher in tumors positive for the EML-ALK fusion than in negative tumors (normalized intensity of 21.99 vs. 0.45, respectively; P = 0.0018. However, expression of EML4 did not differ between the groups. Conclusions The EML4-ALK fusion gene was present at a high frequency in Chinese NSCLC patients, particularly in those with adenocarcinomas lacking EGFR/KRAS mutations. The EML4-ALK fusion appears to be tightly associated with ALK mRNA expression levels. RACE-coupled PCR sequencing is a highly sensitive method that could be used clinically for the identification of EML4-ALK

  11. Identification of ALK germline mutation (3605delG) in pediatric anaplastic medulloblastoma.

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    Coco, Simona; De Mariano, Marilena; Valdora, Francesca; Servidei, Tiziana; Ridola, Vita; Andolfo, Immacolata; Oberthuer, André; Tonini, Gian Paolo; Longo, Luca

    2012-10-01

    The anaplastic lymphoma kinase (ALK) gene has been found either rearranged or mutated in several neoplasms such as anaplastic large-cell lymphoma, non-small-cell lung cancer, neuroblastoma and anaplastic thyroid cancer. Medulloblastoma (MB) is an embryonic pediatric cancer arising from nervous system, a tissue in which ALK is expressed during embryonic development. We performed an ALK mutation screening in 52 MBs and we found a novel heterozygous germline deletion of a single base in exon 23 (3605delG) in a case with marked anaplasia. This G deletion results in a frameshift mutation producing a premature stop codon in exon 25 of ALK tyrosine kinase domain. We also screened three human MB cell lines without finding any mutation of ALK gene. Quantitative expression analysis of 16 out of 52 samples showed overexpression of ALK mRNA in three MBs. In the present study, we report the first mutation of ALK found in MB. Moreover, a deletion of ALK gene producing a stop codon has not been detected in human tumors up to now. Further investigations are now required to elucidate whether the truncated form of ALK may have a role in signal transduction.

  12. Identification of ALK as the Major Familial Neuroblastoma Predisposition Gene

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    Mossë, Yalë P; Laudenslager, Marci; Longo, Luca; Cole, Kristina A; Wood, Andrew; Attiyeh, Edward F; Laquaglia, Michael J; Sennett, Rachel; Lynch, Jill E; Perri, Patrizia; Laureys, Geneviève; Speleman, Frank; Hakonarson, Hakon; Torkamani, Ali; Schork, Nicholas J; Brodeur, Garrett M; Tonini, Gian Paolo; Rappaport, Eric; Devoto, Marcella; Maris, John M

    2009-01-01

    SUMMARY Survival rates for the childhood cancer neuroblastoma have not substantively improved despite dramatic escalation in chemotherapy intensity. Like most human cancers, this embryonal malignancy can be inherited, but the genetic etiology of familial and sporadically occurring neuroblastoma was largely unknown. Here we show that germline mutations in the anaplastic lymphoma kinase gene (ALK) explain the majority of hereditary neuroblastomas, and that activating mutations can also be somatically acquired. We first identified a significant linkage signal at the short arm of chromosome 2 (maximum nonparametric LOD=4.23 at rs1344063) using a whole-genome scan in neuroblastoma pedigrees. Resequencing of regional candidate genes identified three separate missense mutations in the tyrosine kinase domain of ALK (G1128A, R1192P and R1275Q) that segregated with the disease in eight separate families. Examination of 491 sporadically occurring human neuroblastoma samples showed that the ALK locus was gained in 22.8%, and highly amplified in an additional 3.3%, and that these aberrations were highly associated with death from disease (P=0.0003). Resequencing of 194 high-risk neuroblastoma samples showed somatically acquired mutations within the tyrosine kinase domain in 12.4%. Nine of the ten mutations map to critical regions of the kinase domain and were predicted to be oncogenic drivers with high probability. Mutations resulted in constitutive phosphorylation consistent with activation, and targeted knockdown of ALK mRNA resulted in profound growth inhibition of 4 of 4 cell lines harboring mutant or amplified ALK, as well as 2 of 6 wild type for ALK. Our results demonstrate that heritable mutations of ALK are the major cause of familial neuroblastoma, and that germline or acquired activation of this cell surface kinase is a tractable therapeutic target for this lethal pediatric malignancy. PMID:18724359

  13. Genomotyping of Pseudomonas putida strains using P. putida KT2440-based high-density DNA microarrays: Implications for transcriptomics studies

    NARCIS (Netherlands)

    Ballerstedt, H.; Volkers, R.J.M.; Mars, A.E.; Hallsworth, J.E.; Santos, V.A.M.D.; Puchalka, J.; Duuren, J. van; Eggink, G.; Timmis, K.N.; Bont, J.A.M. de; Wery, J.

    2007-01-01

    Pseudomonas putida KT2440 is the only fully sequenced P. putida strain. Thus, for transcriptomics and proteomics studies with other P. putida strains, the P. putida KT2440 genomic database serves as standard reference. The utility of KT2440 whole-genome, high-density oligonucleotide microarrays for

  14. Evaluation of ALK gene rearrangement in central nervous system metastases of non-small-cell lung cancer using two-step RT-PCR technique.

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    Nicoś, M; Krawczyk, P; Wojas-Krawczyk, K; Bożyk, A; Jarosz, B; Sawicki, M; Trojanowski, T; Milanowski, J

    2017-12-01

    RT-PCR technique has showed a promising value as pre-screening method for detection of mRNA containing abnormal ALK sequences, but its sensitivity and specificity is still discussable. Previously, we determined the incidence of ALK rearrangement in CNS metastases of NSCLC using IHC and FISH methods. We evaluated ALK gene rearrangement using two-step RT-PCR method with EML4-ALK Fusion Gene Detection Kit (Entrogen, USA). The studied group included 145 patients (45 females, 100 males) with CNS metastases of NSCLC and was heterogeneous in terms of histology and smoking status. 21% of CNS metastases of NSCLC (30/145) showed presence of mRNA containing abnormal ALK sequences. FISH and IHC tests confirmed the presence of ALK gene rearrangement and expression of ALK abnormal protein in seven patients with positive result of RT-PCR analysis (4.8% of all patients, 20% of RT-PCR positive patients). RT-PCR method compared to FISH analysis achieved 100% of sensitivity and only 82.7% of specificity. IHC method compared to FISH method indicated 100% of sensitivity and 97.8% of specificity. In comparison to IHC, RT-PCR showed identical sensitivity with high number of false positive results. Utility of RT-PCR technique in screening of ALK abnormalities and in qualification patients for molecularly targeted therapies needs further validation.

  15. ALK inhibitors, a pharmaceutical perspective

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    Arturo eGalvani

    2012-02-01

    Full Text Available In 2007, the ALK tyrosine kinase, already known to be translocated and activated in Anaplastic Large Cell Lymphoma, and a few other rare cancers, was described as a potential therapeutic target for a subset of non small-cell lung cancer (NSCLC patients. Clinical proof of concept, culminating in the recent approval by the FDA of the Pfizer drug Xalkori (crizotinib, formerly known as PF-02341066 followed in record time. The drug was approved together with a companion diagnostic, the Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular, Inc. for detection of eligible patients. This remarkable example of the coming of age of personalized medicine in cancer therapy is hopefully only an auspice of things to come in this rapidly developing field. Perhaps unsurprisingly, however, the appearance of clinical acquired resistance to crizotinib has already been observed early on in clinical testing, with the identification of several ALK secondary point mutations which diminish drug efficacy, and which open the way for development of second-generation inhibitors. It is also emerging that acquired resistance to crizotinib may also occur through ALK-independent mechanisms, which still need to be elucidated in detail.

  16. Non-small cell lung cancer with EML4-ALK translocation in Chinese male never-smokers is characterized with early-onset.

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    Guo, Yongjun; Ma, Jie; Lyu, Xiaodong; Liu, Hai; Wei, Bing; Zhao, Jiuzhou; Fu, Shuang; Ding, Lu; Zhang, Jihong

    2014-11-18

    The translocations of the anaplastic lymphoma kinase (ALK) gene with the echinoderm microtubule-associated protein-like 4 (EML4) gene on chromosome 2p have been identified in non-small-cell lung cancers (NSCLCs) as oncogenic driver mutations. It has been suggested that EML4-ALK fusion is associated with the resistance in NSCLCs to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs), such as gefitinib and erlotinib. In contrast, ALK tyrosine kinase inhibitor (ALK TKI) crizotinib has shown superior effects in combating NSCLCs with EML4-ALK. Thus, characterization of EML4-ALK fusion genes and clinical features of resulting carcinomas would be a great benefit to disease diagnosis and designing customized treatment plans. Studies have suggested that EML4-ALK translocation occurs more frequently in never-smokers with NSCLC, especially in female patients. However, it is not clear whether this is the case in male patients, too. In this study, we have determined the frequency of EML4-ALK translocation in male never-smokers with NSCLC in a cohort of Chinese patients. The clinical features associated with EML4-ALK translocation were also investigated. A cohort of 95 Chinese male never-smokers with NSCLC was enrolled in this study. EML4-ALK fusion genes were detected using one-step real time RT-PCR and DNA sequencing. We further determined the expression levels of ALK mRNA by RT-PCR and ALK protein by immunohistochemistry in these specimens. The clinical features of EML4-ALK-positive carcinomas were also determined. We have identified EML4-ALK fusion genes in 8 out of 95 carcinoma cases, accounting for 8.42% in Chinese male never-smokers with NSCLC. It is significantly higher than that in all Chinese male patients (3.44%) regardless smoking habit. It is also significantly higher than that in all Chinese smokers (8/356 or 2.25%) or in smokers worldwide (2.9%) by comparing to published data. Interestingly, EML4-ALK fusion genes are more frequently found in

  17. Small RNA-Controlled Gene Regulatory Networks in Pseudomonas putida

    DEFF Research Database (Denmark)

    Bojanovic, Klara

    evolved numerous mechanisms to controlgene expression in response to specific environmental signals. In addition to two-component systems, small regulatory RNAs (sRNAs) have emerged as major regulators of gene expression. The majority of sRNAs bind to mRNA and regulate their expression. They often have...... multiple targets and are incorporated into large regulatory networks and the RNA chaper one Hfq in many cases facilitates interactions between sRNAs and their targets. Some sRNAs also act by binding to protein targets and sequestering their function. In this PhD thesis we investigated the transcriptional....... Detailed insights into the mechanisms through which P. putida responds to different stress conditions and increased understanding of bacterial adaptation in natural and industrial settings were gained. Additionally, we identified genome-wide transcription start sites, andmany regulatory RNA elements...

  18. Small Rna Regulatory Networks In Pseudomonas Putida

    DEFF Research Database (Denmark)

    Bojanovic, Klara; Long, Katherine

    2015-01-01

    chemicals and has a potential to be used as an efficient cell factory for various products. P. putida KT2240 is a genome-sequenced strain and a well characterized pseudomonad. Our major aim is to identify small RNA molecules (sRNAs) and their regulatory networks. A previous study has identified 37 sRNAs...... in this strain, while in other pseudomonads many more sRNAs have been found so far.P. putida KT2440 has been grown in different conditions which are likely to be encountered in industrial fermentations with the aim of using sRNAs for generation of improved cell factories. For that, cells have been grown in LB......Pseudomonas putida is a ubiquitous Gram-negative soil bacterium with a versatile metabolism and ability to degrade various toxic compounds. It has a high tolerance to different future biobased building blocks and various other stringent conditions. It is used in industry to produce some important...

  19. Detection of circulating tumor cells harboring a unique ALK rearrangement in ALK-positive non-small-cell lung cancer.

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    Pailler, Emma; Adam, Julien; Barthélémy, Amélie; Oulhen, Marianne; Auger, Nathalie; Valent, Alexander; Borget, Isabelle; Planchard, David; Taylor, Melissa; André, Fabrice; Soria, Jean Charles; Vielh, Philippe; Besse, Benjamin; Farace, Françoise

    2013-06-20

    The diagnostic test for ALK rearrangement in non-small-cell lung cancer (NSCLC) for crizotinib treatment is currently done on tumor biopsies or fine-needle aspirations. We evaluated whether ALK rearrangement diagnosis could be performed by using circulating tumor cells (CTCs). The presence of an ALK rearrangement was examined in CTCs of 18 ALK-positive and 14 ALK-negative patients by using a filtration enrichment technique and filter-adapted fluorescent in situ hybridization (FA-FISH), a FISH method optimized for filters. ALK-rearrangement patterns were determined in CTCs and compared with those present in tumor biopsies. ALK-rearranged CTCs and tumor specimens were characterized for epithelial (cytokeratins, E-cadherin) and mesenchymal (vimentin, N-cadherin) marker expression. ALK-rearranged CTCs were monitored in five patients treated with crizotinib. All ALK-positive patients had four or more ALK-rearranged CTCs per 1 mL of blood (median, nine CTCs per 1 mL; range, four to 34 CTCs per 1 mL). No or only one ALK-rearranged CTC (median, one per 1 mL; range, zero to one per 1 mL) was detected in ALK-negative patients. ALK-rearranged CTCs harbored a unique (3'5') split pattern, and heterogeneous patterns (3'5', only 3') of splits were present in tumors. ALK-rearranged CTCs expressed a mesenchymal phenotype contrasting with heterogeneous epithelial and mesenchymal marker expressions in tumors. Variations in ALK-rearranged CTC levels were detected in patients being treated with crizotinib. ALK rearrangement can be detected in CTCs of patients with ALK-positive NSCLC by using a filtration technique and FA-FISH, enabling both diagnostic testing and monitoring of crizotinib treatment. Our results suggest that CTCs harboring a unique ALK rearrangement and mesenchymal phenotype may arise from clonal selection of tumor cells that have acquired the potential to drive metastatic progression of ALK-positive NSCLC.

  20. Advances of naphthalene degradation in Pseudomonas putida ND6

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    Song, Fu; Shi, Yifei; Jia, Shiru; Tan, Zhilei; Zhao, Huabing

    2018-03-01

    Naphthalene is one of the most common and simple polycyclic aromatic hydrocarbons. Degradation of naphthalene has been greatly concerned due to its economic, free-pollution and its fine effect in Pseudomonas putida ND6. This review summarizes the development history of naphthalene degradation, the research progress of naphthalene degrading gene and naphthalene degradation pathway of Pseudomonas putida ND6, and the researching path of this strain. Although the study of naphthalene degradation is not consummate in Pseudomonas putida ND6, there is a potential capability for Pseudomonas putida ND6 to degrade the naphthalene in the further research.

  1. Characterization of starvation-induced dispersion in Pseudomonas putida biofilms

    DEFF Research Database (Denmark)

    Gjermansen, Morten; Ragas, Paula Cornelia; Sternberg, Claus

    2005-01-01

    The biofilm lifestyle, where microbial cells are aggregated because of expression of cell-to-cell interconnecting compounds, is believed to be of paramount importance to microbes in the environment. Because microbes must be able to alternate between sessile and planktonic states, it is anticipated...... that they must be able to regulate their ability to form biofilm and to dissolve biofilm. We present an investigation of a biofilm dissolution process occurring in flow-chamber-grown Pseudomonas putida biofilms. Local starvation-induced biofilm dissolution appears to be an integrated part of P. putida biofilm...... development that causes characteristic structural rearrangements. Rapid global dissolution of entire P. putida biofilms was shown to occur in response to carbon starvation. Genetic analysis suggested that the adjacent P. putida genes PP0164 and PP0165 play a role in P. putida biofilm formation and dissolution...

  2. SUMOylation Confers Posttranslational Stability on NPM-ALK Oncogenic Protein

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    Deeksha Vishwamitra

    2015-09-01

    Full Text Available Nucleophosmin-anaplastic lymphoma kinase–expressing (NPM-ALK+ T-cell lymphoma is an aggressive form of cancer that commonly affects children and adolescents. The expression of NPM-ALK chimeric oncogene results from the chromosomal translocation t(2;5(p23;q35 that causes the fusion of the ALK and NPM genes. This translocation generates the NPM-ALK protein tyrosine kinase that forms the constitutively activated NPM-ALK/NPM-ALK homodimers. In addition, NPM-ALK is structurally associated with wild-type NPM to form NPM/NPM-ALK heterodimers, which can translocate to the nucleus. The mechanisms that sustain the stability of NPM-ALK are not fully understood. SUMOylation is a posttranslational modification that is characterized by the reversible conjugation of small ubiquitin-like modifiers (SUMOs with target proteins. SUMO competes with ubiquitin for substrate binding and therefore, SUMOylation is believed to protect target proteins from proteasomal degradation. Moreover, SUMOylation contributes to the subcellular distribution of target proteins. Herein, we found that the SUMOylation pathway is deregulated in NPM-ALK+ T-cell lymphoma cell lines and primary lymphoma tumors from patients. We also identified Lys24 and Lys32 within the NPM domain as the sites where NPM-ALK conjugates with SUMO-1 and SUMO-3. Importantly, antagonizing SUMOylation by the SENP1 protease decreased the accumulation of NPM-ALK and suppressed lymphoma cell viability, proliferation, and anchorage-independent colony formation. One possible mechanism for the SENP1-mediated decrease in NPM-ALK levels was the increase in NPM-ALK association with ubiquitin, which facilitates its degradation. Our findings propose a model in which aberrancies in SUMOylation contribute to the pathogenesis of NPM-ALK+ T-cell lymphoma. Unraveling such pathogenic mechanisms may lead to devising novel strategies to eliminate this aggressive neoplasm.

  3. Activity of second-generation ALK inhibitors against crizotinib-resistant mutants in an NPM-ALK model compared to EML4-ALK

    International Nuclear Information System (INIS)

    Fontana, Diletta; Ceccon, Monica; Gambacorti-Passerini, Carlo; Mologni, Luca

    2015-01-01

    Anaplastic lymphoma kinase (ALK) is a tyrosine kinase receptor involved in both solid and hematological tumors. About 80% of ALK-positive anaplastic large-cell lymphoma (ALCL) cases are characterized by the t(2;5)(p23;q35) translocation, encoding for the aberrant fusion protein nucleophosmin (NPM)-ALK, whereas 5% of non-small-cell lung cancer (NSCLC) patients carry the inv(2)(p21;p23) rearrangement, encoding for the echinoderm microtubule-associated protein-like 4 (EML4)-ALK fusion. The ALK/c-MET/ROS inhibitor crizotinib successfully improved the treatment of ALK-driven diseases. However, several cases of resistance appeared in NSCLC patients, and ALK amino acid substitutions were identified as a leading cause of resistance to crizotinib. Second-generation ALK inhibitors have been developed in order to overcome crizotinib resistance. In this work, we profiled in vitro the activity of crizotinib, AP26113, ASP3026, alectinib, and ceritinib against six mutated forms of ALK associated with clinical resistance to crizotinib (C1156Y, L1196M, L1152R, G1202R, G1269A, and S1206Y) and provide a classification of mutants according to their level of sensitivity/resistance to the drugs. Since the biological activity of ALK mutations extends beyond the specific type of fusion, both NPM-ALK- and EML4-ALK-positive cellular models were used. Our data revealed that most mutants may be targeted by using different inhibitors. One relevant exception is represented by the G1202R substitution, which was highly resistant to all drugs (>10-fold increased IC 50 compared to wild type) and may represent the most challenging mutation to overcome. These results provide a prediction of cross-resistance of known crizotinib-resistant mutations against all second-generation tyrosine kinase inhibitors (TKIs) clinically available, and therefore could be a useful tool to help clinicians in the management of crizotinib-resistance cases

  4. Oncogenic tyrosine kinase NPM-ALK induces expression of the growth-promoting receptor ICOS

    DEFF Research Database (Denmark)

    Zhang, Qian; Wang, HongYi; Kantekure, Kanchan

    2011-01-01

    Here we report that T-cell lymphoma cells carrying the NPM-ALK fusion protein (ALK(+) TCL) frequently express the cell-stimulatory receptor ICOS. ICOS expression in ALK(+) TCL is moderate and strictly dependent on the expression and enzymatic activity of NPM-ALK. NPM-ALK induces ICOS expression v...

  5. Mechanisms of Acquired Resistance to ALK Inhibitors and the Rationale for Treating ALK-positive Lung Cancer

    Energy Technology Data Exchange (ETDEWEB)

    Isozaki, Hideko [Department of Clinical Pharmaceutics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama 700-8558 (Japan); Takigawa, Nagio, E-mail: ntakigaw@gmail.com [Department of General Internal Medicine 4, Kawasaki Medical School, Okayama 700-8505 (Japan); Kiura, Katsuyuki [Department of Allergy and Respiratory Medicine, Okayama University Hospital, Okayama 700-8558 (Japan)

    2015-04-30

    The discovery of an echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion gene led to improved clinical outcomes in patients with lung cancer after the development of the first ALK-targeting agent, crizotinib. Some second-generation ALK tyrosine kinase inhibitors (TKIs), which might be more potent than crizotinib or effective on crizotinib-resistant patients, have been developed. Although these ALK-TKIs show an excellent response initially, most patients eventually acquire resistance. Therefore, careful consideration of the resistance mechanisms might lead to superior therapeutic strategies. Here, we summarize the history of ALK-TKIs and their underlying resistance mechanisms in both the preclinical and clinical settings. In addition, we discuss potential future treatment strategies in ALK-TKI-naïve and -resistant patients with lung cancer harboring the EML4-ALK fusion gene.

  6. Benzoate transport in Pseudomonas putida CSV86.

    Science.gov (United States)

    Choudhary, Alpa; Purohit, Hemant; Phale, Prashant S

    2017-07-03

    Pseudomonas putida strain CSV86 metabolizes variety of aromatic compounds as the sole carbon source. Genome analysis revealed the presence of genes encoding putative transporters for benzoate, p-hydroxybenzoate, phenylacetate, p-hydroxyphenylacetate and vanillate. Bioinformatic analysis revealed that benzoate transport and metabolism genes are clustered at the ben locus as benK-catA-benE-benF. Protein topology prediction suggests that BenK (aromatic acid-H+ symporter of major facilitator superfamily) has 12 transmembrane α-helices with the conserved motif LADRXGRKX in loop 2, while BenE (benzoate-H+ symporter protein) has 11 predicted transmembrane α-helices. benF and catA encode benzoate specific porin, OprD and catechol 1,2-dioxygenase, respectively. Biochemical studies suggest that benzoate was transported by an inducible and active process. Inhibition (90%-100%) in the presence of dinitrophenol suggests that the energy for the transport process is derived from the proton motive force. The maximum rate of benzoate transport was 484 pmole min-1 mg-1 cells with an affinity constant, Kmof 4.5 μM. Transcriptional analysis of the benzoate and glucose-grown cells showed inducible expression of benF, benK and benE, suggesting that besides outer membrane porin, both inner membrane transporters probably contribute for the benzoate transport in P. putida strain CSV86. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  7. Efficacy of ALK5 inhibition in myelofibrosis

    Science.gov (United States)

    Zhao, Wanke; Ho, Wanting Tina; Han, Ying; Murdun, Cem; Mailloux, Adam W.; Zhang, Ling; Wang, Xuefeng; Budhathoki, Anjali; Pradhan, Kith; Rapaport, Franck; Wang, Huaquan; Shao, Zonghong; Ren, Xiubao; Steidl, Ulrich; Levine, Ross L.; Zhao, Zhizhuang Joe; Verma, Amit; Epling-Burnette, Pearlie K.

    2017-01-01

    Myelofibrosis (MF) is a bone marrow disorder characterized by clonal myeloproliferation, aberrant cytokine production, extramedullary hematopoiesis, and bone marrow fibrosis. Although somatic mutations in JAK2, MPL, and CALR have been identified in the pathogenesis of these diseases, inhibitors of the Jak2 pathway have not demonstrated efficacy in ameliorating MF in patients. TGF-β family members are profibrotic cytokines and we observed significant TGF-β1 isoform overexpression in a large cohort of primary MF patient samples. Significant overexpression of TGF-β1 was also observed in murine clonal MPLW515L megakaryocytic cells. TGF-β1 stimulated the deposition of excessive collagen by mesenchymal stromal cells (MSCs) by activating the TGF-β receptor I kinase (ALK5)/Smad3 pathway. MSCs derived from MPLW515L mice demonstrated sustained overproduction of both collagen I and collagen III, effects that were abrogated by ALK5 inhibition in vitro and in vivo. Importantly, use of galunisertib, a clinically active ALK5 inhibitor, significantly improved MF in both MPLW515L and JAK2V617F mouse models. These data demonstrate the role of malignant hematopoietic stem cell (HSC)/TGF-β/MSC axis in the pathogenesis of MF, and provide a preclinical rationale for ALK5 blockade as a therapeutic strategy in MF. PMID:28405618

  8. The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN

    Directory of Open Access Journals (Sweden)

    J. Guan

    2016-09-01

    Full Text Available The first-in-class inhibitor of ALK, c-MET and ROS1, crizotinib (Xalkori, has shown remarkable clinical efficacy in treatment of ALK-positive non-small cell lung cancer. However, in neuroblastoma, activating mutations in the ALK kinase domain are typically refractory to crizotinib treatment, highlighting the need for more potent inhibitors. The next-generation ALK inhibitor PF-06463922 is predicted to exhibit increased affinity for ALK mutants prevalent in neuroblastoma. We examined PF-06463922 activity in ALK-driven neuroblastoma models in vitro and in vivo. In vitro kinase assays and cell-based experiments examining ALK mutations of increasing potency show that PF-06463922 is an effective inhibitor of ALK with greater activity towards ALK neuroblastoma mutants. In contrast to crizotinib, single agent administration of PF-06463922 caused dramatic tumor inhibition in both subcutaneous and orthotopic xenografts as well as a mouse model of high-risk neuroblastoma driven by Th-ALKF1174L/MYCN. Taken together, our results suggest PF-06463922 is a potent inhibitor of crizotinib-resistant ALK mutations, and highlights an important new treatment option for neuroblastoma patients.

  9. Pseudomonas putida as a microbial cell factory

    DEFF Research Database (Denmark)

    Wigneswaran, Vinoth

    for sustainable production of chemicals, which can be achieved by microbial cell factories. The work presented in this PhD thesis elucidates the application of Pseudomonas putida as a microbial cell factory for production of the biosurfactant rhamnolipid. The rhamnolipid production was achieved by heterologous...... phase. The genomic alterations were identified by genome sequencing and revealed parallel evolution. Glycerol was also shown to be able to support biofilm growth and as a result of this it can be used as an alternative substrate for producing biochemicals in conventional and biofilm reactors. The use...... of biofilm as a production platform and the usage of glycerol as a feedstock show the potential of using microbial cell factories in the transition toward sustainable production of chemicals. Particularly, the applicability of biofilm as a production platform can emerge as a promising alternative...

  10. [Molecular diagnostics of ALK-positive lung cancer].

    Science.gov (United States)

    Tímár, József; Lotz, Gábor; Rásó, Erzsébet; Moldvay, Judit

    2017-09-20

    ALK translocation is the 3rd most frequent genetic aberration in lung adenocarcinoma, and several inhibitors are now clinically available in first and second line settings. Accordingly, molecular diagnostics of ALK-positive lung cancer is very important and can be done with the rational combination of several methods. All international recommendations suggest that, except for cytological samples, screening technology for ALK-positive tumors is immunohistochemistry using a validated test. It is highly recommended that in case of ALK protein positive samples gene translocation must be confirmed by fluorescent in situ hybridization (FISH). In case of cytological samples FISH technique must be used as ALK diagnostics. In equivocal cases the genetic alteration of ALK can be confirmed by alternative molecular techniques such as next generation sequencing or RNAbased PCR methods. Upon administration of ALK inhibitors, acquired resistance is frequent which is mostly due to ALK amplification and/or mutation. It is evident that the diagnostics of these secondary ALK gene alterations must be done from recurrent tumors or circulating nucleic acids.

  11. Development of potent ALK inhibitor and its molecular inhibitory mechanism against NSCLC harboring EML4-ALK proteins

    Energy Technology Data Exchange (ETDEWEB)

    Kang, Chung Hyo [Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600 (Korea, Republic of); College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Yun, Jeong In [Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600 (Korea, Republic of); Lee, Kwangho [Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600 (Korea, Republic of); Medicinal & Pharmaceutical Chemistry, Korea University of Science and Technology, Daejeon 305-350 (Korea, Republic of); Lee, Chong Ock; Lee, Heung Kyoung [Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600 (Korea, Republic of); Yun, Chang-Soo; Hwang, Jong Yeon; Cho, Sung Yun; Jung, Heejung; Kim, Pilho [Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600 (Korea, Republic of); Medicinal & Pharmaceutical Chemistry, Korea University of Science and Technology, Daejeon 305-350 (Korea, Republic of); Ha, Jae Du; Jeon, Jeong Hee; Choi, Sang Un [Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600 (Korea, Republic of); Jeong, Hye Gwang [College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Kim, Hyoung Rae, E-mail: hyungrk@krict.re.kr [Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600 (Korea, Republic of); Park, Chi Hoon, E-mail: chpark@krict.re.kr [Bio & Drug Discovery Division, Korea Research Institute of Chemical Technology, PO Box 107, Daejeon 305-600 (Korea, Republic of); Medicinal & Pharmaceutical Chemistry, Korea University of Science and Technology, Daejeon 305-350 (Korea, Republic of)

    2015-08-28

    Here, we show the newly synthesized and potent ALK inhibitor having similar scaffold to KRCA-0008, which was reported previously, and its molecular mechanism against cancer cells harboring EML4-ALK fusion protein. Through ALK wild type enzyme assay, we selected two compounds, KRCA-0080 and KRCA-0087, which have trifluoromethyl instead of chloride in R2 position. We characterized these newly synthesized compounds by in vitro and in vivo assays. Enzyme assay shows that KRCA-0080 is more potent against various ALK mutants, including L1196M, G1202R, T1151-L1152insT, and C1156Y, which are seen in crizotinib-resistant patients, than KRCA-0008 is. Cell based assays demonstrate our compounds downregulate the cellular signaling, such as Akt and Erk, by suppressing ALK activity to inhibit the proliferation of the cells harboring EML4-ALK. Interestingly, our compounds induced strong G1/S arrest in H3122 cells leading to the apoptosis, which is proved by PARP-1 cleavage. In vivo H3122 xenograft assay, we found that KRCA-0080 shows significant reduction in tumor size compared to crizotinib and KRCA-0008 by 15–20%. Conclusively, we report a potent ALK inhibitor which shows significant in vivo efficacy as well as excellent inhibitory activity against various ALK mutants. - Highlights: • We synthesized KRCA-0008 derivatives having trifluoromethyl instead of chloride. • KRCA-0080 shows superior activity against several ALK mutants to KRCA-0008. • Cellular assays show our ALK inhibitors suppress only EML4-ALK positive cells. • Our ALK inhibitors induce G1/S arrest to lead apoptosis in H3122 cells. • KRCA-0080 has superior in vivo efficacy to crizotinib and KRCA-0008 by 15–20%.

  12. DNA Polymerases ImuC and DinB Are Involved in DNA Alkylation Damage Tolerance in Pseudomonas aeruginosa and Pseudomonas putida.

    Science.gov (United States)

    Jatsenko, Tatjana; Sidorenko, Julia; Saumaa, Signe; Kivisaar, Maia

    2017-01-01

    Translesion DNA synthesis (TLS), facilitated by low-fidelity polymerases, is an important DNA damage tolerance mechanism. Here, we investigated the role and biological function of TLS polymerase ImuC (former DnaE2), generally present in bacteria lacking DNA polymerase V, and TLS polymerase DinB in response to DNA alkylation damage in Pseudomonas aeruginosa and P. putida. We found that TLS DNA polymerases ImuC and DinB ensured a protective role against N- and O-methylation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in both P. aeruginosa and P. putida. DinB also appeared to be important for the survival of P. aeruginosa and rapidly growing P. putida cells in the presence of methyl methanesulfonate (MMS). The role of ImuC in protection against MMS-induced damage was uncovered under DinB-deficient conditions. Apart from this, both ImuC and DinB were critical for the survival of bacteria with impaired base excision repair (BER) functions upon alkylation damage, lacking DNA glycosylases AlkA and/or Tag. Here, the increased sensitivity of imuCdinB double deficient strains in comparison to single mutants suggested that the specificity of alkylated DNA lesion bypass of DinB and ImuC might also be different. Moreover, our results demonstrated that mutagenesis induced by MMS in pseudomonads was largely ImuC-dependent. Unexpectedly, we discovered that the growth temperature of bacteria affected the efficiency of DinB and ImuC in ensuring cell survival upon alkylation damage. Taken together, the results of our study disclosed the involvement of ImuC in DNA alkylation damage tolerance, especially at low temperatures, and its possible contribution to the adaptation of pseudomonads upon DNA alkylation damage via increased mutagenesis.

  13. Whole-cell bio-oxidation of n-dodecane using the alkane hydroxylase system of P. putida GPo1 expressed in E. coli

    DEFF Research Database (Denmark)

    Grant, Chris; Woodley, John; Baganz, Frank

    2011-01-01

    , successful n-dodecane oxidation for the production of 1-dodecanol or dodecanoic acid has proven elusive in the past when using alkB-expressing recombinants. This article demonstrates, for the first time in vivo, by using the Escherichia coli GEC137 pGEc47ΔJ strain, that n-dodecane oxidation using this enzyme......The alkane-1-monoxygenase (alkB) complex of Pseudomonas putida GPo1 has been extensively studied in the past and shown to be capable of oxidising aliphatic C5–C12 alkanes to primary alcohols both in the wild-type organism by growth on C5–C12 alkanes as sole carbon source and in vitro. Despite this...... aqueous phase and 200mL of n-dodecane as a second phase. The maximum volumetric rate of combined alcohol and acid production achieved was 1.9g/Lorganic/h (0.35g/Ltotal/h). The maximum specific activity of combined alcohol and acid production was 7-fold lower on n-dodecane (3.5μmol/min/gdcw) than on n...

  14. Circulating Tumor Cells with Aberrant ALK Copy Number Predict Progression-Free Survival during Crizotinib Treatment in ALK-Rearranged Non-Small Cell Lung Cancer Patients.

    Science.gov (United States)

    Pailler, Emma; Oulhen, Marianne; Borget, Isabelle; Remon, Jordi; Ross, Kirsty; Auger, Nathalie; Billiot, Fanny; Ngo Camus, Maud; Commo, Frédéric; Lindsay, Colin R; Planchard, David; Soria, Jean-Charles; Besse, Benjamin; Farace, Françoise

    2017-05-01

    The duration and magnitude of clinical response are unpredictable in ALK -rearranged non-small cell lung cancer (NSCLC) patients treated with crizotinib, although all patients invariably develop resistance. Here, we evaluated whether circulating tumor cells (CTC) with aberrant ALK -FISH patterns [ ALK -rearrangement, ALK -copy number gain ( ALK -CNG)] monitored on crizotinib could predict progression-free survival (PFS) in a cohort of ALK -rearranged patients. Thirty-nine ALK -rearranged NSCLC patients treated with crizotinib as first ALK inhibitor were recruited prospectively. Blood samples were collected at baseline and at an early time-point (2 months) on crizotinib. Aberrant ALK -FISH patterns were examined in CTCs using immunofluorescence staining combined with filter-adapted FISH after filtration enrichment. CTCs were classified into distinct subsets according to the presence of ALK -rearrangement and/or ALK -CNG signals. No significant association between baseline numbers of ALK -rearranged or ALK -CNG CTCs and PFS was observed. However, we observed a significant association between the decrease in CTC number with ALK -CNG on crizotinib and a longer PFS (likelihood ratio test, P = 0.025). In multivariate analysis, the dynamic change of CTC with ALK -CNG was the strongest factor associated with PFS (HR, 4.485; 95% confidence interval, 1.543-13.030, P = 0.006). Although not dominant, ALK -CNG has been reported to be one of the mechanisms of acquired resistance to crizotinib in tumor biopsies. Our results suggest that the dynamic change in the numbers of CTCs with ALK -CNG may be a predictive biomarker for crizotinib efficacy in ALK -rearranged NSCLC patients. Serial molecular analysis of CTC shows promise for real-time patient monitoring and clinical outcome prediction in this population. Cancer Res; 77(9); 2222-30. ©2017 AACR . ©2017 American Association for Cancer Research.

  15. The Transcriptional Landscape of the Production Organism Pseudomonas putida

    DEFF Research Database (Denmark)

    D'Arrigo, Isotta

    Bacterial cell factories represent a valid alternative to fossil fuel-based production. A promising bacterium that can be optimized as cell factory is Pseudomonas putida. However, its development in bioproduction applications poses some challenges including a clear understanding of the bacterial...... system biology. This thesis has the aim of facilitating the development of P. putida KT2440 as a bacterial cell factory by investigating the transcriptome of the bacterium under different conditions (e.g. growth and stress). The main goals are the identification of differentially expressed genes, which...... provide information on bacterial adaptation to different environments, and the identification of non-coding RNAs, which regulate gene expression. This work focuses on several aspects of P. putida highlighting genomic features such as transcription start sites (TSSs), RNA regulatory elements...

  16. Characterization of starvation-induced dispersion in Pseudomonas putida biofilms: genetic elements and molecular mechanisms

    DEFF Research Database (Denmark)

    Gjermansen, M.; Nilsson, M.; Yang, Liang

    2010-01-01

    P>Pseudomonas putida OUS82 biofilm dispersal was previously shown to be dependent on the gene PP0164 (here designated lapG). Sequence and structural analysis has suggested that the LapG geneproduct belongs to a family of cysteine proteinases that function in the modification of bacterial surface...... proteins. We provide evidence that LapG is involved in P. putida OUS82 biofilm dispersal through modification of the outer membrane-associated protein LapA. While the P. putida lapG mutant formed more biofilm than the wild-type, P. putida lapA and P. putida lapAG mutants displayed decreased surface...

  17. Assessing carbon source-dependent phenotypic variability in Pseudomonas putida

    DEFF Research Database (Denmark)

    Nikel, Pablo Ivan; de Lorenzo, Victor

    2018-01-01

    capacity of single bacteria by means of fluorescence microscopy and flow cytometry, in combination with the analysis of the temporal takeoff of growth in single-cell cultures, is a simple and easy-to-implement approach. It can help to understand the link between macroscopic phenotypes (e.g., microbial......The soil bacterium Pseudomonas putida is rapidly becoming a platform of choice for applications that require a microbial host highly resistant to different types of stresses and elevated rates of reducing power regeneration. P. putida is capable of growing in a wide variety of carbon sources...

  18. Fusion genes with ALK as recurrent partner in ependymoma-like gliomas

    DEFF Research Database (Denmark)

    Olsen, Thale Kristin; Panagopoulos, Ioannis; Meling, Torstein R

    2015-01-01

    , we identified the 2 first ever reported ALK rearrangements in CNS tumors. Such rearrangements may represent the hallmark of a new entity of pediatric glioma characterized by both ependymal and astrocytic features. Our findings are of particular importance because crizotinib, a selective ALK inhibitor......, has demonstrated effect in patients with lung cancer harboring ALK rearrangements. Thus, ALK emerges as an interesting therapeutic target in patients with ependymal tumors carrying ALK fusions....

  19. The Potent ALK Inhibitor Brigatinib (AP26113) Overcomes Mechanisms of Resistance to First- and Second-Generation ALK Inhibitors in Preclinical Models.

    Science.gov (United States)

    Zhang, Sen; Anjum, Rana; Squillace, Rachel; Nadworny, Sara; Zhou, Tianjun; Keats, Jeff; Ning, Yaoyu; Wardwell, Scott D; Miller, David; Song, Youngchul; Eichinger, Lindsey; Moran, Lauren; Huang, Wei-Sheng; Liu, Shuangying; Zou, Dong; Wang, Yihan; Mohemmad, Qurish; Jang, Hyun Gyung; Ye, Emily; Narasimhan, Narayana; Wang, Frank; Miret, Juan; Zhu, Xiaotian; Clackson, Tim; Dalgarno, David; Shakespeare, William C; Rivera, Victor M

    2016-11-15

    Non-small cell lung cancers (NSCLCs) harboring ALK gene rearrangements (ALK + ) typically become resistant to the first-generation anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitor (TKI) crizotinib through development of secondary resistance mutations in ALK or disease progression in the brain. Mutations that confer resistance to second-generation ALK TKIs ceritinib and alectinib have also been identified. Here, we report the structure and first comprehensive preclinical evaluation of the next-generation ALK TKI brigatinib. A kinase screen was performed to evaluate the selectivity profile of brigatinib. The cellular and in vivo activities of ALK TKIs were compared using engineered and cancer-derived cell lines. The brigatinib-ALK co-structure was determined. Brigatinib potently inhibits ALK and ROS1, with a high degree of selectivity over more than 250 kinases. Across a panel of ALK + cell lines, brigatinib inhibited native ALK (IC 50 , 10 nmol/L) with 12-fold greater potency than crizotinib. Superior efficacy of brigatinib was also observed in mice with ALK + tumors implanted subcutaneously or intracranially. Brigatinib maintained substantial activity against all 17 secondary ALK mutants tested in cellular assays and exhibited a superior inhibitory profile compared with crizotinib, ceritinib, and alectinib at clinically achievable concentrations. Brigatinib was the only TKI to maintain substantial activity against the most recalcitrant ALK resistance mutation, G1202R. The unique, potent, and pan-ALK mutant activity of brigatinib could be rationalized by structural analyses. Brigatinib is a highly potent and selective ALK inhibitor. These findings provide the molecular basis for the promising activity being observed in ALK + , crizotinib-resistant patients with NSCLC being treated with brigatinib in clinical trials. Clin Cancer Res; 22(22); 5527-38. ©2016 AACR. ©2016 American Association for Cancer Research.

  20. Efficient recombinant production of prodigiosin in Pseudomonas putida

    Directory of Open Access Journals (Sweden)

    Andreas eDomröse

    2015-09-01

    Full Text Available Serratia marcescens and several other bacteria produce the red-colored pigment prodigiosin which possesses bioactivities as an antimicrobial, anticancer and immunosuppressive agent. Therefore, there is a great interest to produce this natural compound. Efforts aiming at its biotechnological production have so far largely focused on the original producer and opportunistic human pathogen S. marcescens. Here, we demonstrate efficient prodigiosin production in the heterologous host Pseudomonas putida. Random chromosomal integration of the 21 kb prodigiosin biosynthesis gene cluster of S. marcescens in P. putida KT2440 was employed to construct constitutive prodigiosin production strains. Standard cultivation parameters were optimized such that titers of 94 mg/L culture were obtained upon growth of P. putida at 20 °C using rich medium under high aeration conditions. Subsequently, a novel, fast and effective protocol for prodigiosin extraction and purification was established enabling the straightforward isolation of prodigiosin from P. putida growth medium. In summary, we describe here a highly efficient method for the heterologous biosynthetic production of prodigiosin which may serve as a basis to produce large amounts of this bioactive natural compound and may provide a platform for further in-depth studies of prodiginine biosynthesis.

  1. Isolation and characterization of Pseudomonas putida WLY for ...

    African Journals Online (AJOL)

    Using the BMM medium containing 100 mg/L of reactive brilliant red X-3B, a decolorizing bacterium with higher decolorization activity was isolated and it showed a decolorization zone of 10 mm; this decolorizing bacterium was identified as Pseudomonas putida WLY based on physiological and biochemical characteristics ...

  2. The sigma(54) regulon (sigmulon) of Pseudomonas putida

    DEFF Research Database (Denmark)

    Cases, I.; Ussery, David; de Lorenzo, V.

    2003-01-01

    , the sigma(54) regulon has been studied both in Escherichia coli, Salmonella typhimurium and several species of the Rhizobiaceae. Here we present the analysis of the sigma(54) regulon (sigmulon) in the complete genome of Pseudomonas putida KT2440. We have developed an improved method for the prediction...

  3. Comparative evaluation of the efficacy of Pseudomonas putida in ...

    African Journals Online (AJOL)

    This study was carried out to compare the efficacy of Рseudomonas putida, contained in the biopreparation «Pseudomin» in the bioremediation of diesel fuel contaminated derno-podzoluivisolic soil of two different horizons. By analyzing the Total Petroleum Hydrocarbons (TPH) content using IR-spectrometry method under ...

  4. Metabolism of amino acid amides in Pseudomonas putida ATCC 12633

    NARCIS (Netherlands)

    Hermes, H.F.M.; Croes, L.M.; Peeters, W.P.H.; Peters, P.J.H.; Dijkhuizen, L.

    1993-01-01

    The metabolism of the natural amino acid L-valine, the unnatural amino acids D-valine, and D-, L-phenylglycine (D-, L-PG), and the unnatural amino acid amides D-, L-phenylglycine amide (D, L-PG-NH2) and L-valine amide (L-Val-NH2) was studied in Pseudomonas putida ATCC 12633. The organism possessed

  5. Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein Induced Retinal Angiogenesis

    Science.gov (United States)

    Lee, Heon-Woo; Chong, Diana C.; Ola, Roxana; Dunworth, William P.; Meadows, Stryder; Ka, Jun; Kaartinen, Vesa M.; Qyang, Yibing; Cleaver, Ondine; Bautch, Victoria L.; Eichmann, Anne; Jin, Suk-Won

    2017-01-01

    Objective Increasing evidence suggests that Bone Morphogenetic Protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early post-natal angiogenesis by analysis of inducible, endothelial specific deletion of the BMP receptor components Bmpr2, Alk1, Alk2 and Alk3 in mouse retinal vessels. Approach and Results Expression analysis of several BMP ligands showed that pro-angiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of BMP Type 2 receptor (Bmpr2). Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branchpoints behind the front, leading to attenuated radial expansion. To identify critical BMPR1s associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of three BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial specific deletion of either Alk2/acvr1 or Alk3/Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for pro-angiogenic BMP signaling in retinal vessels. Conclusions Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential pro-angiogenic cue for retinal vessels. PMID:28232325

  6. ALK gene amplification is associated with poor prognosis in colorectal carcinoma.

    Science.gov (United States)

    Bavi, P; Jehan, Z; Bu, R; Prabhakaran, S; Al-Sanea, N; Al-Dayel, F; Al-Assiri, M; Al-Halouly, T; Sairafi, R; Uddin, S; Al-Kuraya, K S

    2013-11-12

    Recently, the anaplastic lymphoma kinase (ALK) has been found to be altered in several solid and haematological tumours. ALK gene copy number changes and mutations in colorectal cancers (CRCs) are not well characterised. We aimed to study the prevalence of ALK copy number changes, translocations, gene mutations and protein expression in 770 CRC patients, and correlate these findings with molecular and clinico-pathological data. ALK gene copy number variations and ALK expression were evaluated by fluorescence in situ hybridisation (FISH) and immunohistochemistry, respectively. Translocations of the ALK gene were not observed; 3.4% (26 out of 756) of the CRC patients tested had an increase in ALK gene copy number either amplification or gain. Interestingly, increased ALK gene copy number alteration was associated with poor prognosis (P=0.0135) and was an independent prognostic marker in multivariate Cox proportional hazards model. The study reveals a significant impact of ALK gene copy number alterations on the outcome of patients with CRC. The findings of our study highlight a potential role of targeting ALK in advanced CRCs by using ALK FISH and ALK IHC as a screening tool to detect ALK alterations. Based on these findings, a potential role of ALK inhibitor as a therapeutic agent in a subset of CRC merits further investigation.

  7. A Screening Method for the ALK Fusion Gene in NSCLC

    International Nuclear Information System (INIS)

    Murakami, Yoshiko; Mitsudomi, Tetsuya; Yatabe, Yasushi

    2012-01-01

    Lung cancer research has recently made significant progress in understanding the molecular pathogenesis of lung cancer and in developing treatments for it. Such achievements are directly utilized in clinical practice. Indeed, the echinoderm microtubule-associated protein-like 4–anaplastic lymphoma kinase (ALK) fusion gene was first described in non-small cell lung cancer in 2007, and a molecularly targeted drug against the fusion was approved in 2011. However, lung cancer with the ALK fusion constitutes only a small fraction of lung cancers; therefore, efficient patient selection is crucial for successful treatment using the ALK inhibitor. Currently, RT-PCR, fluorescent in situ hybridization (FISH), and immunohistochemistry are commonly used to detect the ALK fusion. Although FISH is currently the gold standard technique, there are no perfect methods for detecting these genetic alterations. In this article, we discuss the advantages and disadvantages of each method and the possible criteria for selecting patients who are more likely to have the ALK fusion. If we can successfully screen patients, then ALK inhibitor treatment will be the best example of personalized therapy in terms of selecting patients with an uncommon genotype from a larger group with the same tumor phenotype. In other words, the personalized therapy may offer a new challenge for current clinical oncology.

  8. Influence of putative exopolysaccharide genes on Pseudomonas putida KT2440 biofilm stability

    DEFF Research Database (Denmark)

    Nilsson, Martin; Chiang, Wen-Chi; Fazli, Mustafa

    2011-01-01

    We report a study of the role of putative exopolysaccharide gene clusters in the formation and stability of Pseudomonas putida KT2440 biofilm. Two novel putative exopolysaccharide gene clusters, pea and peb, were identified, and evidence is provided that they encode products that stabilize P....... putida KT2440 biofilm. The gene clusters alg and bcs, which code for proteins mediating alginate and cellulose biosynthesis, were found to play minor roles in P. putida KT2440 biofilm formation and stability under the conditions tested. A P. putida KT2440 derivative devoid of any identifiable...

  9. Investigation Of The Primary Transcriptome Of The Production Organism Pseudomonas Putida

    DEFF Research Database (Denmark)

    D'Arrigo, Isotta; Bojanovic, Klara; Long, Katherine

    2015-01-01

    Introduction: Pseudomonas putida is a nonpathogenic, Gram-negative bacterium and an excellent model organism for biotechnological applications. Due to its metabolic versatility, P. putida can grow in different environments including in extreme conditions. It has several genes to degrade xenobiotic....... putida KT2440 transcriptome, in the presence of citrate or glucose as sole carbon source. Results: A total of 7937 putative transcription start sites (TSSs) have been identified. 5’ RACE experiments have been performed to confirm putative TSSs, and 5’ UTR regions have been investigated for conservative......, our study has allowed for the investigation of several biological features of P. putida....

  10. Specific Gene Loci of Clinical Pseudomonas putida Isolates.

    Directory of Open Access Journals (Sweden)

    Lázaro Molina

    Full Text Available Pseudomonas putida are ubiquitous inhabitants of soils and clinical isolates of this species have been seldom described. Clinical isolates show significant variability in their ability to cause damage to hosts because some of them are able to modulate the host's immune response. In the current study, comparisons between the genomes of different clinical and environmental strains of P. putida were done to identify genetic clusters shared by clinical isolates that are not present in environmental isolates. We show that in clinical strains specific genes are mostly present on transposons, and that this set of genes exhibit high identity with genes found in pathogens and opportunistic pathogens. The set of genes prevalent in P. putida clinical isolates, and absent in environmental isolates, are related with survival under oxidative stress conditions, resistance against biocides, amino acid metabolism and toxin/antitoxin (TA systems. This set of functions have influence in colonization and survival within human tissues, since they avoid host immune response or enhance stress resistance. An in depth bioinformatic analysis was also carried out to identify genetic clusters that are exclusive to each of the clinical isolates and that correlate with phenotypical differences between them, a secretion system type III-like was found in one of these clinical strains, a determinant of pathogenicity in Gram-negative bacteria.

  11. BMP15 suppresses progesterone production by down-regulating StAR via ALK3 in human granulosa cells.

    Science.gov (United States)

    Chang, Hsun-Ming; Cheng, Jung-Chien; Klausen, Christian; Leung, Peter C K

    2013-12-01

    In addition to somatic cell-derived growth factors, oocyte-derived growth differentiation factor (GDF)9 and bone morphogenetic protein (BMP)15 play essential roles in female fertility. However, few studies have investigated their effects on human ovarian steroidogenesis, and fewer still have examined their differential effects or underlying molecular determinants. In the present study, we used immortalized human granulosa cells (SVOG) and human granulosa cell tumor cells (KGN) to compare the effects of GDF9 and BMP15 on steroidogenic enzyme expression and investigate potential mechanisms of action. In SVOG cells, neither GDF9 nor BMP15 affects the mRNA levels of P450 side-chain cleavage enzyme or 3β-hydroxysteroid dehydrogenase. However, treatment with BMP15, but not GDF9, significantly decreases steroidogenic acute regulatory protein (StAR) mRNA and protein levels as well as progesterone production. These suppressive effects, along with the induction of Sma and Mad-related protein (SMAD)1/5/8 phosphorylation, are attenuated by cotreatment with 2 different BMP type I receptor inhibitors (dorsomorphin and DMH-1). Furthermore, depletion of activin receptor-like kinase (ALK)3 using small interfering RNA reverses the effects of BMP15 on SMAD1/5/8 phosphorylation and StAR expression. Similarly, knockdown of ALK3 abolishes BMP15-induced SMAD1/5/8 phosphorylation in KGN cells. These results provide evidence that oocyte-derived BMP15 down-regulates StAR expression and decreases progesterone production in human granulosa cells, likely via ALK3-mediated SMAD1/5/8 signaling. Our findings suggest that oocyte may play a critical role in the regulation of progesterone to prevent premature luteinization during the late stage of follicle development.

  12. Inhibition of Axl improves the targeted therapy against ALK-mutated neuroblastoma

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Fei [Department of Neurology, Sichuan Medical Science Institute and Sichuan Provincial Hospital, Chengdu 610072 (China); Li, Hongling [Department of Radiotherapy, Shanghai First People’s Hospital, Shanghai Jiao Tong University, Shanghai 201620 (China); Sun, Yong, E-mail: sunfanqi2010@163.com [Department of Burn and Plastic Surgery, Huai’an First People’s Hospital, Nanjing Medical University, Huai’an 223300 (China)

    2014-11-28

    Highlights: • First reported Axl is co-expressed with ALK in neuroblastoma tissues and cell lines. • Axl activation promotes cell growth and impairs the efficiency of ALK inhibitor. • Further found silence of Axl leads to increased sensitivity to ALK inhibitors. • Axl inhibitor promotes the efficiency of targeted therapy in vitro and in vivo. • Axl activation should be considered in the clinical application of ALK inhibitors. - Abstract: Neuroblastoma (NB) patients harboring mutated ALK can be expected to potentially benefit from targeted therapy based on ALK tyrosine kinase inhibitor (TKI), such as crizotinib and ceritinib. However, the effect of the treatment varies with different individuals, although with the same genic changes. Axl receptor tyrosine kinase is expressed in a variety of human cancers, but little data are reported in NB, particularly in which carrying mutated ALK. In this study, we focus on the roles of Axl in ALK-mutated NB for investigating rational therapeutic strategy. We found that Axl is expressed in ALK-positive NB tissues and cell lines, and could be effectively activated by its ligand GAS6. Ligand-dependent Axl activation obviously rescued crizotinib-mediated suppression of cell proliferation in ALK-mutated NB cells. Genetic inhibition of Axl with specific small interfering RNA markedly increased the sensitivity of cells to ALK-TKIs. Furthermore, a small-molecule inhibitor of Axl significantly enhanced ALK-targeted therapy, as an increased frequency of apoptosis was observed in NB cells co-expressing ALK and Axl. Taken together, our results demonstrated that activation of Axl could lead to insensitivity to ALK inhibitors, and dual inhibition of ALK and Axl might be a potential therapeutic strategy against ALK-mutated NB.

  13. A novel crosstalk between Alk7 and cGMP signaling differentially regulates brown adipocyte function

    Directory of Open Access Journals (Sweden)

    Aileen Balkow

    2015-08-01

    Conclusions: We found a so far unknown crosstalk between cGMP and Alk7 signaling pathways. Tight regulation of Alk7 is required for efficient differentiation of brown adipocytes. Alk7 has differential effects on adipogenic differentiation and the development of the thermogenic program in brown adipocytes.

  14. Lumican Peptides: Rational Design Targeting ALK5/TGFBRI

    Science.gov (United States)

    Gesteira, Tarsis Ferreira; Coulson-Thomas, Vivien J.; Yuan, Yong; Zhang, Jianhua; Nader, Helena B.; Kao, Winston W.-Y.

    2017-02-01

    Lumican, a small leucine rich proteoglycan (SLRP), is a component of extracellular matrix which also functions as a matrikine regulating multiple cell activities. In the cornea, lumican maintains corneal transparency by regulating collagen fibrillogenesis, promoting corneal epithelial wound healing, regulating gene expression and maintaining corneal homeostasis. We have recently shown that a peptide designed from the 13 C-terminal amino acids of lumican (LumC13) binds to ALK5/TGFBR1 (type1 receptor of TGFβ) to promote wound healing. Herein we evaluate the mechanism by which this synthetic C-terminal amphiphilic peptide (LumC13), binds to ALK5. These studies clearly reveal that LumC13-ALK5 form a stable complex. In order to determine the minimal amino acids required for the formation of a stable lumican/ALK5 complex derivatives of LumC13 were designed and their binding to ALK5 investigated in silico. These LumC13 derivatives were tested both in vitro and in vivo to evaluate their ability to promote corneal epithelial cell migration and corneal wound healing, respectively. These validations add to the therapeutic value of LumC13 (Lumikine) and aid its clinical relevance of promoting the healing of corneal epithelium debridement. Moreover, our data validates the efficacy of our computational approach to design active peptides based on interactions of receptor and chemokine/ligand.

  15. Novel targeted therapeutics: inhibitors of MDM2, ALK and PARP

    Directory of Open Access Journals (Sweden)

    Hsueh Chung-Tsen

    2011-04-01

    Full Text Available Abstract We reviewed preclinical data and clinical development of MDM2 (murine double minute 2, ALK (anaplastic lymphoma kinase and PARP (poly [ADP-ribose] polymerase inhibitors. MDM2 binds to p53, and promotes degradation of p53 through ubiquitin-proteasome degradation. JNJ-26854165 and RO5045337 are 2 small-molecule inhibitors of MDM2 in clinical development. ALK is a transmembrane protein and a member of the insulin receptor tyrosine kinases. EML4-ALK fusion gene is identified in approximately 3-13% of non-small cell lung cancer (NSCLC. Early-phase clinical studies with Crizotinib, an ALK inhibitor, in NSCLC harboring EML4-ALK have demonstrated promising activity with high response rate and prolonged progression-free survival. PARPs are a family of nuclear enzymes that regulates the repair of DNA single-strand breaks through the base excision repair pathway. Randomized phase II study has shown adding PARP-1 inhibitor BSI-201 to cytotoxic chemotherapy improves clinical outcome in patients with triple-negative breast cancer. Olaparib, another oral small-molecule PARP inhibitor, demonstrated encouraging single-agent activity in patients with advanced breast or ovarian cancer. There are 5 other PARP inhibitors currently under active clinical investigation.

  16. National Working Group Meeting on ALK diagnostics in lung cancer.

    Science.gov (United States)

    Cooper, Wendy; Fox, Stephen; O'Toole, Sandra; Morey, Adrienne; Frances, Glenn; Pavlakis, Nick; O'Byrne, Kenneth; Dettrick, Andrew; Leong, Trishe; Rathi, Vivek; Spagnolo, Dominic; Hemmings, Chris; Singh, Mahendra; Moffat, David; Tsao, Ming-Sound; Wilner, Keith; Buller, Richard; Pitman Lowenthal, Susan; Arifeen, Shams; Binko, Justin; Alam, Mahmood

    2014-04-01

    The global landscape of molecular testing is rapidly changing, with the recent publication of the International Association for the Study of Lung Cancer (IASLC)/College of American Pathologists (CAP) guidelines and the ALK Atlas. The IASLC/CAP guidelines recommend that tumors from patients with non-small cell lung cancer (NSCLC) be tested for ALK rearrangements in addition to epidermal growth factor receptor (EGFR) mutations. The spur for this recommendation is the availability of novel therapies that target these rearrangements. This article is based on coverage of a Pfizer-sponsored National Working Group Meeting on ALK Diagnostics in Lung Cancer, held around the 15th World Lung Cancer Conference, in Sydney on October 31, 2013. It is based on the presentations given by the authors at the meeting and the discussion that ensued. The content for this article was discussed and agreed on by the authors. © 2014 Wiley Publishing Asia Pty Ltd.

  17. Draft Genome Sequence of the Model Naphthalene-Utilizing Organism Pseudomonas putida OUS82

    DEFF Research Database (Denmark)

    Tay, Martin; Roizman, Dan; Cohen, Yehuda

    2014-01-01

    Pseudomonas putida OUS82 was isolated from petrol- and oil-contaminated soil in 1992, and ever since, it has been used as a model organism to study the microbial assimilation of naphthalene and phenanthrene. Here, we report the 6.7-Mb draft genome sequence of P. putida OUS82 and analyze its...

  18. Activity of EGFR-tyrosine kinase and ALK inhibitors for EML4–ALK-rearranged non–small–cell lung cancer harbored coexisting EGFR mutation

    International Nuclear Information System (INIS)

    Miyanaga, Akihiko; Kawamoto, Masashi; Tsuchiya, Shinichi; Hagiwara, Koichi; Soda, Manabu; Takeuchi, Kengo; Yamamoto, Nobuyuki; Mano, Hiroyuki; Ishikawa, Yuichi; Gemma, Akihiko; Shimizu, Kumi; Noro, Rintaro; Seike, Masahiro; Kitamura, Kazuhiro; Kosaihira, Seiji; Minegishi, Yuji; Shukuya, Takehito; Yoshimura, Akinobu

    2013-01-01

    The EML4–ALK (echinoderm microtubule-associated protein-like 4 gene and the anaplastic lymphoma kinase gene) fusion oncogene represents a novel molecular target in a small subset of non–small–cell lung cancers (NSCLCs). The EML4–ALK fusion gene occurs generally in NSCLC without mutations in epidermal growth factor receptor (EGFR) and KRAS. We report that a case of EML4–ALK-positive NSCLC with EGFR mutation had a response of stable disease to both an EGFR tyrosine kinase inhibitor (EGFR-TKI) and ALK inhibitor. We described the first clinical report of a patient with EML4–ALK-positive NSCLC with EGFR mutation that had a response of stable disease to both single-agent EGFR-TKI and ALK inhibitor. EML4–ALK translocation may be associated with resistance to EGFR-TKI, and EGFR signaling may contribute to resistance to ALK inhibitor in EML4–ALK-positive NSCLC

  19. Repeated batch and continuous degradation of chlorpyrifos by Pseudomonas putida.

    Science.gov (United States)

    Pradeep, Vijayalakshmi; Subbaiah, Usha Malavalli

    2015-01-01

    The present study was undertaken with the objective of studying repeated batch and continuous degradation of chlorpyrifos (O,O-diethyl O-3,5,6-trichloropyridin-2-yl phosphorothioate) using Ca-alginate immobilized cells of Pseudomonas putida isolated from an agricultural soil, and to study the genes and enzymes involved in degradation. The study was carried out to reduce the toxicity of chlorpyrifos by degrading it to less toxic metabolites. Long-term stability of pesticide degradation was studied during repeated batch degradation of chlorpyrifos, which was carried out over a period of 50 days. Immobilized cells were able to show 65% degradation of chlorpyrifos at the end of the 50th cycle with a cell leakage of 112 × 10(3) cfu mL(-1). During continuous treatment, 100% degradation was observed at 100 mL h(-1) flow rate with 2% chlorpyrifos, and with 10% concentration of chlorpyrifos 98% and 80% degradation was recorded at 20 mL h(-1) and 100 mL h(-1) flow rate respectively. The products of degradation detected by liquid chromatography-mass spectrometry analysis were 3,5,6-trichloro-2-pyridinol and chlorpyrifos oxon. Plasmid curing experiments with ethidium bromide indicated that genes responsible for the degradation of chlorpyrifos are present on the chromosome and not on the plasmid. The results of Polymerase chain reaction indicate that a ~890-bp product expected for mpd gene was present in Ps. putida. Enzymatic degradation studies indicated that the enzymes involved in the degradation of chlorpyrifos are membrane-bound. The study indicates that immobilized cells of Ps. putida have the potential to be used in bioremediation of water contaminated with chlorpyrifos.

  20. Microbial production of polyhydroxyalkanoate block copolymer by recombinant Pseudomonas putida.

    Science.gov (United States)

    Li, Shi Yan; Dong, Cui Ling; Wang, Shen Yu; Ye, Hai Mu; Chen, Guo-Qiang

    2011-04-01

    Polyhydroxyalkanoate (PHA) synthesis genes phaPCJ(Ac) cloned from Aeromonas caviae were transformed into Pseudomonas putida KTOY06ΔC, a mutant of P. putida KT2442, resulting in the ability of the recombinant P. putida KTOY06ΔC (phaPCJ(A.c)) to produce a short-chain-length and medium-chain-length PHA block copolymer consisting of poly-3-hydroxybutyrate (PHB) as one block and random copolymer of 3-hydroxyvalerate (3HV) and 3-hydroxyheptanoate (3HHp) as another block. The novel block polymer was studied by differential scanning calorimetry (DSC), nuclear magnetic resonance, and rheology measurements. DSC studies showed the polymer to possess two glass transition temperatures (T(g)), one melting temperature (T(m)) and one cool crystallization temperature (T(c)). Rheology studies clearly indicated a polymer chain re-arrangement in the copolymer; these studies confirmed the polymer to be a block copolymer, with over 70 mol% homopolymer (PHB) of 3-hydroxybutyrate (3HB) as one block and around 30 mol% random copolymers of 3HV and 3HHp as the second block. The block copolymer was shown to have the highest tensile strength and Young's modulus compared with a random copolymer with similar ratio and a blend of homopolymers PHB and PHVHHp with similar ratio. Compared with other commercially available PHA including PHB, PHBV, PHBHHx, and P3HB4HB, the short-chain- and medium-chain-length block copolymer PHB-b-PHVHHp showed differences in terms of mechanical properties and should draw more attentions from the PHA research community. © Springer-Verlag 2010

  1. Engineering Pseudomonas putida KT2440 for efficient ethylene glycol utilization.

    Science.gov (United States)

    Franden, Mary Ann; Jayakody, Lahiru N; Li, Wing-Jin; Wagner, Neil J; Cleveland, Nicholas S; Michener, William E; Hauer, Bernhard; Blank, Lars M; Wierckx, Nick; Klebensberger, Janosch; Beckham, Gregg T

    2018-06-07

    Ethylene glycol is used as a raw material in the production of polyethylene terephthalate, in antifreeze, as a gas hydrate inhibitor in pipelines, and for many other industrial applications. It is metabolized by aerobic microbial processes via the highly toxic intermediates glycolaldehyde and glycolate through C2 metabolic pathways. Pseudomonas putida KT2440, which has been engineered for environmental remediation applications given its high toxicity tolerance and broad substrate specificity, is not able to efficiently metabolize ethylene glycol, despite harboring putative genes for this purpose. To further expand the metabolic portfolio of P. putida, we elucidated the metabolic pathway to enable ethylene glycol via systematic overexpression of glyoxylate carboligase (gcl) in combination with other genes. Quantitative reverse transcription polymerase chain reaction demonstrated that all of the four genes in genomic proximity to gcl (hyi, glxR, ttuD, and pykF) are transcribed as an operon. Where the expression of only two genes (gcl and glxR) resulted in growth in ethylene glycol, improved growth and ethylene glycol utilization were observed when the entire gcl operon was expressed. Both glycolaldehyde and glyoxal inhibit growth in concentrations of ethylene glycol above 50 mM. To overcome this bottleneck, the additional overexpression of the glycolate oxidase (glcDEF) operon removes the glycolate bottleneck and minimizes the production of these toxic intermediates, permitting growth in up to 2 M (~124 g/L) and complete consumption of 0.5 M (31 g/L) ethylene glycol in shake flask experiments. In addition, the engineered strain enables conversion of ethylene glycol to medium-chain-length polyhydroxyalkanoates (mcl-PHAs). Overall, this study provides a robust P. putida KT2440 strain for ethylene glycol consumption, which will serve as a foundational strain for further biocatalyst development for applications in the remediation of waste polyester plastics and

  2. Conversion of levoglucosan and cellobiosan by Pseudomonas putida KT2440

    Directory of Open Access Journals (Sweden)

    Jeffrey G. Linger

    2016-12-01

    Full Text Available Pyrolysis offers a straightforward approach for the deconstruction of plant cell wall polymers into bio-oil. Recently, there has been substantial interest in bio-oil fractionation and subsequent use of biological approaches to selectively upgrade some of the resulting fractions. A fraction of particular interest for biological upgrading consists of polysaccharide-derived substrates including sugars and sugar dehydration products such as levoglucosan and cellobiosan, which are two of the most abundant pyrolysis products of cellulose. Levoglucosan can be converted to glucose-6-phosphate through the use of a levoglucosan kinase (LGK, but to date, the mechanism for cellobiosan utilization has not been demonstrated. Here, we engineer the microbe Pseudomonas putida KT2440 to use levoglucosan as a sole carbon and energy source through LGK integration. Moreover, we demonstrate that cellobiosan can be enzymatically converted to levoglucosan and glucose with β-glucosidase enzymes from both Glycoside Hydrolase Family 1 and Family 3. β-glucosidases are commonly used in both natural and industrial cellulase cocktails to convert cellobiose to glucose to relieve cellulase product inhibition and to facilitate microbial uptake of glucose. Using an exogenous β-glucosidase, we demonstrate that the engineered strain of P. putida can grow on levoglucosan up to 60 g/L and can also utilize cellobiosan. Overall, this study elucidates the biological pathway to co-utilize levoglucosan and cellobiosan, which will be a key transformation for the biological upgrading of pyrolysis-derived substrates. Keywords: Pseudomonas putida KT2440, Levoglucosan kinase, B-glucosidase, Cellobiosan, Pyrolysis, Biofuels

  3. The germacranolide sesquiterpene lactone neurolenin B of the medicinal plant Neurolaena lobata (L.) R.Br. ex Cass inhibits NPM/ALK-driven cell expansion and NF-κB-driven tumour intravasation.

    Science.gov (United States)

    Unger, Christine; Kiss, Izabella; Vasas, Andrea; Lajter, Ildikó; Kramer, Nina; Atanasov, Atanas Georgiev; Nguyen, Chi Huu; Chatuphonprasert, Waranya; Brenner, Stefan; Krieger, Sigurd; McKinnon, Ruxandra; Peschel, Andrea; Kain, Renate; Saiko, Philipp; Szekeres, Thomas; Kenner, Lukas; Hassler, Melanie R; Diaz, Rene; Frisch, Richard; Dirsch, Verena M; Jäger, Walter; de Martin, Rainer; Bochkov, Valery N; Passreiter, Claus M; Peter-Vörösmarty, Barbara; Mader, Robert M; Grusch, Michael; Dolznig, Helmut; Kopp, Brigitte; Zupko, Istvan; Hohmann, Judit; Krupitza, Georg

    2015-08-15

    The t(2;5)(p23;q35) chromosomal translocation results in the expression of the fusion protein NPM/ALK that when expressed in T-lymphocytes gives rise to anaplastic large cell lymphomas (ALCL). In search of new therapy options the dichloromethane extract of the ethnomedicinal plant Neurolaena lobata (L.) R.Br. ex Cass was shown to inhibit NPM/ALK expression. Therefore, we analysed whether the active principles that were recently isolated and found to inhibit inflammatory responses specifically inhibit growth of NPM/ALK+ ALCL, leukaemia and breast cancer cells, but not of normal cells, and the intravasation through the lymphendothelial barrier. ALCL, leukaemia and breast cancer cells, and normal peripheral blood mononuclear cells (PBMCs) were treated with isolated sesquiterpene lactones and analysed for cell cycle progression, proliferation, mitochondrial activity, apoptosis, protein and mRNA expression, NF-κB and cytochrome P450 activity, 12(S)-HETE production and lymphendothelial intravasation. In vitro treatment of ALCL by neurolenin B suppressed NPM/ALK, JunB and PDGF-Rβ expression, inhibited the growth of ALCL cells late in M phase, and induced apoptosis via caspase 3 without compromising mitochondrial activity (as a measure of general exogenic toxicity). Moreover, neurolenin B attenuated tumour spheroid intravasation probably through inhibition of NF-κB and CYP1A1. Neurolenin B specifically decreased pro-carcinogenic NPM/ALK expression in ALK+ ALCL cells and, via the inhibition of NF-kB signalling, attenuated tumour intra/extravasation into the lymphatics. Hence, neurolenin B may open new options to treat ALCL and to manage early metastatic processes to which no other therapies exist. Copyright © 2015 Elsevier GmbH. All rights reserved.

  4. [Lung adenocarcinoma with concomitant EGFR mutation and ALK rearrangement].

    Science.gov (United States)

    Caliez, J; Monnet, I; Pujals, A; Rousseau-Bussac, G; Jabot, L; Boudjemaa, A; Leroy, K; Chouaid, C

    2017-05-01

    Among patients with non-small-cell lung cancer, coexistence of EGFR mutation and ALK rearrangement is rare. We describe the clinical features of two patients with this double anomaly. A 62-year-old Caucasian non-smoking woman was diagnosed with cT4N0M0 lung adenocarcinoma. Initial biopsy showed EGFR mutation and ALK rearrangement. She received cisplatin-gemcitabine, followed by 17 months of gemcitabine. Owing to progression, she received erlotinib for 14 months, then paclitaxel for 6 months and finally crizotinib. A partial response was achieved and maintained for 24 months. A 45-year-old Caucasian woman, light smoker, was diagnosed with cT2N3M0 lung adenocarcinoma. Only EGFR mutation was found on initial analysis. She underwent treatment with cisplatin-gemcitabine and thoracic radiotherapy. Progression occurred after 8 months and afatinbib was started. Eight months later, progression was observed with a neoplasic pleural effusion in which tumor cells expressing ALK rearrangement were found. A new FISH analysis was performed on the initial tumor but did not find this rearrangement. Despite a third line of crizotinib, the patient died one month later. The literature shows 45 other cases of these two abnormalities, observed either from the start or during follow-up. EGFR's TKI were almost always given before ALK's TKI. Therapeutic strategy needs to be clarified in cases of double alteration. With regard to the second patient, appearance of ALK rearrangement may constitute a resistance mechanism to EGFR's TKI. Copyright © 2016 SPLF. Published by Elsevier Masson SAS. All rights reserved.

  5. ALK activation by the CLTC-ALK fusion is a recurrent event in large B-cell lymphoma.

    NARCIS (Netherlands)

    Paepe, P. de; Baens, M; Krieken, J.H.J.M. van; Verhasselt, B.; Stul, M.; Simons, A.; Poppe, B.; Laureys, G.; Brons, P.P.T.; Vandenberghe, P.; Speleman, F.; Praet, M.; Wolf-Peeters, C. de; Marynen, P.; Wlodarska, I.

    2003-01-01

    We present 3 cases of large B-cell lymphoma (LBCL) with a granular cytoplasmic staining for anaplastic lymphoma kinase (ALK). All of the cases showed striking similarities in morphology and immunohistochemical profile characterized by a massive monomorphic proliferation of CD20-/CD138+

  6. Detection of ALK gene rearrangement in non-small cell lung cancer: a comparison of fluorescence in situ hybridization and chromogenic in situ hybridization with correlation of ALK protein expression.

    Science.gov (United States)

    Kim, Hyojin; Yoo, Seol-Bong; Choe, Ji-Young; Paik, Jin Ho; Xu, Xianhua; Nitta, Hiroaki; Zhang, Wenjun; Grogan, Thomas M; Lee, Choon-Taek; Jheon, Sanghoon; Chung, Jin-Haeng

    2011-08-01

    Accurate determination of ALK rearrangement is important in lung cancer patients, especially in determining their eligibility for crizotinib therapy. Fluorescence in situ hybridization (FISH) has been regarded as the gold standard method for detecting ALK rearrangement. However, FISH requires a fluorescence microscope, and the signals are labile and rapidly fade over time. This study evaluates the concordance between ALK gene rearrangement in non-small cell lung cancer assessed by ALK FISH and a newly developed ALK chromogenic in situ hybridization (CISH) and correlates the results with ALK protein expression assessed by immunohistochemistry. A total of 465 formalin-fixed, paraffin-embedded non-small cell lung cancer samples were analyzed by ALK FISH (PathVysion, Vysis, Abbott) and ALK CISH. For comparison, all specimens were stained by immunohistochemistry (clone 5A4, Novocastra) and interobserver reproducibility was assessed. We found that agreement between the pathologists on the CISH-determined ALK status was achieved in 449 patients (96.6%), and ALK rearrangement was identified in 18 patients (4.0%) in CISH method. Among these cases, 443 cases (95.3%) had results matching the corresponding FISH results: 17 rearranged, 425 wild types, and 1 discordant case. There was high concordance in the assessment of ALK gene rearrangement between FISH and CISH techniques (κ = 0.92) and between observers (κ = 0.97). In addition, there was high concordance in the ALK gene status and ALK protein expression between CISH and IHC tests (κ = 0.82). CISH is a highly reproducible and practical method to detect ALK gene rearrangement and correlated well with ALK protein expression. Here, we present a diagnostic algorithm (Chung's SNUBH ALK protocol) to detect lung cancer with ALK rearrangements using IHC, FISH and CISH. Because CISH allows a concurrent analysis of histological features of the tumors and gene rearrangement, it appears to be a useful method in determining ALK gene

  7. EMMPRIN (CD147) is induced by C/EBPβ and is differentially expressed in ALK+ and ALK- anaplastic large-cell lymphoma.

    Science.gov (United States)

    Schmidt, Janine; Bonzheim, Irina; Steinhilber, Julia; Montes-Mojarro, Ivonne A; Ortiz-Hidalgo, Carlos; Klapper, Wolfram; Fend, Falko; Quintanilla-Martínez, Leticia

    2017-09-01

    Anaplastic lymphoma kinase-positive (ALK+) anaplastic large-cell lymphoma (ALCL) is characterized by expression of oncogenic ALK fusion proteins due to the translocation t(2;5)(p23;q35) or variants. Although genotypically a T-cell lymphoma, ALK+ ALCL cells frequently show loss of T-cell-specific surface antigens and expression of monocytic markers. C/EBPβ, a transcription factor constitutively overexpressed in ALK+ ALCL cells, has been shown to play an important role in the activation and differentiation of macrophages and is furthermore capable of transdifferentiating B-cell and T-cell progenitors to macrophages in vitro. To analyze the role of C/EBPβ for the unusual phenotype of ALK+ ALCL cells, C/EBPβ was knocked down by RNA interference in two ALK+ ALCL cell lines, and surface antigen expression profiles of these cell lines were generated using a Human Cell Surface Marker Screening Panel (BD Biosciences). Interesting candidate antigens were further analyzed by immunohistochemistry in primary ALCL ALK+ and ALK- cases. Antigen expression profiling revealed marked changes in the expression of the activation markers CD25, CD30, CD98, CD147, and CD227 after C/EBPβ knockdown. Immunohistochemical analysis confirmed a strong, membranous CD147 (EMMPRIN) expression in ALK+ ALCL cases. In contrast, ALK- ALCL cases showed a weaker CD147 expression. CD274 or PD-L1, an immune inhibitory receptor ligand, was downregulated after C/EBPβ knockdown. PD-L1 also showed stronger expression in ALK+ ALCL compared with ALK- ALCL, suggesting an additional role of C/EBPβ in ALK+ ALCL in generating an immunosuppressive environment. Finally, no expression changes of T-cell or monocytic markers were detected. In conclusion, surface antigen expression profiling demonstrates that C/EBPβ plays a critical role in the activation state of ALK+ ALCL cells and reveals CD147 and PD-L1 as important downstream targets. The multiple roles of CD147 in migration, adhesion, and invasion, as well as

  8. Comparative genomic and functional analyses: unearthing the diversity and specificity of nematicidal factors in Pseudomonas putida strain 1A00316

    Science.gov (United States)

    Guo, Jing; Jing, Xueping; Peng, Wen-Lei; Nie, Qiyu; Zhai, Yile; Shao, Zongze; Zheng, Longyu; Cai, Minmin; Li, Guangyu; Zuo, Huaiyu; Zhang, Zhitao; Wang, Rui-Ru; Huang, Dian; Cheng, Wanli; Yu, Ziniu; Chen, Ling-Ling; Zhang, Jibin

    2016-01-01

    We isolated Pseudomonas putida (P. putida) strain 1A00316 from Antarctica. This bacterium has a high efficiency against Meloidogyne incognita (M. incognita) in vitro and under greenhouse conditions. The complete genome of P. putida 1A00316 was sequenced using PacBio single molecule real-time (SMRT) technology. A comparative genomic analysis of 16 Pseudomonas strains revealed that although P. putida 1A00316 belonged to P. putida, it was phenotypically more similar to nematicidal Pseudomonas fluorescens (P. fluorescens) strains. We characterized the diversity and specificity of nematicidal factors in P. putida 1A00316 with comparative genomics and functional analysis, and found that P. putida 1A00316 has diverse nematicidal factors including protein alkaline metalloproteinase AprA and two secondary metabolites, hydrogen cyanide and cyclo-(l-isoleucyl-l-proline). We show for the first time that cyclo-(l-isoleucyl-l-proline) exhibit nematicidal activity in P. putida. Interestingly, our study had not detected common nematicidal factors such as 2,4-diacetylphloroglucinol (2,4-DAPG) and pyrrolnitrin in P. putida 1A00316. The results of the present study reveal the diversity and specificity of nematicidal factors in P. putida strain 1A00316. PMID:27384076

  9. CCR 20th Anniversary Commentary: Molecular Pathology of ALK-Rearranged Lung Tumors.

    Science.gov (United States)

    Iafrate, A John

    2015-12-01

    The article by Rodig and colleagues, which was published in the August 15, 2009, issue of Clinical Cancer Research, helped detail the clinicopathologic profile of ALK-positive tumors and initiated the development of ALK diagnostics. Subsequent clinical trials utilized these findings, resulting in the approval of effective targeted therapies. The ALK story is a model for the development of therapies in the genomic era. ©2015 American Association for Cancer Research.

  10. Insights into brain metastasis in patients with ALK+ lung cancer: is the brain truly a sanctuary?

    Science.gov (United States)

    Toyokawa, Gouji; Seto, Takashi; Takenoyama, Mitsuhiro; Ichinose, Yukito

    2015-12-01

    Anaplastic lymphoma kinase (ALK) has been identified to exert a potent transforming activity through its rearrangement in non-small cell lung cancer (NSCLC), and patients (pts) with ALK rearrangement can be treated more successfully with ALK inhibitors, such as crizotinib, alectinib, and ceritinib, than with chemotherapy. Despite the excellent efficacy of ALK inhibitors, resistance to these drugs is inevitably encountered in most ALK-rearranged pts. Cases of resistance are subtyped into three groups, i.e., systemic, oligo, and central nervous system (CNS) types, with the CNS being used to be considered a sanctuary. With regard to the management of CNS lesions in pts with ALK+ NSCLC, a growing body of evidence has gradually demonstrated the intracranial (IC) efficacy of ALK inhibitor (ALKi) in ALK+ NSCLC pts with brain metastases (BMs). Although the efficacy of crizotinib for the CNS lesions remains controversial, a recent retrospective investigation of ALK+ pts with BM enrolled in PROFILE 1005 and PROFILE 1007 demonstrated that crizotinib is associated with a high disease control rate for BM. However, BM comprises the most common site of progressive disease in pts with or without baseline BMs, which is a serious problem for crizotinib. Furthermore, alectinib can be used to achieve strong and long-lasting inhibitory effects on BM. In addition to alectinib, the IC efficacy of other next-generation ALK inhibitors, such as ceritinib, AP26113 and PF-06463922, has been demonstrated. In this article, we review the latest evidence regarding the BM and IC efficacy of ALK inhibitors in pts with ALK+ NSCLC.

  11. Functional, genetic and chemical characterization of biosurfactants produced by plant growth-promoting Pseudomonas putida 267

    NARCIS (Netherlands)

    Kruijt, M.; Tran, H.; Raaijmakers, J.M.

    2009-01-01

    Aims: Plant growth-promoting Pseudomonas putida strain 267, originally isolated from the rhizosphere of black pepper, produces biosurfactants that cause lysis of zoospores of the oomycete pathogen Phytophthora capsici. The biosurfactants were characterized, the biosynthesis gene(s) partially

  12. Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans-metabolism

    DEFF Research Database (Denmark)

    Nikel, Pablo I.; de Lorenzo, Víctor

    2018-01-01

    are presented for substantiating the worth of P. putida as one of the favorite workhorses for sustainable manufacturing of fine and bulk chemicals in the current times of the 4th Industrial Revolution. The potential of P. putida to extend its rich native biochemistry beyond existing boundaries is discussed...... biochemistry is naturally geared to generate reductive currency [i.e., NAD(P)H] that makes this bacterium a phenomenal host for redox-intensive reactions. In some cases, genetic editing of the indigenous biochemical network of P. putida (cis-metabolism) has sufficed to obtain target compounds of industrial...... stresses. Over the years, these properties have been capitalized biotechnologically owing to the expanding wealth of genetic tools designed for deep-editing the P. putida genome. A suite of dedicated vectors inspired in the core tenets of synthetic biology have enabled to suppress many of the naturally...

  13. Proteins with GGDEF and EAL domains regulate Pseudomonas putida biofilm formation and dispersal

    DEFF Research Database (Denmark)

    Gjermansen, Morten; Ragas, Paula Cornelia; Tolker-Nielsen, Tim

    2006-01-01

    Microbial biofilm formation often causes problems in medical and industrial settings, and knowledge about the factors that are involved in biofilm development and dispersion is useful for creating strategies to control the processes. In this report, we present evidence that proteins with GGDEF...... and EAL domains are involved in the regulation of biofilm formation and biofilm dispersion in Pseudomonas putida. Overexpression in P. putida of the Escherichia coli YedQ protein, which contains a GGDEF domain, resulted in increased biofilm formation. Overexpression in P. putida of the E. coli Yhj......H protein, which contains an EAL domain, strongly inhibited biofilm formation. Induction of YhjH expression in P. putida cells situated in established biofilms led to rapid dispersion of the biofilms. These results support the emerging theme that GGDEF-domain and EAL-domain proteins are involved...

  14. Targeted Disruption of ALK Reveals a Potential Role in Hypogonadotropic Hypogonadism.

    Directory of Open Access Journals (Sweden)

    Barbara Witek

    Full Text Available Mice lacking ALK activity have previously been reported to exhibit subtle behavioral phenotypes. In this study of ALK of loss of function mice we present data supporting a role for ALK in hypogonadotropic hypogonadism in male mice. We observed lower level of serum testosterone at P40 in ALK knock-out males, accompanied by mild disorganization of seminiferous tubules exhibiting decreased numbers of GATA4 expressing cells. These observations highlight a role for ALK in testis function and are further supported by experiments in which chemical inhibition of ALK activity with the ALK TKI crizotinib was employed. Oral administration of crizotinib resulted in a decrease of serum testosterone levels in adult wild type male mice, which reverted to normal levels after cessation of treatment. Analysis of GnRH expression in neurons of the hypothalamus revealed a significant decrease in the number of GnRH positive neurons in ALK knock-out mice at P40 when compared with control littermates. Thus, ALK appears to be involved in hypogonadotropic hypogonadism by regulating the timing of pubertal onset and testis function at the upper levels of the hypothalamic-pituitary gonadal axis.

  15. Targeted Disruption of ALK Reveals a Potential Role in Hypogonadotropic Hypogonadism

    Science.gov (United States)

    Nord, Christoffer; Ahlgren, Ulf; Eriksson, Maria; Vernersson-Lindahl, Emma; Helland, Åslaug; Alexeyev, Oleg A.; Hallberg, Bengt; Palmer, Ruth H.

    2015-01-01

    Mice lacking ALK activity have previously been reported to exhibit subtle behavioral phenotypes. In this study of ALK of loss of function mice we present data supporting a role for ALK in hypogonadotropic hypogonadism in male mice. We observed lower level of serum testosterone at P40 in ALK knock-out males, accompanied by mild disorganization of seminiferous tubules exhibiting decreased numbers of GATA4 expressing cells. These observations highlight a role for ALK in testis function and are further supported by experiments in which chemical inhibition of ALK activity with the ALK TKI crizotinib was employed. Oral administration of crizotinib resulted in a decrease of serum testosterone levels in adult wild type male mice, which reverted to normal levels after cessation of treatment. Analysis of GnRH expression in neurons of the hypothalamus revealed a significant decrease in the number of GnRH positive neurons in ALK knock-out mice at P40 when compared with control littermates. Thus, ALK appears to be involved in hypogonadotropic hypogonadism by regulating the timing of pubertal onset and testis function at the upper levels of the hypothalamic-pituitary gonadal axis. PMID:25955180

  16. Fluorescence In Situ Hybridization and Immunohistochemistry as Diagnostic Methods for ALK Positive Non-Small Cell Lung Cancer Patients

    Science.gov (United States)

    Martinez, Pablo; Hernández-Losa, Javier; Cedrés, Susana; Castellví, Josep; Martinez-Marti, Alex; Tallada, Natalia; Murtra-Garrell, Nuria; Navarro-Mendivill, Alejandro; Rodriguez-Freixinos, Victor; Canela, Mercedes; Ramon y Cajal, Santiago; Felip, Enriqueta

    2013-01-01

    Background Anaplastic Lymphoma Kinase (ALK) positivity represents a novel molecular target in a subset of Non-Small Cell Lung Cancers (NSCLC). We explore Fluorescence in situ Hybridization (FISH) and Immunohistochemistry (IHC) as diagnostic methods for ALK positive patients and to describe its prevalence and outcomes in a population of NSCLC patients. Methods NSCLC patients previously screened for Epidermal Growth Factor Receptor (EGFR) at our institution were selected. ALK positive patients were identified by FISH and the value of IHC (D5F3) was explored. Results ninety-nine patients were identified. Median age was 61.5 years (range 35–83), all were caucasians, eighty percent were adenocarcinomas, fifty-one percent were male and thirty-eight percent were current smokers. Seven (7.1%) patients were ALK positive by FISH, thirteen (13.1%) were EGFR mutant, and 65 (65.6%) were negative/Wild Type (WT) for both ALK and EGFR. ALK positivity and EGFR mutations were mutually exclusive. ALK positive patients tend to be younger than EGFR mutated or wt patients. ALK positive patients were predominantly never smokers (71.4%) and adenocarcinoma (71.4%). ALK positive and EGFR mutant patients have a better outcome than negative/WT. All patients with ALK FISH negative tumours were negative for ALK IHC. Out of 6 patients positive for ALK FISH with more tissue available, 5 were positive for ALK IHC and 1 negative. Conclusions ALK positive patients represent 7.1% of a population of selected NSCLC. ALK positive patients have different clinical features and a better outcome than EGFR WT and ALK negative patients. IHC is a promising method for detecting ALK positive NSCLC patients. PMID:23359795

  17. ALK signaling and target therapy in anaplastic large cell lymphoma

    Directory of Open Access Journals (Sweden)

    Fabrizio eTabbo

    2012-05-01

    Full Text Available The discovery by Morris SW et al. in 1994 of the genes contributing to the t(2;5(p23;q35 translocation has put the foundation for a molecular based recognition of Anaplastic Large Cell Lymphoma (ALCL and pointed out the need for a further stratification of T-cell neoplasia. Likewise the detection of ALK genetic lesions among many human cancers has defined unique subsets of cancer patients, providing new opportunities for innovative therapeutic interventions. The objective of this review is to appraise the molecular mechanisms driving ALK-mediated transformation, and to maintain the neoplastic phenotype. The understanding of these events will allow the design and implementation of novel tailored strategies for a well-defined subset of cancer patients.

  18. ALK Signaling and Target Therapy in Anaplastic Large Cell Lymphoma

    International Nuclear Information System (INIS)

    Tabbó, Fabrizio; Barreca, Antonella; Piva, Roberto; Inghirami, Giorgio

    2012-01-01

    The discovery by Morris et al. (1994) of the genes contributing to the t(2;5)(p23;q35) translocation has laid the foundation for a molecular based recognition of anaplastic large cell lymphoma and highlighted the need for a further stratification of T-cell neoplasia. Likewise the detection of anaplastic lymphoma kinase (ALK) genetic lesions among many human cancers has defined unique subsets of cancer patients, providing new opportunities for innovative therapeutic interventions. The objective of this review is to appraise the molecular mechanisms driving ALK-mediated transformation, and to maintain the neoplastic phenotype. The understanding of these events will allow the design and implementation of novel tailored strategies for a well-defined subset of cancer patients.

  19. Identification and validation of novel small proteins in Pseudomonas putida

    DEFF Research Database (Denmark)

    Yang, Xiaochen; Ingemann Jensen, Sheila; Wulff, Tune

    2016-01-01

    Small proteins of fifty amino acids or less have been understudied due to difficulties that impede their annotation and detection. In order to obtain information on small open reading frames (sORFs) in P. putida, bioinformatic and proteomic approaches were used to identify putative small open...... reading frames (sORFs) in the well-characterized strain KT2440. A plasmid-based system was established for sORF validation, enabling expression of C-terminal sequential peptide affinity (SPA) tagged variants and their detection via protein immunoblotting. Out of 22 tested putative sORFs, the expression...... of fourteen sORFs was confirmed, where all except one are novel. All of the validated sORFs except one are located adjacent to annotated genes on the same strand and three are in close proximity to genes with known functions. These include an ABC transporter operon and the two transcriptional regulators Fis...

  20. Transcriptome Dynamics of Pseudomonas putida KT2440 under Water Stress

    DEFF Research Database (Denmark)

    Gülez, Gamze; Dechesne, Arnaud; Workman, Christopher

    2012-01-01

    Water deprivation can be a major stressor to microbial life in surface and subsurface soil. In unsaturated soils, the matric potential (Ψm) is often the main component of the water potential, which measures the thermodynamic availability of water. A low matric potential usually translates...... into water forming thin liquid films in the soil pores. Little is known of how bacteria respond to such conditions, where, in addition to facing water deprivation that might impair their metabolism, they have to adapt their dispersal strategy as swimming motility may be compromised. Using the pressurized...... porous surface model (PPSM), which allows creation of thin liquid films by controlling Ψm, we examined the transcriptome dynamics of Pseudomonas putida KT2440. We identified the differentially expressed genes in cells exposed to a mild matric stress (–0.4 MPa) for 4, 24, or 72 h. The major response...

  1. The Ssr protein (T1E_1405) from Pseudomonas putida DOT-T1E enables oligonucleotide-based recombineering in platform strain P. putida EM42

    DEFF Research Database (Denmark)

    Aparicio, Tomás; Ingemann Jensen, Sheila; Nielsen, Alex Toftgaard

    2016-01-01

    Some strains of the soil bacterium Pseudomonas putida have become in recent years platforms of choice for hosting biotransformations of industrial interest. Despite availability of many genetic tools for this microorganism, genomic editing of the cell factory P. putida EM42 (a derivative of refer......Some strains of the soil bacterium Pseudomonas putida have become in recent years platforms of choice for hosting biotransformations of industrial interest. Despite availability of many genetic tools for this microorganism, genomic editing of the cell factory P. putida EM42 (a derivative...

  2. Optimization of Pseudomonas putida KT2440 as host for the production of cis, cis-muconate from benzoate

    OpenAIRE

    Duuren, van, J.B.J.H.

    2011-01-01

    Optimization of Pseudomonas putida KT2440 as host for the production of cis, cis-muconate from benzoate P. putida KT2440 was used as biocatalyst given its versatile and energetically robust metabolism. Therefore, a mutant was generated and a process developed based on which a life cycle assessment (LCA) was performed. Additionally, the growth related parameters were experimentally obtained to constrain the metabolic model iJP815 further. The mutant Pseudomonas putida KT2440-JD1 was deri...

  3. Alk2/ACVR1 and Alk3/BMPR1A Provide Essential Function for Bone Morphogenetic Protein-Induced Retinal Angiogenesis.

    Science.gov (United States)

    Lee, Heon-Woo; Chong, Diana C; Ola, Roxana; Dunworth, William P; Meadows, Stryder; Ka, Jun; Kaartinen, Vesa M; Qyang, Yibing; Cleaver, Ondine; Bautch, Victoria L; Eichmann, Anne; Jin, Suk-Won

    2017-04-01

    Increasing evidence suggests that bone morphogenetic protein (BMP) signaling regulates angiogenesis. Here, we aimed to define the function of BMP receptors in regulating early postnatal angiogenesis by analysis of inducible, endothelial-specific deletion of the BMP receptor components Bmpr2 (BMP type 2 receptor), Alk1 (activin receptor-like kinase 1), Alk2 , and Alk3 in mouse retinal vessels. Expression analysis of several BMP ligands showed that proangiogenic BMP ligands are highly expressed in postnatal retinas. Consistently, BMP receptors are also strongly expressed in retina with a distinct pattern. To assess the function of BMP signaling in retinal angiogenesis, we first generated mice carrying an endothelial-specific inducible deletion of Bmpr2 . Postnatal deletion of Bmpr2 in endothelial cells substantially decreased the number of angiogenic sprouts at the vascular front and branch points behind the front, leading to attenuated radial expansion. To identify critical BMPR1s (BMP type 1 receptors) associated with BMPR2 in retinal angiogenesis, we generated endothelial-specific inducible deletion of 3 BMPR1s abundantly expressed in endothelial cells and analyzed the respective phenotypes. Among these, endothelial-specific deletion of either Alk2 / acvr1 or Alk3 / Bmpr1a caused a delay in radial expansion, reminiscent of vascular defects associated with postnatal endothelial-specific deletion of BMPR2, suggesting that ALK2/ACVR1 and ALK3/BMPR1A are likely to be the critical BMPR1s necessary for proangiogenic BMP signaling in retinal vessels. Our data identify BMP signaling mediated by coordination of ALK2/ACVR1, ALK3/BMPR1A, and BMPR2 as an essential proangiogenic cue for retinal vessels. © 2017 The Authors.

  4. Markerless gene knockout and integration to express heterologous biosynthetic gene clusters in Pseudomonas putida

    DEFF Research Database (Denmark)

    Choi, Kyeong Rok; Cho, Jae Sung; Cho, In Jin

    2018-01-01

    Pseudomonas putida has gained much interest among metabolic engineers as a workhorse for producing valuable natural products. While a few gene knockout tools for P. putida have been reported, integration of heterologous genes into the chromosome of P. putida, an essential strategy to develop stable...... plasmid curing systems, generating final strains free of antibiotic markers and plasmids. This markerless recombineering system for efficient gene knockout and integration will expedite metabolic engineering of P. putida, a bacterial host strain of increasing academic and industrial interest....

  5. Cytomorphological features of ALK-positive lung adenocarcinomas: psammoma bodies and signet ring cells.

    Science.gov (United States)

    Pareja, Fresia; Crapanzano, John P; Mansukhani, Mahesh M; Bulman, William A; Saqi, Anjali

    2015-03-01

    Correlation between histology and genotype has been described in lung adenocarcinomas. For example, studies have demonstrated that adenocarcinomas with an anaplastic lymphoma kinase (ALK) gene rearrangement may have mucinous features. The objective of the current study was to determine whether a similar association can be identified in cytological specimens. A retrospective search for ALK-rearranged cytopathology (CP) and surgical pathology (SP) lung carcinomas was conducted. Additional ALK-negative (-) lung adenocarcinomas served as controls. For CP and SP cases, the clinical data (i.e., age, sex, and smoking history), architecture, nuclear features, presence of mucin-containing cells (including signet ring cells), and any additional salient characteristics were evaluated. The search yielded 20 ALK-positive (+) adenocarcinomas. Compared with patients with ALK(-) lung adenocarcinomas (33 patients; 12 with epidermal growth factor receptor [EGFR]-mutation, 11 with Kristen rat sarcoma [KRAS]-mutation, and 10 wild-type adenocarcinomas), patients with ALK(+) adenocarcinoma presented at a younger age; and there was no correlation noted with sex or smoking status. The most common histological pattern in SP was papillary/micropapillary. Mucinous features were associated with ALK rearrangement in SP specimens. Signet ring cells and psammoma bodies were evident in and significantly associated with ALK(+) SP and CP specimens. However, psammoma bodies were observed in rare adenocarcinomas with an EGFR mutation. Both the ALK(+) and ALK(-) groups had mostly high nuclear grade. Salient features, including signet ring cells and psammoma bodies, were found to be significantly associated with ALK(+) lung adenocarcinomas and are identifiable on CP specimens. Recognizing these may be especially helpful in the molecular triage of scant CP samples. © 2014 American Cancer Society.

  6. Defective processing of methylated single-stranded DNA by E. coli alkB mutants

    Science.gov (United States)

    Dinglay, Suneet; Trewick, Sarah C.; Lindahl, Tomas; Sedgwick, Barbara

    2000-01-01

    Escherichia coli alkB mutants are very sensitive to DNA methylating agents. Despite these mutants being the subject of many studies, no DNA repair or other function has been assigned to the AlkB protein or to its human homolog. Here, we report that reactivation of methylmethanesulfonate (MMS)-treated single-stranded DNA phages, M13, f1, and G4, was decreased dramatically in alkB mutants. No such decrease occurred when using methylated λ phage or M13 duplex DNA. These data show that alkB mutants have a marked defect in processing methylation damage in single-stranded DNA. Recombinant AlkB protein bound more efficiently to single- than double-stranded DNA. The single-strand damage processed by AlkB was primarily cytotoxic and not mutagenic and was induced by SN2 methylating agents, MMS, DMS, and MeI but not by SN1 agent N-methyl-N-nitrosourea or by γ irradiation. Strains lacking other DNA repair activities, alkA tag, xth nfo, uvrA, mutS, and umuC, were not defective in reactivation of methylated M13 phage and did not enhance the defect of an alkB mutant. A recA mutation caused a small but additive defect. Thus, AlkB functions in a novel pathway independent of these activities. We propose that AlkB acts on alkylated single-stranded DNA in replication forks or at transcribed regions. Consistent with this theory, stationary phase alkB cells were less MMS sensitive than rapidly growing cells. PMID:10950872

  7. Degradation of phenanthrene and pyrene using genetically engineered dioxygenase producing Pseudomonas putida in soil

    Directory of Open Access Journals (Sweden)

    Mardani Gashtasb

    2016-01-01

    Full Text Available Bioremediation use to promote degradation and/or removal of contaminants into nonhazardous or less-hazardous substances from the environment using microbial metabolic ability. Pseudomonas spp. is one of saprotrophic soil bacterium and can be used for biodegradation of polycyclic aromatic hydrocarbons (PAHs but this activity in most species is weak. Phenanthrene and pyrene could associate with a risk of human cancer development in exposed individuals. The aim of the present study was application of genetically engineered P. putida that produce dioxygenase for degradation of phenanthrene and pyrene in spiked soil using high-performance liquid chromatography (HPLC method. The nahH gene that encoded catechol 2,3-dioxygenase (C23O was cloned into pUC18 and pUC18-nahH recombinant vector was generated and transformed into wild P. putida, successfully. The genetically modified and wild types of P. putida were inoculated in soil and pilot plan was prepared. Finally, degradation of phenanthrene and pyrene by this bacterium in spiked soil were evaluated using HPLC measurement technique. The results were showed elimination of these PAH compounds in spiked soil by engineered P. putida comparing to dishes containing natural soil with normal microbial flora and inoculated autoclaved soil by wild type of P. putida were statistically significant (p0.05 but it was few impact on this process (more than 2%. Additional and verification tests including catalase, oxidase and PCR on isolated bacteria from spiked soil were indicated that engineered P. putida was alive and functional as well as it can affect on phenanthrene and pyrene degradation via nahH gene producing. These findings indicated that genetically engineered P. putida generated in this work via producing C23O enzyme can useful and practical for biodegradation of phenanthrene and pyrene as well as petroleum compounds in polluted environments.

  8. The Chemistry of Alk-1-yn-1-yl DisulfidesA Review

    DEFF Research Database (Denmark)

    Senning, Alexander Erich Eugen

    2009-01-01

    The preparation and the properties of the elusive alk-1-yn-1-yl disulfides are reviewed, including the most recent quantum chemical findings with regard to their reactivity.......The preparation and the properties of the elusive alk-1-yn-1-yl disulfides are reviewed, including the most recent quantum chemical findings with regard to their reactivity....

  9. ALK1 heterozygosity delays development of late normal tissue damage in the irradiated mouse kidney

    International Nuclear Information System (INIS)

    Scharpfenecker, Marion; Floot, Ben; Korlaar, Regina; Russell, Nicola S.; Stewart, Fiona A.

    2011-01-01

    Background and Purpose: Activin receptor-like kinase 1 (ALK1) is a transforming growth factor β (TGF-β) receptor, which is mainly expressed in endothelial cells regulating proliferation and migration in vitro and angiogenesis in vivo. Endothelial cells also express the co-receptor endoglin, which modulates ALK1 effects on endothelial cells. Our previous studies showed that mice with reduced endoglin levels develop less irradiation-induced vascular damage and fibrosis, caused by an impaired inflammatory response. This study was aimed at investigating the role of ALK1 in late radiation toxicity. Material and Methods: Kidneys of ALK +/+ and ALK1 +/- mice were irradiated with 14 Gy. Mice were sacrificed at 10, 20, and 30 weeks after irradiation and gene expression and protein levels were analyzed. Results: Compared to wild type littermates, ALK1 +/- mice developed less inflammation and fibrosis at 20 weeks after irradiation, but displayed an increase in pro-inflammatory and pro-fibrotic gene expression at 30 weeks. In addition, ALK1 +/- mice showed superior vascular integrity at 10 and 20 weeks after irradiation which deteriorated at 30 weeks coinciding with changes in the VEGF pathway. Conclusions: ALK1 +/- mice develop a delayed normal tissue response by modulating the inflammatory response and growth factor expression after irradiation.

  10. Identification of CARS-ALK fusion in primary and metastatic lesions of an inflammatory myofibroblastic tumor

    NARCIS (Netherlands)

    Debelenko, LV; Arthur, DC; Pack, SD; Helman, LJ; Schrump, DS; Tsokos, M

    Inflammatory myofibroblastic tumor (IMT) is a rare childhood neoplasm. The natural history of this disease is poorly understood. Recently chromosomal rearrangements involving the anaplastic lymphoma kinase (ALK) gene have been implicated in this tumor. We have studied a case of ALK-positive soft

  11. Crizotinib-Resistant Mutants of EML4-ALK Identified Through an Accelerated Mutagenesis Screen

    Science.gov (United States)

    Zhang, Sen; Wang, Frank; Keats, Jeffrey; Zhu, Xiaotian; Ning, Yaoyu; Wardwell, Scott D; Moran, Lauren; Mohemmad, Qurish K; Anjum, Rana; Wang, Yihan; Narasimhan, Narayana I; Dalgarno, David; Shakespeare, William C; Miret, Juan J; Clackson, Tim; Rivera, Victor M

    2011-01-01

    Activating gene rearrangements of anaplastic lymphoma kinase (ALK) have been identified as driver mutations in non-small-cell lung cancer, inflammatory myofibroblastic tumors, and other cancers. Crizotinib, a dual MET/ALK inhibitor, has demonstrated promising clinical activity in patients with non-small-cell lung cancer and inflammatory myofibroblastic tumors harboring ALK translocations. Inhibitors of driver kinases often elicit kinase domain mutations that confer resistance, and such mutations have been successfully predicted using in vitro mutagenesis screens. Here, this approach was used to discover an extensive set of ALK mutations that can confer resistance to crizotinib. Mutations at 16 residues were identified, structurally clustered into five regions around the kinase active site, which conferred varying degrees of resistance. The screen successfully predicted the L1196M, C1156Y, and F1174L mutations, recently identified in crizotinib-resistant patients. In separate studies, we demonstrated that crizotinib has relatively modest potency in ALK-positive non-small-cell lung cancer cell lines. A more potent ALK inhibitor, TAE684, maintained substantial activity against mutations that conferred resistance to crizotinib. Our study identifies multiple novel mutations in ALK that may confer clinical resistance to crizotinib, suggests that crizotinib's narrow selectivity window may underlie its susceptibility to such resistance and demonstrates that a more potent ALK inhibitor may be effective at overcoming resistance. PMID:22034911

  12. ALK1 signaling inhibits angiogenesis by cooperating with the Notch pathway.

    Science.gov (United States)

    Larrivée, Bruno; Prahst, Claudia; Gordon, Emma; del Toro, Raquel; Mathivet, Thomas; Duarte, Antonio; Simons, Michael; Eichmann, Anne

    2012-03-13

    Activin receptor-like kinase 1 (ALK1) is an endothelial-specific member of the TGF-β/BMP receptor family that is inactivated in patients with hereditary hemorrhagic telangiectasia (HHT). How ALK1 signaling regulates angiogenesis remains incompletely understood. Here we show that ALK1 inhibits angiogenesis by cooperating with the Notch pathway. Blocking Alk1 signaling during postnatal development in mice leads to retinal hypervascularization and the appearance of arteriovenous malformations (AVMs). Combined blockade of Alk1 and Notch signaling further exacerbates hypervascularization, whereas activation of Alk1 by its high-affinity ligand BMP9 rescues hypersprouting induced by Notch inhibition. Mechanistically, ALK1-dependent SMAD signaling synergizes with activated Notch in stalk cells to induce expression of the Notch targets HEY1 and HEY2, thereby repressing VEGF signaling, tip cell formation, and endothelial sprouting. Taken together, these results uncover a direct link between ALK1 and Notch signaling during vascular morphogenesis that may be relevant to the pathogenesis of HHT vascular lesions. Copyright © 2012 Elsevier Inc. All rights reserved.

  13. Inhibition of Rac controls NPM–ALK-dependent lymphoma development and dissemination

    Energy Technology Data Exchange (ETDEWEB)

    Colomba, A [INSERM, U1048, Université Toulouse III, Toulouse, Institut des Maladies Métaboliques et Cardiovasculaires, Toulouse (France); Giuriato, S; Dejean, E [Centre de Recherches en Cancérologie de Toulouse, UMR1037-Université Toulouse III, IFR150-IFRBMT, Toulouse (France); Thornber, K [INSERM, U1048, Université Toulouse III, Toulouse, Institut des Maladies Métaboliques et Cardiovasculaires, Toulouse (France); Delsol, G [Centre de Recherches en Cancérologie de Toulouse, UMR1037-Université Toulouse III, IFR150-IFRBMT, Toulouse (France); Tronchère, H [INSERM, U1048, Université Toulouse III, Toulouse, Institut des Maladies Métaboliques et Cardiovasculaires, Toulouse (France); Meggetto, F [Centre de Recherches en Cancérologie de Toulouse, UMR1037-Université Toulouse III, IFR150-IFRBMT, Toulouse (France); Payrastre, B; Gaits-Iacovoni, F [INSERM, U1048, Université Toulouse III, Toulouse, Institut des Maladies Métaboliques et Cardiovasculaires, Toulouse (France)

    2011-06-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM–ALK) is a tyrosine kinase oncogene responsible for the pathogenesis of the majority of human ALK-positive lymphomas. We recently reported that it activated the Rac1 GTPase in anaplastic large-cell lymphoma (ALCL), leading to Rac-dependent formation of active invadopodia required for invasiveness. Herein, we went further into the study of this pathway and used the inhibitor of Rac, NSC23766, to validate its potential as a molecular target in ALCL in vitro and in vivo in a xenograft model and in a conditional model of NPM–ALK transgenic mice. Our data demonstrate that Rac regulates important effectors of NPM–ALK-induced transformation such as Erk1/2, p38 and Akt. Moreover, inhibition of Rac signaling abrogates NPM–ALK-elicited disease progression and metastasis in mice, highlighting the potential of small GTPases and their regulators as additional therapic targets in lymphomas.

  14. Inhibition of Rac controls NPM–ALK-dependent lymphoma development and dissemination

    International Nuclear Information System (INIS)

    Colomba, A; Giuriato, S; Dejean, E; Thornber, K; Delsol, G; Tronchère, H; Meggetto, F; Payrastre, B; Gaits-Iacovoni, F

    2011-01-01

    Nucleophosmin-anaplastic lymphoma kinase (NPM–ALK) is a tyrosine kinase oncogene responsible for the pathogenesis of the majority of human ALK-positive lymphomas. We recently reported that it activated the Rac1 GTPase in anaplastic large-cell lymphoma (ALCL), leading to Rac-dependent formation of active invadopodia required for invasiveness. Herein, we went further into the study of this pathway and used the inhibitor of Rac, NSC23766, to validate its potential as a molecular target in ALCL in vitro and in vivo in a xenograft model and in a conditional model of NPM–ALK transgenic mice. Our data demonstrate that Rac regulates important effectors of NPM–ALK-induced transformation such as Erk1/2, p38 and Akt. Moreover, inhibition of Rac signaling abrogates NPM–ALK-elicited disease progression and metastasis in mice, highlighting the potential of small GTPases and their regulators as additional therapic targets in lymphomas

  15. Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3

    International Nuclear Information System (INIS)

    Amano, Yosuke; Ishikawa, Rie; Sakatani, Toshio; Ichinose, Junji; Sunohara, Mitsuhiro; Watanabe, Kousuke; Kage, Hidenori; Nakajima, Jun; Nagase, Takahide; Ohishi, Nobuya; Takai, Daiya

    2015-01-01

    Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation. - Highlights: • TPM3-ALK fusion protein dimerizes through the coiled-coil structure of TPM3. • Longer coiled-coil structure of TPM3 leads to higher TPM3-ALK dimer formation. • Presence of TPM3-ALK dimer leads to ALK, STAT3, and ERK1/2 phosphorylation. • Presence of TPM3-ALK leads to loss of contact inhibition. • BN-PAGE is a simple technique for visualizing oncogenic dimerization

  16. Oncogenic TPM3-ALK activation requires dimerization through the coiled-coil structure of TPM3

    Energy Technology Data Exchange (ETDEWEB)

    Amano, Yosuke; Ishikawa, Rie; Sakatani, Toshio [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Ichinose, Junji [Department of Cardiothoracic Surgery, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Sunohara, Mitsuhiro; Watanabe, Kousuke; Kage, Hidenori [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Nakajima, Jun [Department of Cardiothoracic Surgery, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Nagase, Takahide; Ohishi, Nobuya [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Takai, Daiya, E-mail: dtakai-ind@umin.ac.jp [Department of Respiratory Medicine, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan); Department of Clinical Laboratory, The University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655 (Japan)

    2015-02-13

    Inflammatory myofibroblastic tumor (IMT) is a mesenchymal tumor that can arise from anywhere in the body. Anaplastic lymphoma kinase (ALK) gene rearrangements, most often resulting in the tropomyosin 3 (TPM3)-ALK fusion gene, are the main causes of IMT. However, the mechanism of malignant transformation in IMT has yet to be elucidated. The purpose of this study was to clarify the role of the TPM3 region in the transformation of IMT via TPM3-ALK. Lentivirus vectors containing a TPM3-ALK fusion gene lacking various lengths of TPM3 were constructed and expressed in HEK293T and NIH3T3 cell lines. Focus formation assay revealed loss of contact inhibition in NIH3T3 cells transfected with full-length TPM3-ALK, but not with ALK alone. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) revealed that TPM3-ALK dimerization increased in proportion to the length of TPM3. Western blot showed phosphorylation of ALK, ERK1/2, and STAT3 in HEK293T cells transfected with TPM3-ALK. Thus, the coiled-coil structure of TPM3 contributes to the transforming ability of the TPM3-ALK fusion protein, and longer TPM3 region leads to higher dimer formation. - Highlights: • TPM3-ALK fusion protein dimerizes through the coiled-coil structure of TPM3. • Longer coiled-coil structure of TPM3 leads to higher TPM3-ALK dimer formation. • Presence of TPM3-ALK dimer leads to ALK, STAT3, and ERK1/2 phosphorylation. • Presence of TPM3-ALK leads to loss of contact inhibition. • BN-PAGE is a simple technique for visualizing oncogenic dimerization.

  17. Colony morphology and transcriptome profiling of Pseudomonas putida KT2440 and its mutants deficient in alginate or all EPS synthesis under controlled matric potentials

    DEFF Research Database (Denmark)

    Gülez, Gamze; Altintas, Ali; Fazli, Mustafa

    2014-01-01

    Pseudomonas putida is a versatile bacterial species adapted to soil and its fluctuations. Like many other species living in soil, P. putida often faces water limitation. Alginate, an exopolysaccharide (EPS) produced by P. putida, is known to create hydrated environments and alleviate the effect o...

  18. Prevalence and natural history of ALK positive non-small-cell lung cancer and the clinical impact of targeted therapy with ALK inhibitors

    Directory of Open Access Journals (Sweden)

    Chia PL

    2014-11-01

    Full Text Available Puey Ling Chia,1 Paul Mitchell,1 Alexander Dobrovic,2–4 Thomas John1,2,4 1Department of Medical Oncology, Olivia-Newton John Cancer and Wellness Centre, Victoria, Australia; 2Ludwig Institute for Cancer Research, Austin Health, Victoria, Australia; 3Department of Pathology, University of Melbourne, Victoria, Australia; 4School of Cancer Medicine, La Trobe University, Victoria, Australia Abstract: Improved understanding of molecular drivers of carcinogenesis has led to significant progress in the management of lung cancer. Patients with non-small-cell lung cancer (NSCLC with anaplastic lymphoma kinase (ALK gene rearrangements constitute about 4%–5% of all NSCLC patients. ALK+ NSCLC cells respond well to small molecule ALK inhibitors such as crizotinib; however, resistance invariably develops after several months of treatment. There are now several newer ALK inhibitors, with the next generation of agents targeting resistance mutations. In this review, we will discuss the prevalence and clinical characteristics of ALK+ lung cancer, current treatment options, and future directions in the management of this subset of NSCLC patients. Keywords: anaplastic lymphoma kinase (ALK, gene rearrangements, lung cancer, kinase inhibitors, lung adenocarcinoma

  19. Effect of additional carbon source on naphthalene biodegradation by Pseudomonas putida G7

    International Nuclear Information System (INIS)

    Lee, Kangtaek; Park, Jin-Won; Ahn, Ik-Sung

    2003-01-01

    Addition of a carbon source as a nutrient into soil is believed to enhance in situ bioremediation by stimulating the growth of microorganisms that are indigenous to the subsurface and are capable of degrading contaminants. However, it may inhibit the biodegradation of organic contaminants and result in diauxic growth. The objective of this work is to study the effect of pyruvate as another carbon source on the biodegradation of polynuclear aromatic hydrocarbons (PAHs). In this study, naphthalene was used as a model PAH, ammonium sulfate as a nitrogen source, and oxygen as an electron acceptor. Pseudomonas putida G7 was used as a model naphthalene-degrading microorganism. From a chemostat culture, the growth kinetics of P. putida G7 on pyruvate was determined. At concentrations of naphthalene and pyruvate giving similar growth rates of P. putida G7, diauxic growth of P. putida G7 was not observed. It is suggested that pyruvate does not inhibit naphthalene biodegradation and can be used as an additional carbon source to stimulate the growth of P. putida G7 that can degrade polynuclear aromatic hydrocarbons

  20. Benzoxazinoids in root exudates of maize attract Pseudomonas putida to the rhizosphere.

    Directory of Open Access Journals (Sweden)

    Andrew L Neal

    Full Text Available Benzoxazinoids, such as 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H-one (DIMBOA, are secondary metabolites in grasses. In addition to their function in plant defence against pests and diseases above-ground, benzoxazinoids (BXs have also been implicated in defence below-ground, where they can exert allelochemical or antimicrobial activities. We have studied the impact of BXs on the interaction between maize and Pseudomonas putida KT2440, a competitive coloniser of the maize rhizosphere with plant-beneficial traits. Chromatographic analyses revealed that DIMBOA is the main BX compound in root exudates of maize. In vitro analysis of DIMBOA stability indicated that KT2440 tolerance of DIMBOA is based on metabolism-dependent breakdown of this BX compound. Transcriptome analysis of DIMBOA-exposed P. putida identified increased transcription of genes controlling benzoate catabolism and chemotaxis. Chemotaxis assays confirmed motility of P. putida towards DIMBOA. Moreover, colonisation essays in soil with Green Fluorescent Protein (GFP-expressing P. putida showed that DIMBOA-producing roots of wild-type maize attract significantly higher numbers of P. putida cells than roots of the DIMBOA-deficient bx1 mutant. Our results demonstrate a central role for DIMBOA as a below-ground semiochemical for recruitment of plant-beneficial rhizobacteria during the relatively young and vulnerable growth stages of maize.

  1. The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) is enhanced by NPM-ALK

    DEFF Research Database (Denmark)

    Boccalatte, Francesco E; Voena, Claudia; Riganti, Chiara

    2009-01-01

    . A well-defined set of ALK-associated tyrosine phospho-peptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins was identified. Validation studies confirmed that VASP and ATIC associated with NPM-ALK and their phosphorylation required ALK activity. ATIC phosphorylation was also...... documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampering the methotrexate-mediated transformylase activity inhibition...

  2. Optimization of Pseudomonas putida KT2440 as host for the production of cis, cis-muconate from benzoate

    NARCIS (Netherlands)

    Duuren, van J.B.J.H.

    2011-01-01

    Optimization of Pseudomonas putida KT2440 as host for the production of cis, cis-muconate

    from benzoate P. putida KT2440 was used as biocatalyst given its versatile and energetically robust metabolism.

    Therefore, a mutant was generated and a process developed based on which a

  3. ALK and TGF-Beta Resistance in Breast Cancer

    Science.gov (United States)

    2017-10-01

    Award Number: W81XWH‐15‐1‐0650 TITLE: ALK and TGF-Beta Resistance in Breast Cancer PRINCIPAL INVESTIGATOR: Xin-Hua Feng CONTRACTING...and TGF-Beta Resistance in Breast Cancer 5b. GRANT NUMBER W81XWH‐15‐1‐0650 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Xin-Hua Feng...response is a hallmark in human cancer . However, the mechanisms underlying TGF- resistance in breast cancer have not been elucidated. Anaplastic

  4. Cometabolic degradation kinetics of TCE and phenol by Pseudomonas putida.

    Science.gov (United States)

    Chen, Yan-Min; Lin, Tsair-Fuh; Huang, Chih; Lin, Jui-Che

    2008-08-01

    Modeling of cometabolic kinetics is important for better understanding of degradation reaction and in situ application of bio-remediation. In this study, a model incorporated cell growth and decay, loss of transformation activity, competitive inhibition between growth substrate and non-growth substrate and self-inhibition of non-growth substrate was proposed to simulate the degradation kinetics of phenol and trichloroethylene (TCE) by Pseudomonas putida. All the intrinsic parameters employed in this study were measured independently, and were then used for predicting the batch experimental data. The model predictions conformed well to the observed data at different phenol and TCE concentrations. At low TCE concentrations (TCE concentrations (>6 mg l(-1)), only the model considering self-inhibition can describe the experimental data, suggesting that a self-inhibition of TCE was present in the system. The proposed model was also employed in predicting the experimental data conducted in a repeated batch reactor, and good agreements were observed between model predictions and experimental data. The results also indicated that the biomass loss in the degradation of TCE below 2 mg l(-1) can be totally recovered in the absence of TCE for the next cycle, and it could be used for the next batch experiment for the degradation of phenol and TCE. However, for higher concentration of TCE (>6 mg l(-1)), the recovery of biomass may not be as good as that at lower TCE concentrations.

  5. Responses of KT2440 Pseudomonas putida to mild water stress

    DEFF Research Database (Denmark)

    Gülez, Gamze

    betydningen af flagellær motilitet og produktion af EPS under matric stess undersøgt ved brug af den såkaldte Porous Surface Model (PSM), som genererer væske-filmens effekter ved kontrol af !m. Det blev vist, at flagellær motilitet var begrænset under matric stress; Pseudomonas putida KT2440 kolonier udviste...... væsentlig højere vækstrater tæt ved mætningsbetingelser (-0.5 kPa !m) end kolonier dyrket under matric stress (ved -3.6 kPa !m). Desuden viste 1:1 kompettitive eksperimenter med begge phenotyper ved mættede betingelser, at den naturligt forekomne KT2440 udkonkurrerer dens ikke-flagellare mutant med hensyn...... til overflade-kolonivækst, hvorimod begge phenotyper sameksisterer under matric stress (-3.6 kPa). Denne afhandling har endvidere vist at bakterierne kunne drage fordel af pludseligt forekomne mættede betingelser (5 minutter, to gange dagligt) idet flagellær motilitet blev observeret. Dette resultat...

  6. Pseudomonas putida CSV86: a candidate genome for genetic bioaugmentation.

    Directory of Open Access Journals (Sweden)

    Vasundhara Paliwal

    Full Text Available Pseudomonas putida CSV86, a plasmid-free strain possessing capability to transfer the naphthalene degradation property, has been explored for its metabolic diversity through genome sequencing. The analysis of draft genome sequence of CSV86 (6.4 Mb revealed the presence of genes involved in the degradation of naphthalene, salicylate, benzoate, benzylalcohol, p-hydroxybenzoate, phenylacetate and p-hydroxyphenylacetate on the chromosome thus ensuring the stability of the catabolic potential. Moreover, genes involved in the metabolism of phenylpropanoid and homogentisate, as well as heavy metal resistance, were additionally identified. Ability to grow on vanillin, veratraldehyde and ferulic acid, detection of inducible homogentisate dioxygenase and growth on aromatic compounds in the presence of heavy metals like copper, cadmium, cobalt and arsenic confirm in silico observations reflecting the metabolic versatility. In silico analysis revealed the arrangement of genes in the order: tRNA(Gly, integrase followed by nah operon, supporting earlier hypothesis of existence of a genomic island (GI for naphthalene degradation. Deciphering the genomic architecture of CSV86 for aromatic degradation pathways and identification of elements responsible for horizontal gene transfer (HGT suggests that genetic bioaugmentation strategies could be planned using CSV86 for effective bioremediation.

  7. The Crc protein inhibits the production of polyhydroxyalkanoates in Pseudomonas putida under balanced carbon/nitrogen growth conditions.

    Science.gov (United States)

    La Rosa, Ruggero; de la Peña, Fernando; Prieto, María Axiliadora; Rojo, Fernando

    2014-01-01

    Pseudomonas putida synthesizes polyhydroxyalkanoates (PHAs) as storage compounds. PHA synthesis is more active when the carbon source is in excess and the nitrogen source is limiting, but can also occur at a lower rate under balanced carbon/nitrogen ratios. This work shows that PHA synthesis is controlled by the Crc global regulator, a protein that optimizes carbon metabolism by inhibiting the expression of genes involved in the use of non-preferred carbon sources. Crc acts post-transcriptionally. The mRNAs of target genes contain characteristic catabolite activity (CA) motifs near the ribosome binding site. Sequences resembling CA motifs can be predicted for the phaC1 gene, which codes for a PHA polymerase, and for phaI and phaF, which encode proteins associated to PHA granules. Our results show that Crc inhibits the translation of phaC1 mRNA, but not that of phaI or phaF, reducing the amount of PHA accumulated in the cell. Crc inhibited PHA synthesis during exponential growth in media containing a balanced carbon/nitrogen ratio. No inhibition was seen when the carbon/nitrogen ratio was imbalanced. This extends the role of Crc beyond that of controlling the hierarchical utilization of carbon sources and provides a link between PHA synthesis and the global regulatory networks controlling carbon flow. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

  8. KLC1-ALK: a novel fusion in lung cancer identified using a formalin-fixed paraffin-embedded tissue only.

    Directory of Open Access Journals (Sweden)

    Yuki Togashi

    Full Text Available The promising results of anaplastic lymphoma kinase (ALK inhibitors have changed the significance of ALK fusions in several types of cancer. These fusions are no longer mere research targets or diagnostic markers, but they are now directly linked to the therapeutic benefit of patients. However, most available tumor tissues in clinical settings are formalin-fixed and paraffin-embedded (FFPE, and this significantly limits detailed genetic studies in many clinical cases. Although recent technical improvements have allowed the analysis of some known mutations in FFPE tissues, identifying unknown fusion genes by using only FFPE tissues remains difficult. We developed a 5'-rapid amplification of cDNA ends-based system optimized for FFPE tissues and evaluated this system on a lung cancer tissue with ALK rearrangement and without the 2 known ALK fusions EML4-ALK and KIF5B-ALK. With this system, we successfully identified a novel ALK fusion, KLC1-ALK. The result was confirmed by reverse transcription-polymerase chain reaction and fluorescence in situ hybridization. Then, we synthesized the putative full-length cDNA of KLC1-ALK and demonstrated the transforming potential of the fusion kinase with assays using mouse 3T3 cells. To the best of our knowledge, KLC1-ALK is the first novel oncogenic fusion identified using only FFPE tissues. This finding will broaden the potential value of archival FFPE tissues and provide further biological and clinical insights into ALK-positive lung cancer.

  9. Epithelioid fibrous histiocytoma: molecular characterization of ALK fusion partners in 23 cases.

    Science.gov (United States)

    Dickson, Brendan C; Swanson, David; Charames, George S; Fletcher, Christopher Dm; Hornick, Jason L

    2018-05-01

    Epithelioid fibrous histiocytoma is a rare and distinctive cutaneous neoplasm. Most cases harbor ALK rearrangement and show ALK overexpression, which distinguish this neoplasm from conventional cutaneous fibrous histiocytoma and variants. SQSTM1 and VCL have previously been shown to partner with ALK in one case each of epithelioid fibrous histiocytoma. The purpose of this study was to examine a large cohort of epithelioid fibrous histiocytomas by next-generation sequencing to characterize the nature and prevalence of ALK fusion partners. A retrospective archival review was performed to identify cases of epithelioid fibrous histiocytoma (2012-2016). Immunohistochemistry was performed to confirm ALK expression. Targeted next-generation sequencing was applied on RNA extracted from formalin-fixed paraffin-embedded tissue to identify the fusion partners. Twenty-three cases fulfilled inclusion criteria. The mean patient age was 39 years (range, 8-74), there was no sex predilection, and >75% of cases involved the lower extremities. The most common gene fusions were SQSTM1-ALK (N=12; 52%) and VCL-ALK (N=7; 30%); the other four cases harbored novel fusion partners (DCTN1, ETV6, PPFIBP1, and SPECC1L). The pattern of ALK immunoreactivity was usually granular cytoplasmic (N=12; 52%) or granular cytoplasmic and nuclear (N=10; 43%); the case containing an ETV6 fusion partner showed nuclear staining alone. There was no apparent relationship between tumor morphology and the ALK fusion partner. In summary, SQSTM1 and VCL are the most common ALK fusion partners in epithelioid fibrous histiocytoma; DCTN1, ETV6, PPFIBP1, and SPECC1L represent rare fusion partners. The proteins encoded by these genes play diverse roles in scaffolding, cell adhesion, signaling, and transcription (among others) without clear commonalities. These findings expand the oncogenic promiscuity of many of these ALK fusion genes, which drive neoplasia in tumors of diverse lineages with widely varied clinical

  10. Marine Pseudomonas putida: a potential source of antimicrobial substances against antibiotic-resistant bacteria

    Directory of Open Access Journals (Sweden)

    Palloma Rodrigues Marinho

    2009-08-01

    Full Text Available Bacteria isolated from marine sponges found off the coast of Rio de Janeiro, Brazil, were screened for the production of antimicrobial substances. We report a new Pseudomonas putida strain (designated P. putida Mm3 isolated from the sponge Mycale microsigmatosa that produces a powerful antimicrobial substance active against multidrug-resistant bacteria. P. putida Mm3 was identified on the basis of 16S rRNA gene sequencing and phenotypic tests. Molecular typing for Mm3 was performed by RAPD-PCR and comparison of the results to other Pseudomonas strains. Our results contribute to the search for new antimicrobial agents, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.

  11. Biotransformation of isoeugenol to vanillin by Pseudomonas putida IE27 cells.

    Science.gov (United States)

    Yamada, Mamoru; Okada, Yukiyoshi; Yoshida, Toyokazu; Nagasawa, Toru

    2007-01-01

    The ability to produce vanillin and/or vanillic acid from isoeugenol was screened using resting cells of various bacteria. The vanillin- and/or vanillic-acid-producing activities were observed in strains belonging to the genera Achromobacter, Aeromonas, Agrobacerium, Alcaligenes, Arthrobacter, Bacillus, Micrococcus, Pseudomonas, Rhodobacter, and Rhodococcus. Strain IE27, a soil isolate showing the highest vanillin-producing activity, was identified as Pseudomonas putida. We optimized the culture and reaction conditions for vanillin production from isoeugenol using P. putida IE27 cells. The vanillin-producing activity was induced by adding isoeugenol to the culture medium but not vanillin or eugenol. Under the optimized reaction conditions, P. putida IE27 cells produced 16.1 g/l vanillin from 150 mM isoeugenol, with a molar conversion yield of 71% at 20 degrees C after a 24-h incubation in the presence of 10% (v/v) dimethyl sulfoxide.

  12. Marine Pseudomonas putida: a potential source of antimicrobial substances against antibiotic-resistant bacteria.

    Science.gov (United States)

    Marinho, Palloma Rodrigues; Moreira, Ana Paula Barbosa; Pellegrino, Flávia Lúcia Piffano Costa; Muricy, Guilherme; Bastos, Maria do Carmo de Freire; Santos, Kátia Regina Netto dos; Giambiagi-deMarval, Marcia; Laport, Marinella Silva

    2009-08-01

    Bacteria isolated from marine sponges found off the coast of Rio de Janeiro, Brazil, were screened for the production of antimicrobial substances. We report a new Pseudomonas putida strain (designated P. putida Mm3) isolated from the sponge Mycale microsigmatosa that produces a powerful antimicrobial substance active against multidrug-resistant bacteria. P. putida Mm3 was identified on the basis of 16S rRNA gene sequencing and phenotypic tests. Molecular typing for Mm3 was performed by RAPD-PCR and comparison of the results to other Pseudomonas strains. Our results contribute to the search for new antimicrobial agents, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.

  13. Novel covalent modification of human anaplastic lymphoma kinase (ALK and potentiation of crizotinib-mediated inhibition of ALK activity by BNP7787

    Directory of Open Access Journals (Sweden)

    Parker AR

    2015-02-01

    Full Text Available Aulma R Parker,1 Pavankumar N Petluru,1 Vicki L Nienaber,2 Min Zhao,1 Philippe Y Ayala,1 John Badger,2 Barbara Chie-Leon,2 Vandana Sridhar,2 Cheyenne Logan,2 Harry Kochat,1 Frederick H Hausheer1 1BioNumerik Pharmaceuticals, Inc., San Antonio, TX, USA; 2Zenobia Therapeutics, Inc., La Jolla, CA, USA Abstract: BNP7787 (Tavocept, disodium 2,2’-dithio-bis-ethanesulfonate is a novel, investigational, water-soluble disulfide that is well-tolerated and nontoxic. In separate randomized multicenter Phase II and Phase III clinical trials in non-small-cell lung cancer (NSCLC patients, treatment with BNP7787 in combination with standard chemotherapy resulted in substantial increases in the overall survival of patients with advanced adenocarcinoma of the lung in the first-line treatment setting. We hypothesized that BNP7787 might interact with and modify human anaplastic lymphoma kinase (ALK. At least seven different variants of ALK fusions with the gene encoding the echinoderm microtubule-associated protein-like 4 (EML4 are known to occur in NSCLC. EML4–ALK fusions are thought to account for approximately 3% of NSCLC cases. Herein, we report the covalent modification of the kinase domain of human ALK by a BNP7787-derived mesna moiety and the functional consequences of this modification in ALK assays evaluating kinase activity. The kinase domain of the ALK protein crystallizes as a monomer, and BNP7787-derived mesna-cysteine adducts were observed at Cys 1235 and Cys 1156. The BNP7787-derived mesna adduct at Cys 1156 is located in close proximity to the active site and results in substantial disorder of the P-loop and activation loop (A-loop. Comparison with the P-loop of apo-ALK suggests that the BNP7787-derived mesna adduct at Cys 1156 interferes with the positioning of Phe 1127 into a small pocket now occupied by mesna, resulting in a destabilization of the loop's binding orientation. Additionally, in vitro kinase activity assays indicate that BNP7787

  14. Performance of a RT-PCR Assay in Comparison to FISH and Immunohistochemistry for the Detection of ALK in Non-Small Cell Lung Cancer.

    Science.gov (United States)

    Hout, David R; Schweitzer, Brock L; Lawrence, Kasey; Morris, Stephan W; Tucker, Tracy; Mazzola, Rosetta; Skelton, Rachel; McMahon, Frank; Handshoe, John; Lesperance, Mary; Karsan, Aly; Saltman, David L

    2017-08-01

    Patients with lung cancers harboring an activating anaplastic lymphoma kinase ( ALK ) rearrangement respond favorably to ALK inhibitor therapy. Fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) are validated and widely used screening tests for ALK rearrangements but both methods have limitations. The ALK RGQ RT-PCR Kit (RT-PCR) is a single tube quantitative real-time PCR assay for high throughput and automated interpretation of ALK expression. In this study, we performed a direct comparison of formalin-fixed paraffin-embedded (FFPE) lung cancer specimens using all three ALK detection methods. The RT-PCR test (diagnostic cut-off Δ C t of ≤8) was shown to be highly sensitive (100%) when compared to FISH and IHC. Sequencing of RNA detected full-length ALK transcripts or EML4-ALK and KIF5B-ALK fusion variants in discordant cases in which ALK expression was detected by the ALK RT-PCR test but negative by FISH and IHC. The overall specificity of the RT-PCR test for the detection of ALK in cases without full-length ALK expression was 94% in comparison to FISH and sequencing. These data support the ALK RT-PCR test as a highly efficient and reliable diagnostic screening approach to identify patients with non-small cell lung cancer whose tumors are driven by oncogenic ALK.

  15. Automation of ALK gene rearrangement testing with fluorescence in situ hybridization (FISH): a feasibility study.

    Science.gov (United States)

    Zwaenepoel, Karen; Merkle, Dennis; Cabillic, Florian; Berg, Erica; Belaud-Rotureau, Marc-Antoine; Grazioli, Vittorio; Herelle, Olga; Hummel, Michael; Le Calve, Michele; Lenze, Dido; Mende, Stefanie; Pauwels, Patrick; Quilichini, Benoit; Repetti, Elena

    2015-02-01

    In the past several years we have observed a significant increase in our understanding of molecular mechanisms that drive lung cancer. Specifically in the non-small cell lung cancer sub-types, ALK gene rearrangements represent a sub-group of tumors that are targetable by the tyrosine kinase inhibitor Crizotinib, resulting in significant reductions in tumor burden. Phase II and III clinical trials were performed using an ALK break-apart FISH probe kit, making FISH the gold standard for identifying ALK rearrangements in patients. FISH is often considered a labor and cost intensive molecular technique, and in this study we aimed to demonstrate feasibility for automation of ALK FISH testing, to improve laboratory workflow and ease of testing. This involved automation of the pre-treatment steps of the ALK assay using various protocols on the VP 2000 instrument, and facilitating automated scanning of the fluorescent FISH specimens for simplified enumeration on various backend scanning and analysis systems. The results indicated that ALK FISH can be automated. Significantly, both the Ikoniscope and BioView system of automated FISH scanning and analysis systems provided a robust analysis algorithm to define ALK rearrangements. In addition, the BioView system facilitated consultation of difficult cases via the internet. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Comparative analysis of clinicoradiologic characteristics of lung adenocarcinomas with ALK rearrangements or EGFR mutations

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, J.Y.; Zheng, J.; Chen, X.; Zhou, J.Y. [Zhejiang University, Department of Respiratory Disease, Thoracic Disease Center, First Affiliated Hospital, College of Medicine, Hangzhou (China); Yu, Z.F.; Xiao, W.B.; Jiang, L.N. [Zhejiang University, Department of Radiology, First Affiliated Hospital, College of Medicine, Hangzhou (China); Zhao, J.; Sun, K.; Wang, B.; Ding, W. [Zhejiang University, Department of Pathology, First Affiliated Hospital, College of Medicine, Hangzhou (China)

    2015-05-01

    To compare the clinicoradiologic features of tumours with echinoderm anaplastic lymphoma kinase (ALK) rearrangements, epidermal growth factor receptor (EGFR) mutations, or wild type (WT) for both genes in a cohort of patients with lung adenocarcinoma to identify useful characteristics of different gene statuses. In 346 lung adenocarcinoma patients, ALK rearrangements were confirmed with fluorescence in situ hybridisation, and EGFR mutations were determined by pyrosequencing assay. Patients were divided into three groups: ALK rearrangement (ALK+ group, n = 48), EGFR mutation (EGFR+ group, n = 166), and WT for both genes (WT group, n = 132). Chest computed tomography (CT) examinations were performed in all patients. The percentages of ground-glass opacity volume (pGGO) and tumour shadow disappearance rate (TDR) were measured using semi-automated nodule assessment software. The pGGO was significantly lower in the ALK+ group (25.1 % ± 24.3) than in the EGFR+ group (37.2 % ± 25.7, p < 0.001) and the WT group (36.1 % ± 24.6, p = 0.001). The TDR in the ALK+ group (17.3 % ± 25.1) was significantly lower than in the EGFR+ group (26.8 % ± 24.9, p = 0.002) and the WT group (25.7 % ± 24.6, p = 0.003). Solid pattern with lower incidence of lobulated border, finely spiculated margins, pleural retraction, and bubble-like lucency on CT imaging are the main characteristics of ALK rearrangement tumours. (orig.)

  17. Expression analysis of the fpr (ferredoxin-NADP+ reductase) gene in Pseudomonas putida KT2440

    International Nuclear Information System (INIS)

    Lee, Yunho; Pena-Llopis, Samuel; Kang, Yoon-Suk; Shin, Hyeon-Dong; Demple, Bruce; Madsen, Eugene L.; Jeon, Che Ok; Park, Woojun

    2006-01-01

    The ferredoxin-NADP + reductase (fpr) participates in cellular defense against oxidative damage. The fpr expression in Pseudomonas putida KT2440 is induced by oxidative and osmotic stresses. FinR, a LysR-type transcriptional factor near the fpr gene in the P. putida KT2440 genome, is required for induction of the fpr under both conditions. We have shown that the fpr and finR gene products can counteract the effects of oxidative and osmotic stresses. Interestingly, FinR-independent expression occurs either during a long period of incubation with paraquat or with high concentrations of oxidative stress agent. This result indicates that there may be additional regulators present in the P. putida KT2440 genome. In contrast to in vivo expression kinetics of fpr from the plant pathogen, Pseudomonas syringae, the fpr gene from P. putida KT2440 exhibited unusually prolonged expression after oxidative stress. Transcriptional fusion and Northern blot analysis studies indicated that the FinR is negatively autoregulated. Expression of the fpr promoter was higher in minimal media than in rich media during exponential phase growth. Consistent with this result, the fpr and finR mutants had a long lag phase in minimal media in contrast to wild-type growth characteristics. Antioxidants such as ascorbate could increase the growth rate of all tested strains in minimal media. This result confirmed that P. putida KT2440 experienced more oxidative stress during exponential growth in minimal media than in rich media. Endogenous promoter activity of the fpr gene is much higher during exponential growth than during stationary growth. These findings demonstrate new relationships between fpr, finR, and the physiology of oxidative stress in P. putida KT2440

  18. Involvement of Pseudomonas putida RpoN sigma factor in regulation of various metabolic functions.

    OpenAIRE

    Köhler, T; Harayama, S; Ramos, J L; Timmis, K N

    1989-01-01

    The RpoN protein was originally identified in Escherichia coli as a sigma (sigma) factor essential for the expression of nitrogen regulons. In the present study we cloned the Pseudomonas putida rpoN gene and identified its gene product as a protein with an apparent molecular weight of 78,000. A mutant rpoN gene was constructed by in vitro insertion mutagenesis with a kanamycin cassette. A P. putida rpoN mutant was then isolated by replacement of the intact chromosomal rpoN gene by the mutant ...

  19. Metal Inhibition of Growth and Manganese Oxidation in Pseudomonas putida GB-1

    Science.gov (United States)

    Pena, J.; Sposito, G.

    2009-12-01

    Biogenic manganese oxides (MnO2) are ubiquitous nanoparticulate minerals that contribute to the adsorption of nutrient and toxicant metals, the oxidative degradation of various organic compounds, and the respiration of metal-reducing bacteria in aquatic and terrestrial environments. The formation of these minerals is catalyzed by a diverse and widely-distributed group of bacteria and fungi, often through the enzymatic oxidation of aqueous Mn(II) to Mn(IV). In metal-impacted ecosystems, toxicant metals may alter the viability and metabolic activity of Mn-oxidizing organisms, thereby limiting the conditions under which biogenic MnO2 can form and diminishing their potential as adsorbent materials. Pseudomonas putida GB-1 (P. putida GB-1) is a model Mn-oxidizing laboratory culture representative of freshwater and soil biofilm-forming bacteria. Manganese oxidation in P. putida GB-1 occurs via two single-electron-transfer reactions, involving a multicopper oxidase enzyme found on the bacterial outer membrane surface. Near the onset of the stationary phase of growth, dark brown MnO2 particles are deposited in a matrix of bacterial cells and extracellular polymeric substances, thus forming heterogeneous biomineral assemblages. In this study, we assessed the influence of various transition metals on microbial growth and manganese oxidation capacity in a P. putida GB-1 culture propagated in a nutrient-rich growth medium. The concentration-response behavior of actively growing P. putida GB-1 cells was investigated for Fe, Co, Ni, Cu and Zn at pH ≈ 6 in the presence and absence of 1 mM Mn. Toxicity parameters such as EC0, EC50 and Hillslope, and EC100 were obtained from the sigmoidal concentration-response curves. The extent of MnO2 formation in the presence of the various metal cations was documented 24, 50, 74 and 104 h after the metal-amended medium was inoculated. Toxicity values were compared to twelve physicochemical properties of the metals tested. Significant

  20. The heat shock protein-90 co-chaperone, Cyclophilin 40, promotes ALK-positive, anaplastic large cell lymphoma viability and its expression is regulated by the NPM-ALK oncoprotein

    International Nuclear Information System (INIS)

    Pearson, Joel D; Mohammed, Zubair; Bacani, Julinor T C; Lai, Raymond; Ingham, Robert J

    2012-01-01

    Anaplastic lymphoma kinase-positive, anaplastic large cell lymphoma (ALK+ ALCL) is a T cell lymphoma defined by the presence of chromosomal translocations involving the ALK tyrosine kinase gene. These translocations generate fusion proteins (e.g. NPM-ALK) with constitutive tyrosine kinase activity, which activate numerous signalling pathways important for ALK+ ALCL pathogenesis. The molecular chaperone heat shock protein-90 (Hsp90) plays a critical role in allowing NPM-ALK and other signalling proteins to function in this lymphoma. Co-chaperone proteins are important for helping Hsp90 fold proteins and for directing Hsp90 to specific clients; however the importance of co-chaperone proteins in ALK+ ALCL has not been investigated. Our preliminary findings suggested that expression of the immunophilin co-chaperone, Cyclophilin 40 (Cyp40), is up-regulated in ALK+ ALCL by JunB, a transcription factor activated by NPM-ALK signalling. In this study we examined the regulation of the immunophilin family of co-chaperones by NPM-ALK and JunB, and investigated whether the immunophilin co-chaperones promote the viability of ALK+ ALCL cell lines. NPM-ALK and JunB were knocked-down in ALK+ ALCL cell lines with siRNA, and the effect on the expression of the three immunophilin co-chaperones: Cyp40, FK506-binding protein (FKBP) 51, and FKBP52 examined. Furthermore, the effect of knock-down of the immunophilin co-chaperones, either individually or in combination, on the viability of ALK+ ALCL cell lines and NPM-ALK levels and activity was also examined. We found that NPM-ALK promoted the transcription of Cyp40 and FKBP52, but only Cyp40 transcription was promoted by JunB. We also observed reduced viability of ALK+ ALCL cell lines treated with Cyp40 siRNA, but not with siRNAs directed against FKBP52 or FKBP51. Finally, we demonstrate that the decrease in the viability of ALK+ ALCL cell lines treated with Cyp40 siRNA does not appear to be due to a decrease in NPM-ALK levels or the

  1. Inconsistent results in the analysis of ALK rearrangements in non-small cell lung cancer

    International Nuclear Information System (INIS)

    Mattsson, Johanna S. M.; Brunnström, Hans; Jabs, Verena; Edlund, Karolina; Jirström, Karin; Mindus, Stephanie; Fleur, Linnéa la; Pontén, Fredrik; Karlsson, Mats G.; Karlsson, Christina; Koyi, Hirsh; Brandén, Eva; Botling, Johan; Helenius, Gisela; Micke, Patrick; Svensson, Maria A.

    2016-01-01

    Identification of targetable EML4-ALK fusion proteins has revolutionized the treatment of a minor subgroup of non-small cell lung cancer (NSCLC) patients. Although fluorescence in situ hybridization (FISH) is regarded as the gold standard for detection of ALK rearrangements, ALK immunohistochemistry (IHC) is often used as screening tool in clinical practice. In order to unbiasedly analyze the diagnostic impact of such a screening strategy, we compared ALK IHC with ALK FISH in three large representative Swedish NSCLC cohorts incorporating clinical parameters and gene expression data. ALK rearrangements were detected using FISH on tissue microarrays (TMAs), including tissue from 851 NSCLC patients. In parallel, ALK protein expression was detected using IHC, applying the antibody clone D5F3 with two different protocols (the FDA approved Ventana CDx assay and our in house Dako IHC protocol). Gene expression microarray data (Affymetrix) was available for 194 patients. ALK rearrangements were detected in 1.7 % in the complete cohort and 2.0 % in the non-squamous cell carcinoma subgroup. ALK protein expression was observed in 1.8 and 1.4 % when applying the Ventana assay or the in house Dako protocol, respectively. The specificity and accuracy of IHC was high (> 98 %), while the sensitivity was between 69 % (Ventana) and 62 % (in house Dako protocol). Furthermore, only 67 % of the ALK IHC positive cases were positive with both IHC assays. Gene expression analysis revealed that 6/194 (3 %) tumors showed high ALK gene expression (≥ 6 AU) and of them only three were positive by either FISH or IHC. The overall frequency of ALK rearrangements based on FISH was lower than previously reported. The sensitivity of both IHC assays was low, and the concordance between the FISH and the IHC assays poor, questioning current strategies to screen with IHC prior to FISH or completely replace FISH by IHC. The online version of this article (doi:10.1186/s12885-016-2646-x) contains

  2. Identification of Driving ALK Fusion Genes and Genomic Landscape of Medullary Thyroid Cancer.

    Directory of Open Access Journals (Sweden)

    Jun Ho Ji

    2015-08-01

    Full Text Available The genetic landscape of medullary thyroid cancer (MTC is not yet fully understood, although some oncogenic mutations have been identified. To explore genetic profiles of MTCs, formalin-fixed, paraffin-embedded tumor tissues from MTC patients were assayed on the Ion AmpliSeq Cancer Panel v2. Eighty-four sporadic MTC samples and 36 paired normal thyroid tissues were successfully sequenced. We discovered 101 hotspot mutations in 18 genes in the 84 MTC tissue samples. The most common mutation was in the ret proto-oncogene, which occurred in 47 cases followed by mutations in genes encoding Harvey rat sarcoma viral oncogene homolog (N = 14, serine/threonine kinase 11 (N = 11, v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (N = 6, mutL homolog 1 (N = 4, Kiesten rat sarcoma viral oncogene homolog (N = 3 and MET proto-oncogene (N = 3. We also evaluated anaplastic lymphoma kinase (ALK rearrangement by immunohistochemistry and break-apart fluorescence in situ hybridization (FISH. Two of 98 screened cases were positive for ALK FISH. To identify the genomic breakpoint and 5' fusion partner of ALK, customized targeted cancer panel sequencing was performed using DNA from tumor samples of the two patients. Glutamine:fructose-6-phosphate transaminase 1 (GFPT1-ALK and echinoderm microtubule-associated protein-like 4 (EML4-ALK fusions were identified. Additional PCR analysis, followed by Sanger sequencing, confirmed the GFPT1-ALK fusion, indicating that the fusion is a result of intra-chromosomal translocation or deletion. Notably, a metastatic MTC case harboring the EML4-ALK fusion showed a dramatic response to an ALK inhibitor, crizotinib. In conclusion, we found several genetic mutations in MTC and are the first to identify ALK fusions in MTC. Our results suggest that the EML4-ALK fusion in MTC may be a potential driver mutation and a valid target of ALK inhibitors. Furthermore, the GFPT1-ALK fusion may be a potential candidate for molecular

  3. Physical forces shape group identity of swimming Pseudomonas putida cells

    Directory of Open Access Journals (Sweden)

    David Rodriguez-Espeso

    2016-09-01

    Full Text Available The often striking macroscopic patterns developed by motile bacterial populations on agar plates are a consequence of the environmental conditions where the cells grow and spread. Parameters such as medium stiffness and nutrient concentration have been reported to alter cell swimming behavior, while mutual interactions among populations shape collective patterns. One commonly observed occurrence is the mutual inhibition of clonal bacteria when moving towards each other, which results in a distinct halt at a finite distance on the agar matrix before having direct contact. The dynamics behind this phenomenon (i.e. intolerance to mix in time and space with otherwise identical others has been traditionally explained in terms of cell-to-cell competition/cooperation regarding nutrient availability. In this work, the same scenario has been revisited from an alternative perspective: the effect of the physical mechanics that frame the process, in particular the consequences of collisions between moving bacteria and the semi-solid matrix of the swimming medium. To this end we set up a simple experimental system in which the swimming patterns of Pseudomonas putida were tested with different geometries and agar concentrations. A computational analysis framework that highlights cell-to-medium interactions was developed to fit experimental observations. Simulated outputs suggested that the medium is compressed in the direction of the bacterial front motion. This phenomenon generates what was termed a compression wave that goes through the medium preceding the swimming population and that determines the visible high-level pattern. Taken together, the data suggested that the mechanical effects of the bacteria moving through the medium created a factual barrier that impedes to merge with neighboring cells swimming from a different site. The resulting divide between otherwise clonal bacteria is thus brought about by physical forces –not genetic or metabolic

  4. Platform comparison for evaluation of ALK protein immunohistochemical expression, genomic copy number and hotspot mutation status in neuroblastomas.

    Directory of Open Access Journals (Sweden)

    Benedict Yan

    Full Text Available ALK is an established causative oncogenic driver in neuroblastoma, and is likely to emerge as a routine biomarker in neuroblastoma diagnostics. At present, the optimal strategy for clinical diagnostic evaluation of ALK protein, genomic and hotspot mutation status is not well-studied. We evaluated ALK immunohistochemical (IHC protein expression using three different antibodies (ALK1, 5A4 and D5F3 clones, ALK genomic status using single-color chromogenic in situ hybridization (CISH, and ALK hotspot mutation status using conventional Sanger sequencing and a next-generation sequencing platform (Ion Torrent Personal Genome Machine (IT-PGM, in archival formalin-fixed, paraffin-embedded neuroblastoma samples. We found a significant difference in IHC results using the three different antibodies, with the highest percentage of positive cases seen on D5F3 immunohistochemistry. Correlation with ALK genomic and hotspot mutational status revealed that the majority of D5F3 ALK-positive cases did not possess either ALK genomic amplification or hotspot mutations. Comparison of sequencing platforms showed a perfect correlation between conventional Sanger and IT-PGM sequencing. Our findings suggest that D5F3 immunohistochemistry, single-color CISH and IT-PGM sequencing are suitable assays for evaluation of ALK status in future neuroblastoma clinical trials.

  5. Evaluation of NGS and RT-PCR methods for ALK rearrangement in European NSCLC patients

    DEFF Research Database (Denmark)

    Letovanec, Igor; Finn, Stephen; Zygoura, Panagiota

    2018-01-01

    INTRODUCTION: The reported prevalence of ALK rearrangement in NSCLC ranges from 2%-7%. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proven to be a reproducible and sensitive technique. Reverse transcriptase...

  6. Biological production of hydroxylated aromatics : Optimization strategies for Pseudomonas putida S12

    NARCIS (Netherlands)

    Verhoef, A.

    2010-01-01

    To replace environmentally unfriendly petrochemical production processes, the demand for bio-based production of organic chemicals is increasing. This thesis focuses on the biological production of hydroxylated aromatics from renewable substrates by engineered P. putida S12 including several cases

  7. Establishment of pseudomonas putida strains for sensitive detection of heavy metals in effluents

    International Nuclear Information System (INIS)

    Genthe, B.

    1987-09-01

    The objective of this study was to isolate a mutant of Pseudomonas putida that is more sensitive to heavy metal toxicants in water than the wild type. P. putida was the organism chosen in this study as it occurs naturally in unpolluted waters, is nonpathogenic, aerobic and because it is commonly applied in bacterial toxicity assays due to its sensitivity to toxicants. Three methods of mutagenesis were employed, which included N-methyl-N'-nitro-N-nitrosoguanidine (NG) ; ultraviolet light and transposon-mediated mutagenesis in order to generate as wide a range of mutants as possible. Four mutants, which were more sensitive to mercury, copper, lead, zinc, cadmium and silver were isolated using the NG method of mutagenesis. These mutants were designated strains 53, 56, 60 and 61 and were characterized as P. putida strains on the basis of Gram staining, biochemical reactions and immunological properties. The sensitivity of the mutants to a variety of industrial effluents was compared to that of the parent strain using a bacterial growth test. Using industrial effluents, one of the mutants, namely strain 56 was found to be more sensitive than the parent strain on 71.4% of the tests. Strains 60 and 61 were also both more sensitive than the parent strain on 42.9% of the occasions using industrial effluents. The uptake rates of radioactive mercury were measured for the parent strain of P. putida and the mutants that were found to be more sensitive to mercury

  8. Testosterone 15β-hydroxylation by solvent tolerant Pseudomonas putida S12

    NARCIS (Netherlands)

    Ruijssenaars, H.J.; Sperling, E.M.G.M.; Wiegerinck, P.H.G.; Brands, F.T.L.; Wery, J.; Bont, J.A.M.de

    2007-01-01

    A steroid 15β-hydroxylating whole-cell solvent tolerant biocatalyst was constructed by expressing the Bacillus megaterium steroid hydroxylase CYP106A2 in the solvent tolerant Pseudomonas putida S12. Testosterone hydroxylation was improved by a factor 16 by co-expressing Fer, a putative Fe-S protein

  9. In-silico-driven metabolic engineering of Pseudomonas putida for enhanced production of poly-hydroxyalkanoates

    NARCIS (Netherlands)

    Poblete-Castro, I.; Binger, D.; Rodrigues, A.; Becker, J.; Martins Dos Santos, V.A.P.; Wittmann, C.

    2013-01-01

    Here, we present systems metabolic engineering driven by in-silico modeling to tailor Pseudomonas putida for synthesis of medium chain length PHAs on glucose. Using physiological properties of the parent wild type as constraints, elementary flux mode analysis of a large-scale model of the metabolism

  10. Draft Genome Sequences of Four Hospital-Associated Pseudomonas putida Isolates.

    Science.gov (United States)

    Mustapha, Mustapha M; Marsh, Jane W; Ezeonwuka, Chinelo D; Pasculle, Anthony W; Pacey, Marissa P; Querry, Ashley M; Muto, Carlene A; Harrison, Lee H

    2016-09-29

    We present here the draft genome sequences of four Pseudomonas putida isolates belonging to a single clone suspected for nosocomial transmission between patients and a bronchoscope in a tertiary hospital. The four genome sequences belong to a single lineage but contain differences in their mobile genetic elements. Copyright © 2016 Mustapha et al.

  11. Physiological response of Pseudomonas putida S12 subjected to reduced water activity.

    NARCIS (Netherlands)

    Kets, E.P.W.; Bont, de J.A.M.; Heipieper, H.J.

    1996-01-01

    The effect of osmotic stress, given as decreased water activity (aw), on growth and the accumulation of potassium and the compatible solute betaine by Pseudomonas putida S12 was investigated. Reduced aw was imposed by addition of sodium chloride, sucrose, glycerol or polyethylene glycol to the

  12. Establishment of oxidative D-xylose metabolism in Pseudomonas putida S12

    NARCIS (Netherlands)

    Meijnen, J.P.; Winde, J.H. de; Ruijssenaars, H.J.

    2009-01-01

    The oxidative D-xylose catabolic pathway of Caulobacter crescentus, encoded by the xylXABCD operon, was expressed in the gram-negative bacterium Pseudomonas putida S12. This engineered transformant strain was able to grow on D-xylose as a sole carbon source with a biomass yield of 53% (based on g

  13. Using "Pseudomonas Putida xylE" Gene to Teach Molecular Cloning Techniques for Undergraduates

    Science.gov (United States)

    Dong, Xu; Xin, Yi; Ye, Li; Ma, Yufang

    2009-01-01

    We have developed and implemented a serial experiment in molecular cloning laboratory course for undergraduate students majored in biotechnology. "Pseudomonas putida xylE" gene, encoding catechol 2, 3-dioxygenase, was manipulated to learn molecular biology techniques. The integration of cloning, expression, and enzyme assay gave students…

  14. Effect of Pseudomonas putida on Growth and Anthocyanin Pigment in Two Poinsettia (Euphorbia pulcherrima Cultivars

    Directory of Open Access Journals (Sweden)

    Ramon Zulueta-Rodriguez

    2014-01-01

    Full Text Available Pseudomonas putida is plant growth promoting rhizobacteria (PGPR that have the capacity to improve growth in plants. The purpose of this study was to determine growth and anthocyanin pigmentation of the bracts in two poinsettia Euphorbia pulcherrima cultivars (Prestige and Sonora Marble using three strains of P. putida, as well as a mixture of the three (MIX. Comparison with the control group indicated for the most part that Prestige grew better than the Sonora Marble cultivars with the PGPR strains. Prestige with the MIX strain grew better compared to control for the number of cyathia (83 versus 70.4, volume of roots (45 versus 35 cm3, number of leaves (78 versus 58, and area of leaf (1,788 versus 1,331 cm2, except for the number of flowers (8.8 versus 11.6. To the naked eye, coloration of plants appeared identical in color compared to the control group. For all plants with P. putida strains, there was less anthocyanin pigment, but biomass was always greater with PGPR strains. Nevertheless, to the naked eye, the coloration of the plants appeared identical in color compared to the control group. This is the first study reporting the positive effects of P. putida rhizobacteria treatments on growth of poinsettia cultivars.

  15. Rhizoplane colonisation of peas by Rhizobium leguminosarum bv. viceae and a deleterious Pseudomonas putida

    NARCIS (Netherlands)

    Berggren, I.; Alstrom, S.; Vuurde, van J.W.L.; Martensson, A.M.

    2005-01-01

    Pseudomonas putida strain angstrom 313, a deleterious rhizosphere bacterium, reduced pea nitrogen content when inoculated alone or in combination with Rhizobium leguminosarum bv. viceae on plants in the presence of soil under greenhouse conditions. When plants were grown gnotobiotically in liquid

  16. Stereospecific hydroxylation of indan by Escherichia coli containing the cloned toluene dioxygenase genes from Pseudomonas putida F1.

    Science.gov (United States)

    Brand, J M; Cruden, D L; Zylstra, G J; Gibson, D T

    1992-01-01

    Escherichia coli JM109(pDTG601), containing the todC1C2BA genes encoding toluene dioxygenase from Pseudomonas putida F1, oxidizes indan to (-)-(1R)-indanol (83% R) and trans-1,3-indandiol. Under similar conditions, P. putida F39/D oxidizes indan to (-)-(1R)-indanol (96% R), 1-indanone, and trans-1,3-indandiol. The differences in the enantiomeric composition of the 1-indanols formed by the two organisms are due to the presence of a 1-indanol dehydrogenase in P. putida F39/D that preferentially oxidizes (+)-(1S)-indanol. PMID:1444374

  17. Epithelioid inflammatory myofibroblastic sarcoma responsive to surgery and an ALK inhibitor in a patient with panhypopituitarism.

    Science.gov (United States)

    Kurihara-Hosokawa, Kotomi; Kawasaki, Isao; Tamai, Anna; Yoshida, Yoko; Yakushiji, Yosuke; Ueno, Hiroki; Fukumoto, Mariko; Fukushima, Hiroko; Inoue, Takeshi; Hosoi, Masayuki

    2014-01-01

    We encountered a case of epithelioid inflammatory myofibroblastic sarcoma (EIMS) originating from an abdominal organ that rapidly regrew twice. The patient underwent two surgeries. Large tumors grew within three months after the second surgery. The patient subsequently received chemotherapy with an anaplastic lymphoma kinase (ALK) inhibitor. Although EIMS has a poor prognosis, the patient continues to be alive with disease 14 months after surgical treatment and the administration of the ALK inhibitor.

  18. Effects of Pharmacologic and Genetic Inhibition of Alk on Cognitive Impairments in NF1 Mutant Mice

    Science.gov (United States)

    2015-06-01

    in the general population. Specific learning disabilities in reading, spelling and math occur in 20% of children without overt central nervous...Fusion of a kinase gene , ALK, to a nucleolar protein gene , NPM, in non-Hodgkin’s lymphoma. Science 263, 1281-1284 (1994). 12 Mossâe, Y. P. et al...Identification of ALK as a major familial neuroblastoma predisposition gene . Nature 455, 930-935 (2008). 13 Janoueix-Lerosey, I. et al. Somatic and

  19. The tyrosine phosphatase Shp2 interacts with NPM-ALK and regulates anaplastic lymphoma cell growth and migration

    DEFF Research Database (Denmark)

    Voena, Claudia; Conte, Chiara; Ambrogio, Chiara

    2007-01-01

    Anaplastic large cell lymphomas (ALCL) are mainly characterized by the reciprocal translocation t(2;5)(p23;q35) that involves the anaplastic lymphoma kinase (ALK) gene and generates the fusion protein NPM-ALK with intrinsic tyrosine kinase activity. NPM-ALK triggers several signaling cascades......, leading to increased cell growth, resistance to apoptosis, and changes in morphology and migration of transformed cells. To search for new NPM-ALK interacting molecules, we developed a mass spectrometry-based proteomic approach in HEK293 cells expressing an inducible NPM-ALK and identified the tyrosine...... phosphatase Shp2 as a candidate substrate. We found that NPM-ALK was able to bind Shp2 in coprecipitation experiments and to induce its phosphorylation in the tyrosine residues Y542 and Y580 both in HEK293 cells and ALCL cell lines. In primary lymphomas, antibodies against the phosphorylated tyrosine Y542...

  20. Targeting brain metastases in ALK-rearranged non-small-cell lung cancer.

    Science.gov (United States)

    Zhang, Isabella; Zaorsky, Nicholas G; Palmer, Joshua D; Mehra, Ranee; Lu, Bo

    2015-10-01

    The incidence of brain metastases has increased as a result of improved systemic control and advances in imaging. However, development of novel therapeutics with CNS activity has not advanced at the same rate. Research on molecular markers has revealed many potential targets for antineoplastic agents, and a particularly important aberration is translocation in the ALK gene, identified in non-small-cell lung cancer (NSCLC). ALK inhibitors have shown systemic efficacy against ALK-rearranged NSCLC in many clinical trials, but the effectiveness of crizotinib in CNS disease is limited by poor blood-brain barrier penetration and acquired drug resistance. In this Review, we discuss potential pathways to target ALK-rearranged brain metastases, including next generation ALK inhibitors with greater CNS penetration and mechanisms to overcome resistance. Other important mechanisms to control CNS disease include targeting pathways downstream of ALK phosphorylation, increasing the permeability of the blood-brain barrier, modifying the tumour microenvironment, and adding concurrent radiotherapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. ALK7 Gene Polymorphism is Associated with Metabolic Syndrome Risk and Cardiovascular Remodeling

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Wenchao; Wang, Hui; Zhang, Wei [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital of Shandong University, Jinan (China); Lv, Ruijuan [Department of Emergency, Qilu Hospital of Shandong University, Jinan (China); Wang, Zhihao [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital of Shandong University, Jinan (China); Department of Geriatrics, Qilu Hospital of Shandong University, Jinan (China); Shang, Yuanyuan; Zhang, Yun; Zhong, Ming [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital of Shandong University, Jinan (China); Chen, Yuguo; Tang, Mengxiong, E-mail: tangmengxiongsdu8@163.com [Key Laboratory of Cardiovascular Remodeling and Function Research Chinese Ministry of Education and Chinese Ministry of Public Health, Qilu Hospital of Shandong University, Jinan (China); Department of Emergency, Qilu Hospital of Shandong University, Jinan (China)

    2013-08-15

    Activin receptor-like kinase 7 (ALK7) is a type I receptor for the TGF-β superfamily and has recently been demonstrated to play an important role in the maintenance of metabolic homeostasis. To investigate the association of the ALK7 gene polymorphism with metabolic syndrome (MetS) and cardiovascular remodeling in MetS patients. The single nucleotide polymorphism rs13010956 in the ALK7 gene was genotyped in 351 Chinese subjects undergoing carotid and cardiac ultrasonography. The associations of the ALK7 gene polymorphism with the MetS phenotype, MetS parameters, and cardiovascular ultrasonic features were analyzed. The rs13010956 polymorphism in the ALK7 gene was found to be significantly associated with the MetS phenotype in females (p < 0.05) and was also significantly associated with blood pressure in the total (p < 0.05) and female populations (p < 0.01). Further analysis revealed that rs13010956 was associated with mean intima-media thickness of the carotid arteries in females (p < 0.05). After control for body mass index, blood pressure, fasting blood glucose, and triglycerides, rs13010956 was also found to be significantly associated with left ventricular mass index in the total (p < 0.05) and female populations (p < 0.05). Our findings suggested that the ALK7 gene polymorphism rs13010956 was significantly associated with MetS risk in females and may be involved in cardiovascular remodeling in MetS patients.

  2. Biomarkers for ALK and ROS1 in Lung Cancer: Immunohistochemistry and Fluorescent In Situ Hybridization.

    Science.gov (United States)

    Luk, Peter P; Selinger, Christina I; Mahar, Annabelle; Cooper, Wendy A

    2018-06-14

    - A small proportion of non-small cell lung cancers harbor rearrangements of ALK or ROS1 genes, and these tumors are sensitive to targeted tyrosine kinase inhibitors. It is crucial for pathologists to accurately identify tumors with these genetic alterations to enable patients to access optimal treatments and avoid unnecessary side effects of less effective agents. Although a number of different techniques can be used to identify ALK- and ROS1-rearranged lung cancers, immunohistochemistry and fluorescence in situ hybridization are the mainstays. - To review the role of immunohistochemistry in assessment of ALK and ROS1 rearrangements in lung cancer, focusing on practical issues in comparison with other modalities such as fluorescence in situ hybridization. - This manuscript reviews the current literature on ALK and ROS1 detection using immunohistochemistry and fluorescence in situ hybridization as well as current recommendations. - Although fluorescence in situ hybridization remains the gold standard for detecting ALK and ROS1 rearrangement in non-small cell lung cancer, immunohistochemistry plays an important role and can be an effective screening method for detection of these genetic alterations, or a diagnostic test in the setting of ALK.

  3. ALK7 Gene Polymorphism is Associated with Metabolic Syndrome Risk and Cardiovascular Remodeling

    International Nuclear Information System (INIS)

    Zhang, Wenchao; Wang, Hui; Zhang, Wei; Lv, Ruijuan; Wang, Zhihao; Shang, Yuanyuan; Zhang, Yun; Zhong, Ming; Chen, Yuguo; Tang, Mengxiong

    2013-01-01

    Activin receptor-like kinase 7 (ALK7) is a type I receptor for the TGF-β superfamily and has recently been demonstrated to play an important role in the maintenance of metabolic homeostasis. To investigate the association of the ALK7 gene polymorphism with metabolic syndrome (MetS) and cardiovascular remodeling in MetS patients. The single nucleotide polymorphism rs13010956 in the ALK7 gene was genotyped in 351 Chinese subjects undergoing carotid and cardiac ultrasonography. The associations of the ALK7 gene polymorphism with the MetS phenotype, MetS parameters, and cardiovascular ultrasonic features were analyzed. The rs13010956 polymorphism in the ALK7 gene was found to be significantly associated with the MetS phenotype in females (p < 0.05) and was also significantly associated with blood pressure in the total (p < 0.05) and female populations (p < 0.01). Further analysis revealed that rs13010956 was associated with mean intima-media thickness of the carotid arteries in females (p < 0.05). After control for body mass index, blood pressure, fasting blood glucose, and triglycerides, rs13010956 was also found to be significantly associated with left ventricular mass index in the total (p < 0.05) and female populations (p < 0.05). Our findings suggested that the ALK7 gene polymorphism rs13010956 was significantly associated with MetS risk in females and may be involved in cardiovascular remodeling in MetS patients

  4. Functional, genetic and chemical characterization of biosurfactants produced by plant growth-promoting Pseudomonas putida 267.

    Science.gov (United States)

    Kruijt, Marco; Tran, Ha; Raaijmakers, Jos M

    2009-08-01

    Plant growth-promoting Pseudomonas putida strain 267, originally isolated from the rhizosphere of black pepper, produces biosurfactants that cause lysis of zoospores of the oomycete pathogen Phytophthora capsici. The biosurfactants were characterized, the biosynthesis gene(s) partially identified, and their role in control of Phytophthora damping-off of cucumber evaluated. The biosurfactants were shown to lyse zoospores of Phy. capsici and inhibit growth of the fungal pathogens Botrytis cinerea and Rhizoctonia solani. In vitro assays further showed that the biosurfactants of strain 267 are essential in swarming motility and biofilm formation. In spite of the zoosporicidal activity, the biosurfactants did not play a significant role in control of Phytophthora damping-off of cucumber, since both wild type strain 267 and its biosurfactant-deficient mutant were equally effective, and addition of the biosurfactants did not provide control. Genetic characterization revealed that surfactant biosynthesis in strain 267 is governed by homologues of PsoA and PsoB, two nonribosomal peptide synthetases involved in production of the cyclic lipopeptides (CLPs) putisolvin I and II. The structural relatedness of the biosurfactants of strain 267 to putisolvins I and II was supported by LC-MS and MS-MS analyses. The biosurfactants produced by Ps. putida 267 were identified as putisolvin-like CLPs; they are essential in swarming motility and biofilm formation, and have zoosporicidal and antifungal activities. Strain 267 provides excellent biocontrol activity against Phytophthora damping-off of cucumber, but the lipopeptide surfactants are not involved in disease suppression. Pseudomonas putida 267 suppresses Phy. capsici damping-off of cucumber and provides a potential supplementary strategy to control this economically important oomycete pathogen. The putisolvin-like biosurfactants exhibit zoosporicidal and antifungal activities, yet they do not contribute to biocontrol of Phy

  5. Molecular and biochemical characterization of Pseudomonas putida isolated from bottled uncarbonated mineral drinking water

    Directory of Open Access Journals (Sweden)

    Tasić S.

    2014-01-01

    Full Text Available Pseudomonas putida belongs to a group of opportunistic pathogens that can cause disease in people with weakened or damaged immune systems. Some strains have medical significance, and for most ingestion is not the primary route of infection. If water used by predisposed subjects is contaminated by P. putida, they may become ill. The aim of this work was the biochemical and molecular characterization of strain ST3 of P. putida isolated from non-carbonated bottled drinking water from Jakov Do 4 on Mt. Vlasina. Characterization of P. putida was performed to assess the risk to human health of the indigenous strains present in the water. Biochemical characterization of strains was performed using the manual identification system ID 32 GN (BioMérieux. Identification was obtained using the database identification software ATB System (Bio-Mérieux. Molecular characterization was performed by PCR amplification and 16S rDNA “thermal cycling sequencing”. Biochemical identification of the strain ST3 was accurate (Id = 99.8%. Comparing the sequences obtained for strain ST3 with NCBI gene bank sequences for 16S rRNA, the highest similarity of our strain (96% identity with a strain of P. putida, designated as biotype A (gi|18076625|emb|AJ308311.1|.PPU308311 isolated in New Zealand, was obtained. While comparison with the NCBI collection of all deposited sequences showed that the 16S rRNA gene sequence of strain ST3 has very high homology, it is not identical, indicating indirectly that strain ST3 is an indigenous strain.

  6. Dynamics of development and dispersal in sessile microbial communities: examples from Pseudomonas aeruginosa and Pseudomonas putida model biofilms

    DEFF Research Database (Denmark)

    Klausen, M.; Gjermansen, Morten; Kreft, J.-U.

    2006-01-01

    Surface-associated microbial communities in many cases display dynamic developmental patterns. Model biofilms formed by Pseudomonas aeruginosa and Pseudomonas putida in laboratory flow-chamber setups represent examples of such behaviour. Dependent on the experimental conditions the bacteria...

  7. Assessing storage of stability and mercury reduction of freeze-dried Pseudomonas putida within different types of lyoprotectant

    Science.gov (United States)

    Azoddein, Abdul Aziz Mohd; Nuratri, Yana; Azli, Faten Ahada Mohd; Bustary, Ahmad Bazli

    2017-12-01

    Pseudomonas putida is a potential strain in biological treatment to remove mercury contained in the effluent of petrochemical industry due to its mercury reductase enzyme that able to reduce ionic mercury to elementary mercury. Freeze-dried P. putida allows easy, inexpensive shipping, handling and high stability of the product. This study was aimed to freeze dry P. putida cells with addition of lyoprotectant. Lyoprotectant was added into the cells suspension prior to freezing. Dried P. putida obtained was then mixed with synthetic mercury. Viability of recovery P. putida after freeze dry was significantly influenced by the type of lyoprotectant. Among the lyoprotectants, tween 80/ sucrose was found to be the best lyoprotectant. Sucrose was able to recover more than 78% (6.2E+09 CFU/ml) of the original cells (7.90E+09CFU/ml) after freeze dry and able to retain 5.40E+05 viable cells after 4 weeks storage at 4 °C without vacuum. Polyethylene glycol (PEG) pre-treated freeze dried cells and broth pre-treated freeze dried cells after the freeze-dry process recovered more than 64% (5.0 E+09CFU/ml) and >0.1% (5.60E+07CFU/ml). Freeze-dried P. putida cells in PEG and broth cannot survive after 4 weeks storage. Freeze dry also does not really change the pattern of growth P. putida but extension of lag time was found 1 hour after 3 weeks of storage. Additional time was required for freeze-dried P. putida cells to recover before introducing freeze-dried cells to more complicated condition such as mercury solution. The maximum mercury reduction of PEG pre-treated freeze-dried cells after freeze dry and after storage of 3 weeks was 17.91 %. The maximum of mercury reduction of tween 80/sucrose pre-treated freeze-dried cells after freeze dry and after storage 3 weeks was 25.03%. Freeze dried P. putida was found to have lower mercury reduction compare to the fresh P. putida that has been grown in agar. Result from this study may be beneficial and useful as initial reference before

  8. In situ biosurfactant production and hydrocarbon removal by Pseudomonas putida CB-100 in bioaugmented and biostimulated oil-contaminated soil.

    Science.gov (United States)

    Ángeles, Martínez-Toledo; Refugio, Rodríguez-Vázquez

    2013-01-01

    In situ biosurfactant (rhamnolipid) production by Pseudomonas putida CB-100 was achieved during a bioaugmented and biostimulated treatment to remove hydrocarbons from aged contaminated soil from oil well drilling operations. Rhamnolipid production and contaminant removal were determined for several treatments of irradiated and non-irradiated soils: nutrient addition (nitrogen and phosphorus), P. putida addition, and addition of both (P. putida and nutrients). The results were compared against a control treatment that consisted of adding only sterilized water to the soils. In treatment with native microorganisms (non-irradiated soils) supplemented with P. putida, the removal of total petroleum hydrocarbons (TPH) was 40.6%, the rhamnolipid production was 1.54 mg/kg, and a surface tension of 64 mN/m was observed as well as a negative correlation (R = -0.54; p soil treated with P. putida, TPH removal was 24.5% with rhamnolipid generation of 1.79 mg/kg and 65.6 mN/m of surface tension, and a correlation between bacterial growth and biosurfactant production (R = -0.64; p soils, in situ rhamnolipid production by P. putida enhanced TPH decontamination of the soil.

  9. ALK-1-negative anaplastic large cell lymphoma associated with breast implants: a new clinical entity.

    Science.gov (United States)

    Lazzeri, Davide; Agostini, Tommaso; Bocci, Guido; Giannotti, Giordano; Fanelli, Giovanni; Naccarato, Antonio Giuseppe; Danesi, Romano; Tuccori, Marco; Pantaloni, Marcello; D'Aniello, Carlo

    2011-10-01

    Concerns have been raised recently regarding the increasing number of reports of non-Hodgkin lymphoma (NHL) that developed in close proximity to silicone or saline breast implants. In particular, an increased risk of anaplastic large cell lymphoma (ALCL) in patients with breast prostheses has been proposed. We reviewed clinical and pathologic findings in 40 women who received a diagnosis of breast NHL arising in association with breast implants and of 27 patients who had a diagnosis of ALCL with breast involvement reported in the published literature. Among the 40 reported cases of prosthesis-associated breast lymphomas, 28 were anaplastic lymphoma kinase-1-negative (ALK-1(-)) ALCLs, whereas of 27 ALCLs in patients without implants found in the literature, only 10 were ALK-1(-). The finding of 28 cases of breast ALK-1(-) ALCL occurring in patients with implants compared with 10 cases in women without implants is in favor of an association between silicone breast prostheses and ALK-1(-) ALCL. Although the incidence of this type of lymphoma remains remarkably low given that breast prostheses have been widely used for decades, clinical and pathologic evidence for a causative role is becoming dramatically strong. The histologic, phenomenologic, and clinical similarities of the majority of implant-related ALK-1(-) ALCLs suggest a common mechanism, especially when compared with the counterpart of patients without implants in which very few and highly dishomogeneous cases of the same malignancy were detected. There is convincing evidence that primary implant-related ALK-1(-) ALCL represents a distinct clinicopathologic entity that has been inappropriately fitted into the category of systemic ALK-1(-) ALCL. Thus it should be recognized as a separate category and classified on its own. Copyright © 2011 Elsevier Inc. All rights reserved.

  10. Meta-analysis of neuroblastomas reveals a skewed ALK mutation spectrum in tumors with MYCN amplification.

    Science.gov (United States)

    De Brouwer, Sara; De Preter, Katleen; Kumps, Candy; Zabrocki, Piotr; Porcu, Michaël; Westerhout, Ellen M; Lakeman, Arjan; Vandesompele, Jo; Hoebeeck, Jasmien; Van Maerken, Tom; De Paepe, Anne; Laureys, Geneviève; Schulte, Johannes H; Schramm, Alexander; Van Den Broecke, Caroline; Vermeulen, Joëlle; Van Roy, Nadine; Beiske, Klaus; Renard, Marleen; Noguera, Rosa; Delattre, Olivier; Janoueix-Lerosey, Isabelle; Kogner, Per; Martinsson, Tommy; Nakagawara, Akira; Ohira, Miki; Caron, Huib; Eggert, Angelika; Cools, Jan; Versteeg, Rogier; Speleman, Frank

    2010-09-01

    Activating mutations of the anaplastic lymphoma kinase (ALK) were recently described in neuroblastoma. We carried out a meta-analysis of 709 neuroblastoma tumors to determine their frequency and mutation spectrum in relation to genomic and clinical parameters, and studied the prognostic significance of ALK copy number and expression. The frequency and type of ALK mutations, copy number gain, and expression were analyzed in a new series of 254 neuroblastoma tumors. Data from 455 published cases were used for further in-depth analysis. ALK mutations were present in 6.9% of 709 investigated tumors, and mutations were found in similar frequencies in favorable [International Neuroblastoma Staging System (INSS) 1, 2, and 4S; 5.7%] and unfavorable (INSS 3 and 4; 7.5%) neuroblastomas (P = 0.087). Two hotspot mutations, at positions R1275 and F1174, were observed (49% and 34.7% of the mutated cases, respectively). Interestingly, the F1174 mutations occurred in a high proportion of MYCN-amplified cases (P = 0.001), and this combined occurrence was associated with a particular poor outcome, suggesting a positive cooperative effect between both aberrations. Furthermore, the F1174L mutant was characterized by a higher degree of autophosphorylation and a more potent transforming capacity as compared with the R1275Q mutant. Chromosome 2p gains, including the ALK locus (91.8%), were associated with a significantly increased ALK expression, which was also correlated with poor survival. ALK mutations occur in equal frequencies across all genomic subtypes, but F1174L mutants are observed in a higher frequency of MYCN-amplified tumors and show increased transforming capacity as compared with the R1275Q mutants.

  11. ALK1 expression in oral lichen planus: a possible relation to microvessel density.

    Science.gov (United States)

    Hazzaa, Hala H A; El-Wakeel, Naglaa M; Attia, Enas A S; Abo Hager, Eman A

    2016-05-01

    To assess the expression of activin receptor-like kinase 1 (ALK1) and investigate its possible relationship with microvessel density (MVD) in different forms of oral lichen planus (OLP) compared to controls' biopsies. Biopsies from 20 reticular/papular OLP (R/PLP), 20 atrophic/erosive OLP (A/ELP) patients, and 20 healthy subjects were immunohistochemically analyzed and statistically compared and correlated for ALK1 expression and MVD as assessed by CD34 expression. All OLP specimens revealed the presence of positive cytoplasmic CD34 immunostaining in endothelial cells, with statistically high significant MVD in each of R/PLP (Median; M = 4.40) and A/ELP (M = 7.69) compared to controls (M = 1.16) (P < 0.001). Statistically significant MVD was found in A/ELP compared to R/PLP (P < 0.001). All control specimens revealed negative ALK1 immunostaining of the few inflammatory cells found, while 85% of A/ELP cases and 70% of R/PLP cases showed positively immunostained sections for ALK-1, with statistically significant higher ALK1 expression In A/ELP (M = 1.95) compared to R/PLP (M = 0.86) (P = 0.005). No significant correlation between CD34 and ALK1 was detected in R/PLP (r = 0.081), while a barely moderate positive correlation was found in A/ELP (r = 0.396). ALK1 expression and MVD are increased in OLP, particularly in A/ELP type. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  12. Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans-metabolism.

    Science.gov (United States)

    Nikel, Pablo I; de Lorenzo, Víctor

    2018-05-16

    The itinerary followed by Pseudomonas putida from being a soil-dweller and plant colonizer bacterium to become a flexible and engineer-able platform for metabolic engineering stems from its natural lifestyle, which is adapted to harsh environmental conditions and all sorts of physicochemical stresses. Over the years, these properties have been capitalized biotechnologically owing to the expanding wealth of genetic tools designed for deep-editing the P. putida genome. A suite of dedicated vectors inspired in the core tenets of synthetic biology have enabled to suppress many of the naturally-occurring undesirable traits native to this species while enhancing its many appealing properties, and also to import catalytic activities and attributes from other biological systems. Much of the biotechnological interest on P. putida stems from the distinct architecture of its central carbon metabolism. The native biochemistry is naturally geared to generate reductive currency [i.e., NAD(P)H] that makes this bacterium a phenomenal host for redox-intensive reactions. In some cases, genetic editing of the indigenous biochemical network of P. putida (cis-metabolism) has sufficed to obtain target compounds of industrial interest. Yet, the main value and promise of this species (in particular, strain KT2440) resides not only in its capacity to host heterologous pathways from other microorganisms, but also altogether artificial routes (trans-metabolism) for making complex, new-to-Nature molecules. A number of examples are presented for substantiating the worth of P. putida as one of the favorite workhorses for sustainable manufacturing of fine and bulk chemicals in the current times of the 4th Industrial Revolution. The potential of P. putida to extend its rich native biochemistry beyond existing boundaries is discussed and research bottlenecks to this end are also identified. These aspects include not just the innovative genetic design of new strains but also the incorporation of

  13. Evaluation of EML4-ALK Fusion Proteins in Non–Small Cell Lung Cancer Using Small Molecule Inhibitors

    Directory of Open Access Journals (Sweden)

    Yongjun Li

    2011-01-01

    Full Text Available The echinoderm microtubule–associated protein-like 4–anaplastic lymphoma kinase (EML4-ALK fusion gene resulting from an inversion within chromosome 2p occurs in approximately 5% of non–small cell lung cancer and is mu-tually exclusive with Ras and EGFR mutations. In this study, we have used a potent and selective ALK small molecule inhibitor, NPV-TAE684, to assess the oncogenic role of EML4-ALK in non–small cell lung cancer (NSCLC. We show here that TAE684 inhibits proliferation and induces cell cycle arrest, apoptosis, and tumor regression in two NSCLC models that harbor EML4-ALK fusions. TAE684 inhibits EML4-ALK activation and its downstream signaling including ERK, AKT, and STAT3. We used microarray analysis to carry out targeted pathway studies of gene expression changes in H2228 NSCLC xenograft model after TAE684 treatment and identified a gene signature of EML4-ALK inhibition. The gene signature represents 1210 known human genes, and the top biologic processes represented by these genes are cell cycle, DNA synthesis, cell proliferation, and cell death. We also compared the effect of TAE684 with PF2341066, a c-Met and ALK small molecule inhibitor currently in clinical trial in cancers harboring ALK fusions, and demonstrated that TAE684 is a much more potent inhibitor of EML4-ALK. Our data demonstrate that EML4-ALK plays an important role in the pathogenesis of a subset of NSCLC and provides insight into the mech-anism of EML4-ALK inhibition by a small molecule inhibitor.

  14. Therapeutic strategies to overcome crizotinib resistance in non-small cell lung cancers harboring the fusion oncogene EML4-ALK

    Science.gov (United States)

    Katayama, Ryohei; Khan, Tahsin M.; Benes, Cyril; Lifshits, Eugene; Ebi, Hiromichi; Rivera, Victor M.; Shakespeare, William C.; Iafrate, A. John; Engelman, Jeffrey A.; Shaw, Alice T.

    2011-01-01

    The echinoderm microtubule-associated protein-like 4 (EML4)-anaplastic lymphoma kinase (ALK) fusion oncogene represents a molecular target in a small subset of non-small cell lung cancers (NSCLCs). This fusion leads to constitutive ALK activation with potent transforming activity. In a pivotal phase 1 clinical trial, the ALK tyrosine kinase inhibitor (TKI) crizotinib (PF-02341066) demonstrated impressive antitumor activity in the majority of patients with NSCLC harboring ALK fusions. However, despite these remarkable initial responses, cancers eventually develop resistance to crizotinib, usually within 1 y, thereby limiting the potential clinical benefit. To determine how cancers acquire resistance to ALK inhibitors, we established a model of acquired resistance to crizotinib by exposing a highly sensitive EML4-ALK–positive NSCLC cell line to increasing doses of crizotinib until resistance emerged. We found that cells resistant to intermediate doses of crizotinib developed amplification of the EML4-ALK gene. Cells resistant to higher doses (1 μM) also developed a gatekeeper mutation, L1196M, within the kinase domain, rendering EML4-ALK insensitive to crizotinib. This gatekeeper mutation was readily detected using a unique and highly sensitive allele-specific PCR assay. Although crizotinib was ineffectual against EML4-ALK harboring the gatekeeper mutation, we observed that two structurally different ALK inhibitors, NVP-TAE684 and AP26113, were highly active against the resistant cancer cells in vitro and in vivo. Furthermore, these resistant cells remained highly sensitive to the Hsp90 inhibitor 17-AAG. Thus, we have developed a model of acquired resistance to ALK inhibitors and have shown that second-generation ALK TKIs or Hsp90 inhibitors are effective in treating crizotinib-resistant tumors harboring secondary gatekeeper mutations. PMID:21502504

  15. Pf16 and phiPMW: Expanding the realm of Pseudomonas putida bacteriophages.

    Directory of Open Access Journals (Sweden)

    Damian J Magill

    Full Text Available We present the analysis of two novel Pseudomonas putida phages, pf16 and phiPMW. Pf16 represents a peripherally related T4-like phage, and is the first of its kind infecting a Pseudomonad, with evidence suggesting cyanophage origins. Extensive divergence has resulted in pf16 occupying a newly defined clade designated as the pf16-related phages, lying at the interface of the Schizo T-Evens and Exo T-Evens. Recombination with an ancestor of the P. putida phage AF is likely responsible for the tropism of this phage. phiPMW represents a completely novel Pseudomonas phage with a genome containing substantial genetic novelty through its many hypothetical proteins. Evidence suggests that this phage has been extensively shaped through gene transfer events and vertical evolution. Phylogenetics shows that this phage has an evolutionary history involving FelixO1-related viruses but is in itself highly distinct from this group.

  16. Global Transcriptional Responses to Osmotic, Oxidative, and Imipenem Stress Conditions in Pseudomonas putida

    DEFF Research Database (Denmark)

    Bojanovic, Klara; D'Arrigo, Isotta; Long, Katherine

    2017-01-01

    functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which P. putida responds to different stress conditions...... intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous......Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification...

  17. Crystallization and preliminary X-ray diffraction analysis of alanine racemase from Pseudomonas putida YZ-26

    International Nuclear Information System (INIS)

    Liu, Junlin; Feng, Lei; Shi, Yawei; Feng, Wei

    2012-01-01

    A recombinant alanine racemase from the Pseudomonas putida YZ-26, has been crystallized by the sitting-drop vapor-diffusion method and X-ray diffraction data were collected to 2.4 Å. A recombinant form of alanine racemase (Alr) from Pseudomonas putida YZ-26 has been crystallized by the sitting-drop vapour diffusion method. X-ray diffraction data were collected to 2.4 Å resolution. The crystals belong to the space group C222 1 , with unit-cell parameters a = 118.08, b = 141.86, c = 113.83 Å, and contain an Alr dimer in the asymmetric unit. The Matthews coefficient and the solvent content were calculated to be 2.8 Å 3 Da −1 and approximately 50%, respectively

  18. Diabetic foot gangrene patient with multi-drug resistant Pseudomonas putida infection in Karawaci District, Indonesia

    Directory of Open Access Journals (Sweden)

    Nata Pratama Hardjo Lugito

    2015-01-01

    Full Text Available Pseudomonas putida is a rod-shaped, non fermenting Gram-negative organism frequently found in the environment that utilizes aerobic metabolism, previously thought to be of low pathogenicity. It had been reported as cause of skin and soft tissue infection, especially in immunocompromised patients. A female green grocer, 51 year-old came to internal medicine out-patient clinic with gangrene and osteomyelitis on her 1 st , 2 nd and 3 rd digit and wound on the sole of the right foot since 1 month prior. The patient had history of uncontrolled diabetes since a year ago. She was given ceftriaxone 2 grams b.i.d, metronidazole 500 mg t.i.d empirically and then amikacin 250 mg b.i.d, followed by amputation of the digits and wound debridement. The microorganism′s culture from pus revealed multi drug resistant Pseudomonas putida. She recovered well after antibiotics and surgery.

  19. A molecular dynamics investigation on the crizotinib resistance mechanism of C1156Y mutation in ALK

    International Nuclear Information System (INIS)

    Sun, Hui-Yong; Ji, Feng-Qin

    2012-01-01

    Highlights: ► The study revealed the detailed resistance mechanism of the non-active mutation C1156Y in ALK. ► C1156Y leads to crizotinib displacement and conformational changes in the binding cavity. ► The conformations cause a decline in the vdW and electrostatic energy between crizotinib and ALK. -- Abstract: Crizotinib is an anaplastic lymphoma kinase (ALK) inhibitor that has recently been approved in the US for the treatment of non-small cell lung carcinoma (NSCLC). Despite its outstanding safety and efficacy, several resistant mutations against crizotinib have been detected in the treatment of NSCLC. However, in contrast to the widely accepted mechanism of steric hindrance by mutations at the active site, the mechanism by which the C1156Y non-active site mutation confers resistance against crizotinib remains unclear. In the present study, the resistance mechanism of C1156Y in ALK was investigated using molecular dynamics simulations. The results suggest that despite the non-active site mutation, C1156Y causes the dislocation of crizotinib as well as the indirect conformational changes in the binding cavity, which results in a marked decrease in the van der Waals and electrostatic interactions between crizotinib and ALK. The obtained results provide a detailed explanation of the resistance caused by C1156Y and may give a vital clue for the design of drugs to combat crizotinib resistance.

  20. A molecular dynamics investigation on the crizotinib resistance mechanism of C1156Y mutation in ALK

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Hui-Yong [Shandong University of Technology, Zibo 255049 (China); Ji, Feng-Qin, E-mail: fengqinji@mail.hzau.edu.cn [National Key Laboratory of Crop Genetic Improvement, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070 (China); Center for Bioinformatics, Huazhong Agricultural University, Wuhan 430070 (China)

    2012-06-29

    Highlights: Black-Right-Pointing-Pointer The study revealed the detailed resistance mechanism of the non-active mutation C1156Y in ALK. Black-Right-Pointing-Pointer C1156Y leads to crizotinib displacement and conformational changes in the binding cavity. Black-Right-Pointing-Pointer The conformations cause a decline in the vdW and electrostatic energy between crizotinib and ALK. -- Abstract: Crizotinib is an anaplastic lymphoma kinase (ALK) inhibitor that has recently been approved in the US for the treatment of non-small cell lung carcinoma (NSCLC). Despite its outstanding safety and efficacy, several resistant mutations against crizotinib have been detected in the treatment of NSCLC. However, in contrast to the widely accepted mechanism of steric hindrance by mutations at the active site, the mechanism by which the C1156Y non-active site mutation confers resistance against crizotinib remains unclear. In the present study, the resistance mechanism of C1156Y in ALK was investigated using molecular dynamics simulations. The results suggest that despite the non-active site mutation, C1156Y causes the dislocation of crizotinib as well as the indirect conformational changes in the binding cavity, which results in a marked decrease in the van der Waals and electrostatic interactions between crizotinib and ALK. The obtained results provide a detailed explanation of the resistance caused by C1156Y and may give a vital clue for the design of drugs to combat crizotinib resistance.

  1. A molecular dynamics investigation into the mechanisms of alectinib resistance of three ALK mutants.

    Science.gov (United States)

    He, Muyang; Li, Weikang; Zheng, Qingchuan; Zhang, Hongxing

    2018-01-11

    Alectinib, a highly selective next-genetation anaplastic lymphoma kinase (ALK) inhibitor, has demonstrated promising antitumor activity in patients with ALK-positive non-small cell lung carcinomas (NSCLC). However, the therapeutic benefits of alectinib is inescapably hampered by the development of acquired resistant mutations in ALK. Despite the availability of ample experimental mutagenesis data, the molecular origin and the structural motifs under alectinib binding affinity deficiencies are still ambiguous. Here, molecular dynamics (MD) simulations and molecular mechanics generalized born surface area (MM-GBSA) calculation approaches were employed to elucidate the mechanisms of alectinib resistance induced by the mutations I1171N, V1180L, and L1198F. The MD results reveal that the studied mutations could trigger the dislocation of alectinib as well as conformational changes at the inhibitor binding site, thus induce the interactional changes between alectinib and mutants. The most influenced regions are the ligand binding entrance and the hinge region, which are considered to be the dominant binding motifs accounting for the binding affinity loss in mutants. The "key and lock mechanism" between the ethyl group at position 9 of alectinib and a recognition cavity in the hinge region of ALK is presented to illustrate the major molecular origin of drug resistance. Our results provide mechanistic insight into the effect of ALK mutations resistant to alectinib, which could contribute to further rational design of inhibitors to combat the acquired resistance. © 2018 Wiley Periodicals, Inc.

  2. Clinical Utility of Circulating Tumor Cells in ALK-Positive Non-Small-Cell Lung Cancer.

    Science.gov (United States)

    Faugeroux, Vincent; Pailler, Emma; Auger, Nathalie; Taylor, Melissa; Farace, Françoise

    2014-01-01

    The advent of rationally targeted therapies such as small-molecule tyrosine kinase inhibitors (TKIs) has considerably transformed the therapeutic management of a subset of patients with non-small-cell lung cancer (NSCLC) harboring defined molecular abnormalities. When such genetic molecular alterations are detected the use of specific TKI has demonstrated better results (overall response rate, progression free survival) compared to systemic therapy. However, the detection of such molecular abnormalities is complicated by the difficulty in obtaining sufficient tumor material, in terms of quantity and quality, from a biopsy. Here, we described how circulating tumor cells (CTCs) can have a clinical utility in anaplastic lymphoma kinase (ALK) positive NSCLC patients to diagnose ALK-EML4 gene rearrangement and to guide therapeutic management of these patients. The ability to detect genetic abnormalities such ALK rearrangement in CTCs shows that these cells could offer new perspectives both for the diagnosis and the monitoring of ALK-positive patients eligible for treatment with ALK inhibitors.

  3. Aerobic TCE degradation by encapsulated toluene-oxidizing bacteria, Pseudomonas putida and Bacillus spp.

    Science.gov (United States)

    Kim, Seungjin; Bae, Wookeun; Hwang, Jungmin; Park, Jaewoo

    2010-01-01

    The degradation rates of toluene and trichloroethylene (TCE) by Pseudomonas putida and Bacillus spp. that were encapsulated in polyethylene glycol (PEG) polymers were evaluated in comparison with the results of exposure to suspended cultures. PEG monomers were polymerized together with TCE-degrading microorganisms, such that the cells were encapsulated in and protected by the matrices of the PEG polymers. TCE concentrations were varied from 0.1 to 1.5 mg/L. In the suspended cultures of P. putida, the TCE removal rate decreased as the initial TCE concentration increased, revealing TCE toxicity or a limitation of reducing power, or both. When the cells were encapsulated, an initial lag period of about 10-20 h was observed for toluene degradation. Once acclimated, the encapsulated P. putida cultures were more tolerant to TCE at an experimental range of 0.6-1.0 mg/L and gave higher transfer efficiencies (mass TCE transformed/mass toluene utilized). When the TCE concentration was low (e.g., 0.1 mg/L) the removal of TCE per unit mass of cells (specific removal) was significantly lower, probably due to a diffusion limitation into the PEG pellet. Encapsulated Bacillus spp. were able to degrade TCE cometabolically. The encapsulated Bacillus spp. gave significantly higher values than did P. putida in the specific removal and the transfer efficiency, particularly at relatively high TCE concentration of approximately 1.0±0.5 mg/L. The transfer efficiency by encapsulated Bacillus spp. in this study was 0.27 mgTCE/mgToluene, which was one to two orders of magnitude greater than the reported values.

  4. Pseudomonas putida and Pseudomonas fluorescens Species Group Recovery from Human Homes Varies Seasonally and by Environment.

    Directory of Open Access Journals (Sweden)

    Susanna K Remold

    Full Text Available By shedding light on variation in time as well as in space, long-term biogeographic studies can help us define organisms' distribution patterns and understand their underlying drivers. Here we examine distributions of Pseudomonas in and around 15 human homes, focusing on the P. putida and P. fluorescens species groups. We describe recovery from 10,941 samples collected during up to 8 visits per home, occurring on average 2.6 times per year. We collected a mean of 141 samples per visit, from sites in most rooms of the house, from the surrounding yards, and from human and pet occupants. We recovered Pseudomonas in 9.7% of samples, with the majority of isolates being from the P. putida and P. fluorescens species groups (approximately 62% and 23% of Pseudomonas samples recovered respectively. Although representatives of both groups were recovered from every season, every house, and every type of environment sampled, recovery was highly variable across houses and samplings. Whereas recovery of P. putida group was higher in summer and fall than in winter and spring, P. fluorescens group isolates were most often recovered in spring. P. putida group recovery from soils was substantially higher than its recovery from all other environment types, while higher P. fluorescens group recovery from soils than from other sites was much less pronounced. Both species groups were recovered from skin and upper respiratory tract samples from healthy humans and pets, although this occurred infrequently. This study indicates that even species that are able to survive under a broad range of conditions can be rare and variable in their distributions in space and in time. For such groups, determining patterns and causes of stochastic and seasonal variability may be more important for understanding the processes driving their biogeography than the identity of the types of environments in which they can be found.

  5. Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase.

    Science.gov (United States)

    Blank, Lars M; Ionidis, Georgios; Ebert, Birgitta E; Bühler, Bruno; Schmid, Andreas

    2008-10-01

    A key limitation of whole-cell redox biocatalysis for the production of valuable, specifically functionalized products is substrate/product toxicity, which can potentially be overcome by using solvent-tolerant micro-organisms. To investigate the inter-relationship of solvent tolerance and energy-dependent biocatalysis, we established a model system for biocatalysis in the presence of toxic low logP(ow) solvents: recombinant solvent-tolerant Pseudomonas putida DOT-T1E catalyzing the stereospecific epoxidation of styrene in an aqueous/octanol two-liquid phase reaction medium. Using (13)C tracer based metabolic flux analysis, we investigated the central carbon and energy metabolism and quantified the NAD(P)H regeneration rate in the presence of toxic solvents and during redox biocatalysis, which both drastically increased the energy demands of solvent-tolerant P. putida. According to the driven by demand concept, the NAD(P)H regeneration rate was increased up to eightfold by two mechanisms: (a) an increase in glucose uptake rate without secretion of metabolic side products, and (b) reduced biomass formation. However, in the presence of octanol, only approximately 1% of the maximally observed NAD(P)H regeneration rate could be exploited for styrene epoxidation, of which the rate was more than threefold lower compared with operation with a non-toxic solvent. This points to a high energy and redox cofactor demand for cell maintenance, which limits redox biocatalysis in the presence of octanol. An estimated upper bound for the NAD(P)H regeneration rate available for biocatalysis suggests that cofactor availability does not limit redox biocatalysis under optimized conditions, for example, in the absence of toxic solvent, and illustrates the high metabolic capacity of solvent-tolerant P. putida. This study shows that solvent-tolerant P. putida have the remarkable ability to compensate for high energy demands by boosting their energy metabolism to levels up to an order of

  6. Selective base excision repair of DNA damage by the non-base-flipping DNA glycosylase AlkC

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Rongxin; Mullins, Elwood A.; Shen, Xing; #8208; Xing; Lay, Kori T.; Yuen, Philip K.; David, Sheila S.; Rokas, Antonis; Eichman, Brandt F. (UCD); (Vanderbilt)

    2017-10-20

    DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT-like repeat (HLR) fold. AlkD uses a unique non-base-flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3-methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non-base-flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin-like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA. This active site can accommodate and excise N3-methylcytosine (3mC) and N1-methyladenine (1mA), which are also repaired by AlkB-catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.

  7. Redundant and nonredundant roles for Cdc42 and Rac1 in lymphomas developed in NPM-ALK transgenic mice

    DEFF Research Database (Denmark)

    Choudhari, Ramesh; Minero, Valerio Giacomo; Menotti, Matteo

    2016-01-01

    Increasing evidence suggests that Rho family GTPases could have a critical role in the biology of T-cell lymphoma. In ALK-rearranged anaplastic large cell lymphoma (ALCL), a specific subtype of T-cell lymphoma, the Rho family GTPases Cdc42 and Rac1 are activated by the ALK oncogenic activity. In ...

  8. Combination of pseudomonas putida and EK method to reduce the amount of mercury on landfill soil

    Science.gov (United States)

    Nabila, A. T. A.; Azhar, A. T. S.; Nurshuhaila, M. S.; Azim, M. A. M.; Amirah, S. N.

    2017-11-01

    Landfills usually lack of environment measures especially on soil. There are no guarantee that the landfill soil is free from being contaminated. It may cause harm for humans, animals and plants at surrounding area. In order to solve this problem, advance remediation technique is essential such as the electrokinetic combined with microorganisms known as electrokinetic bioremediation technique. The aim of this study is to investigate the performance of P.putida with 15 volt electric current supply (Ek-bio) and without electric current (Bio) in removal of mercury in landfill soil. Both treatments were running throughout 14 days. The P.putida was placed at anode compartment meanwhile sterile distilled water poured at cathode compartment. According to the both results, Ek-bio was removed mercury up to 48 % but by using standard bioremediation treatment, the removal only 32 %. Besides that, the migration of P.putida react more aggressively during the present of electric current compared with bioremediation. As the results, it was proven that by using Ek-bio technique can increase the activity of bacteria beside and the removal of mercury. Therefore, Ek-bio method can be commercialized to the parties concerned to solve the contaminated soil by mercury.

  9. Antibiotic resistance determinants in a Pseudomonas putida strain isolated from a hospital.

    Directory of Open Access Journals (Sweden)

    Lázaro Molina

    Full Text Available Environmental microbes harbor an enormous pool of antibiotic and biocide resistance genes that can impact the resistance profiles of animal and human pathogens via horizontal gene transfer. Pseudomonas putida strains are ubiquitous in soil and water but have been seldom isolated from humans. We have established a collection of P. putida strains isolated from in-patients in different hospitals in France. One of the isolated strains (HB3267 kills insects and is resistant to the majority of the antibiotics used in laboratories and hospitals, including aminoglycosides, ß-lactams, cationic peptides, chromoprotein enediyne antibiotics, dihydrofolate reductase inhibitors, fluoroquinolones and quinolones, glycopeptide antibiotics, macrolides, polyketides and sulfonamides. Similar to other P. putida clinical isolates the strain was sensitive to amikacin. To shed light on the broad pattern of antibiotic resistance, which is rarely found in clinical isolates of this species, the genome of this strain was sequenced and analysed. The study revealed that the determinants of multiple resistance are both chromosomally-borne as well as located on the pPC9 plasmid. Further analysis indicated that pPC9 has recruited antibiotic and biocide resistance genes from environmental microorganisms as well as from opportunistic and true human pathogens. The pPC9 plasmid is not self-transmissible, but can be mobilized by other bacterial plasmids making it capable of spreading antibiotic resistant determinants to new hosts.

  10. Bioremediation Of Heavy Metals By Pseudomonas Putida Isolated From Groundwater In Egypt.

    Directory of Open Access Journals (Sweden)

    Fawazy

    2015-08-01

    Full Text Available In this present study total four bacterial isolates were obtained from 34 collected groundwater samples in 10th of Ramadan Sharkia governorate Egypt. These isolate were grown on nutrient agar supplemented with 1mgl of iron manganese and combination between them VV. Further testing of the bacterial isolates were grown on nutrient agar supplemented with different concentrations 2 4 5 6 7 8 and 9 mgl of iron and manganese. Out of four isolates one bacterial isolate no.83 has shown the resistance to heavy metals at maximum concentration of 8mgl. Selected isolate no.S83 was identified as Pseudomonas putida S83 according to Bergeys manual depending on morphological and biochemical characteristics. Transmission electron microscopy study of P. putida isolate no. S83 showed accumulation of heavy metal salts within and external to bacterial cells. P. putida S83 have higher removal efficiency of Fe2 94.5 and Mn2 94 at concentration 2 mgl and 96 hours.

  11. Identification and elucidation of in vivo function of two alanine racemases from Pseudomonas putida KT2440.

    Science.gov (United States)

    Duque, Estrella; Daddaoua, Abdelali; Cordero, Baldo F; De la Torre, Jesús; Antonia Molina-Henares, Maria; Ramos, Juan-Luis

    2017-10-01

    The genome of Pseudomonas putida KT2440 contains two open reading frames (ORFs), PP_3722 and PP_5269, that encode proteins with a Pyridoxal phosphate binding motif and a high similarity to alanine racemases. Alanine racemases play a key role in the biosynthesis of D-alanine, a crucial amino acid in the peptidoglycan layer. For these ORFs, we generated single and double mutants and found that inactivation of PP_5269 resulted in D-alanine auxotrophy, while inactivation of PP_3722 did not. Furthermore, as expected, the PP_3722/PP_5269 double mutant was a strict auxotroph for D-alanine. These results indicate that PP_5269 is an alr allele and that it is the essential alanine racemase in P. putida. We observed that the PP_5269 mutant grew very slowly, while the double PP_5269/PP_3722 mutant did not grow at all. This suggests that PP_3722 may replace PP_5269 in vivo. In fact, when the ORF encoding PP_3772 was cloned into a wide host range expression vector, ORF PP_3722 successfully complemented P. putida PP_5269 mutants. We purified both proteins to homogeneity and while they exhibit similar K M values, the V max of PP_5269 is fourfold higher than that of PP_3722. Here, we propose that PP_5269 and PP_3722 encode functional alanine racemases and that these genes be named alr-1 and alr-2 respectively. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  12. The AlkB Family of Fe(II)/α-Ketoglutarate-dependent Dioxygenases: Repairing Nucleic Acid Alkylation Damage and Beyond.

    Science.gov (United States)

    Fedeles, Bogdan I; Singh, Vipender; Delaney, James C; Li, Deyu; Essigmann, John M

    2015-08-21

    The AlkB family of Fe(II)- and α-ketoglutarate-dependent dioxygenases is a class of ubiquitous direct reversal DNA repair enzymes that remove alkyl adducts from nucleobases by oxidative dealkylation. The prototypical and homonymous family member is an Escherichia coli "adaptive response" protein that protects the bacterial genome against alkylation damage. AlkB has a wide variety of substrates, including monoalkyl and exocyclic bridged adducts. Nine mammalian AlkB homologs exist (ALKBH1-8, FTO), but only a subset functions as DNA/RNA repair enzymes. This minireview presents an overview of the AlkB proteins including recent data on homologs, structural features, substrate specificities, and experimental strategies for studying DNA repair by AlkB family proteins. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Occurrence of diverse alkane hydroxylase alkB genes in indigenous oil-degrading bacteria of Baltic Sea surface water.

    Science.gov (United States)

    Viggor, Signe; Jõesaar, Merike; Vedler, Eve; Kiiker, Riinu; Pärnpuu, Liis; Heinaru, Ain

    2015-12-30

    Formation of specific oil degrading bacterial communities in diesel fuel, crude oil, heptane and hexadecane supplemented microcosms of the Baltic Sea surface water samples was revealed. The 475 sequences from constructed alkane hydroxylase alkB gene clone libraries were grouped into 30 OPFs. The two largest groups were most similar to Pedobacter sp. (245 from 475) and Limnobacter sp. (112 from 475) alkB gene sequences. From 56 alkane-degrading bacterial strains 41 belonged to the Pseudomonas spp. and 8 to the Rhodococcus spp. having redundant alkB genes. Together 68 alkB gene sequences were identified. These genes grouped into 20 OPFs, half of them being specific only to the isolated strains. Altogether 543 diverse alkB genes were characterized in the brackish Baltic Sea water; some of them representing novel lineages having very low sequence identities with corresponding genes of the reference strains. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. In situ biosurfactant production and hydrocarbon removal by Pseudomonas putida CB-100 in bioaugmented and biostimulated oil-contaminated soil

    Directory of Open Access Journals (Sweden)

    Martínez-Toledo Ángeles

    2013-01-01

    Full Text Available In situ biosurfactant (rhamnolipid production by Pseudomonas putida CB-100 was achieved during a bioaugmented and biostimulated treatment to remove hydrocarbons from aged contaminated soil from oil well drilling operations. Rhamnolipid production and contaminant removal were determined for several treatments of irradiated and non-irradiated soils: nutrient addition (nitrogen and phosphorus, P. putida addition, and addition of both (P. putida and nutrients. The results were compared against a control treatment that consisted of adding only sterilized water to the soils. In treatment with native microorganisms (non-irradiated soils supplemented with P. putida, the removal of total petroleum hydrocarbons (TPH was 40.6%, the rhamnolipid production was 1.54 mg/kg, and a surface tension of 64 mN/m was observed as well as a negative correlation (R = -0.54; p < 0.019 between TPH concentration (mg/kg and surface tension (mN/m, When both bacteria and nutrients were involved, TPH levels were lowered to 33.7%, and biosurfactant production and surface tension were 2.03 mg/kg and 67.3 mN/m, respectively. In irradiated soil treated with P. putida, TPH removal was 24.5% with rhamnolipid generation of 1.79 mg/kg and 65.6 mN/m of surface tension, and a correlation between bacterial growth and biosurfactant production (R = -0.64; p < 0.009 was observed. When the nutrients and P. putida were added, TPH removal was 61.1%, 1.85 mg/kg of biosurfactants were produced, and the surface tension was 55.6 mN/m. In summary, in irradiated and non-irradiated soils, in situ rhamnolipid production by P. putida enhanced TPH decontamination of the soil.

  15. Positive effects of bio-nano Pd (0) toward direct electron transfer in Pseudomona putida and phenol biodegradation.

    Science.gov (United States)

    Niu, Zhuyu; Jia, Yating; Chen, Yuancai; Hu, Yongyou; Chen, Junfeng; Lv, Yuancai

    2018-06-08

    This study constructed a biological-inorganic hybrid system including Pseudomonas putida (P. putida) and bioreduced Pd (0) nanoparticles (NPs), and inspected the influence of bio-nano Pd (0) on the direct electron transfer and phenol biodegradation. Scanning electron microscopy and energy-dispersive X-ray spectroscopy (SEM-EDX) showed that bio-nano Pd (0) (~10 nm) were evenly dispersed on the surface and in the periplasm of P. putida. With the incorporation of bio-nano Pd (0), the redox currents of bacteria in the cyclic voltammetry (CV) became higher and the oxidation current increased as the addition of lactate, while the highest increase rates of two electron transfer system (ETS) rates were 63.97% and 33.79%, respectively. These results indicated that bio-nano Pd (0) could directly promote the electron transfer of P. putida. In phenol biodegradation process, P. putida-Pd (0)- 2 showed the highest k (0.2992 h -1 ), μ m (0.035 h -1 ) and K i (714.29 mg/L) and the lowest apparent K s (76.39 mg/L). The results of kinetic analysis indicated that bio-nano Pd (0) markedly enhanced the biocatalytic efficiency, substrate affinity and the growth of cells compared to native P. putida. The positive effects of bio-nano Pd (0) to the electron transfer of P. putida would promote the biodegradation of phenol. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Next-generation sequencing identifies deregulation of microRNAs involved in both innate and adaptive immune response in ALK+ ALCL.

    Directory of Open Access Journals (Sweden)

    Julia Steinhilber

    Full Text Available Anaplastic large cell lymphoma (ALCL is divided into two systemic diseases according to the expression of the anaplastic lymphoma kinase (ALK. We investigated the differential expression of miRNAs between ALK+ ALCL, ALK- ALCL cells and normal T-cells using next generation sequencing (NGS. In addition, a C/EBPβ-dependent miRNA profile was generated. The data were validated in primary ALCL cases. NGS identified 106 miRNAs significantly differentially expressed between ALK+ and ALK- ALCL and 228 between ALK+ ALCL and normal T-cells. We identified a signature of 56 miRNAs distinguishing ALK+ ALCL, ALK- ALCL and T-cells. The top candidates significant differentially expressed between ALK+ and ALK- ALCL included 5 upregulated miRNAs: miR-340, miR-203, miR-135b, miR-182, miR-183; and 7 downregulated: miR-196b, miR-155, miR-146a, miR-424, miR-503, miR-424*, miR-542-3p. The miR-17-92 cluster was also upregulated in ALK+ cells. Additionally, we identified a signature of 3 miRNAs significantly regulated by the transcription factor C/EBPβ, which is specifically overexpressed in ALK+ ALCL, including the miR-181 family. Of interest, miR-181a, which regulates T-cell differentiation and modulates TCR signalling strength, was significantly downregulated in ALK+ ALCL cases. In summary, our data reveal a miRNA signature linking ALK+ ALCL to a deregulated immune response and may reflect the abnormal TCR antigen expression known in ALK+ ALCL.

  17. Overcoming drug-tolerant cancer cell subpopulations showing AXL activation and epithelial–mesenchymal transition is critical in conquering ALK-positive lung cancer

    Science.gov (United States)

    Nakamichi, Shinji; Seike, Masahiro; Miyanaga, Akihiko; Chiba, Mika; Zou, Fenfei; Takahashi, Akiko; Ishikawa, Arimi; Kunugi, Shinobu; Noro, Rintaro; Kubota, Kaoru; Gemma, Akihiko

    2018-01-01

    Anaplastic lymphoma kinase tyrosine kinase inhibitors (ALK-TKIs) induce a dramatic response in non–small cell lung cancer (NSCLC) patients with the ALK fusion gene. However, acquired resistance to ALK-TKIs remains an inevitable problem. In this study, we aimed to discover novel therapeutic targets to conquer ALK-positive lung cancer. We established three types of ALK-TKI (crizotinib, alectinib and ceritinib)-resistant H2228 NSCLC cell lines by high exposure and stepwise methods. We found these cells showed a loss of ALK signaling, overexpressed AXL with epithelial-mesenchymal transition (EMT), and had cancer stem cell-like (CSC) properties, suggesting drug-tolerant cancer cell subpopulations. Similarly, we demonstrated that TGF-β1 treated H2228 cells also showed AXL overexpression with EMT features and ALK-TKI resistance. The AXL inhibitor, R428, or HSP90 inhibitor, ganetespib, were effective in reversing ALK-TKI resistance and EMT changes in both ALK-TKI-resistant and TGF-β1-exposed H2228 cells. Tumor volumes of xenograft mice implanted with established H2228-ceritinib-resistant (H2228-CER) cells were significantly reduced after treatment with ganetespib, or ganetespib in combination with ceritinib. Some ALK-positive NSCLC patients with AXL overexpression showed a poorer response to crizotinib therapy than patients with a low expression of AXL. ALK signaling-independent AXL overexpressed in drug-tolerant cancer cell subpopulations with EMT and CSC features may be commonly involved commonly involved in intrinsic and acquired resistance to ALK-TKIs. This suggests AXL and HSP90 inhibitors may be promising therapeutic drugs to overcome drug-tolerant cancer cell subpopulations in ALK-positive NSCLC patients for the reason that ALK-positive NSCLC cells do not live through ALK-TKI therapy. PMID:29930762

  18. Data driven polypharmacological drug design for lung cancer: analyses for targeting ALK, MET, and EGFR

    DEFF Research Database (Denmark)

    Narayanan, Dilip; Gani, Osman ABSM; Gruber, Franz XE

    2017-01-01

    encoded into molecular mechanics force fields. Cheminformatics analyses of binding data show EGFR to be dissimilar to ALK and MET, but its structure shows how it may be co-targeted with the addition of a covalent trap. This suggests a strategy for the design of a focussed chemical library based on a pan......Drug design of protein kinase inhibitors is now greatly enabled by thousands of publicly available X-ray structures, extensive ligand binding data, and optimized scaffolds coming off patent. The extensive data begin to enable design against a spectrum of targets (polypharmacology); however...... consider polypharmacological targeting of protein kinases ALK, MET, and EGFR (and its drug resistant mutant T790M) in non small cell lung cancer as an example. Both EGFR and ALK represent sources of primary oncogenic lesions, while drug resistance arises from MET amplification and EGFR mutation. A drug...

  19. ALK receptor activation, ligands and therapeutic targeting in glioblastoma and in other cancers

    International Nuclear Information System (INIS)

    Wellstein, Anton

    2012-01-01

    The intracellular anaplastic lymphoma kinase (ALK) fragment shows striking homology with members of the insulin receptor family and was initially identified as an oncogenic fusion protein resulting from a translocation in lymphoma and more recently in a range of cancers. The full-length ALK transmembrane receptor of ~220 kDa was identified based on this initial work. This tyrosine kinase receptor and its ligands, the growth factors pleiotrophin (PTN) and midkine (MK) are highly expressed during development of the nervous system and other organs. Each of these genes has been implicated in malignant progression of different tumor types and shown to alter phenotypes as well as signal transduction in cultured normal and tumor cells. Beyond its role in cancer, the ALK receptor pathway is thought to contribute to nervous system development, function, and repair, as well as metabolic homeostasis and the maintenance of tissue regeneration. ALK receptor activity in cancer can be up-regulated by amplification, overexpression, ligand binding, mutations in the intracellular domain of the receptor and by activity of the receptor tyrosine phosphatase PTPRz. Here we discuss the evidence for ligand control of ALK activity as well as the potential prognostic and therapeutic implications from gene expression and functional studies. An analysis of 18 published gene expression data sets from different cancers shows that overexpression of ALK, its smaller homolog LTK (leukocyte tyrosine kinase) and the ligands PTN and MK in cancer tissues from patients correlate significantly with worse course and outcome of the disease. This observation together with preclinical functional studies suggests that this pathway could be a valid therapeutic target for which complementary targeting strategies with small molecule kinase inhibitors as well as antibodies to ligands or the receptors may be used.

  20. Clinical and CT characteristics of surgically resected lung adenocarcinomas harboring ALK rearrangements or EGFR mutations

    International Nuclear Information System (INIS)

    Wang, Hua; Schabath, Matthew B.; Liu, Ying; Han, Ying; Li, Qi; Gillies, Robert J.; Ye, Zhaoxiang

    2016-01-01

    Purpose: To determine if clinical and CT characteristics of surgically resected lung adenocarcinomas can distinguish those harboring ALK rearrangements from EGFR mutations. Materials and methods: Patients who had surgical resection and histologically confirmed lung adenocarcinoma were enrolled, including 41 patients with ALK rearrangements and 66 patients with EGFR mutations. Eighteen categorical and six quantitative CT characteristics were used to evaluate the tumors. Differences in clinical and CT characteristics between the two groups were investigated. Results: Age (P = 0.003), histological subtypes (P < 0.001), pathological stage (P = 0.007), and five CT characteristics, including size (P < 0.001), GGO (P = 0.001), bubble-like lucency (P = 0.048), lymphadenopathy (P = 0.001), and tumor shadow disappearance rate (P = 0.005) were significantly different between patients harboring ALK rearrangements compared to patients with EGFR mutations. When we compared histologic components, a solid pattern was more common (P = 0.009) in tumors with ALK rearrangements, and lepidic and acinar patterns were more common (P < 0.001 and P = 0.040, respectively) in those with EGFR mutations. Backward elimination analyses revealed that age (OR = 0.93; 95% CI 0.89–0.98), GGO (OR = 0.14; 95% CI 0.03–0.67), and lymphadenopathy (OR = 4.15; 95% CI 1.49–11.60) were significantly associated with ALK rearrangement status. Conclusion: Our analyses revealed that clinical and CT characteristics of lung adenocarcinomas harboring ALK rearrangements were significantly different, compared with those with EGFR mutations. These differences may be related to the molecular pathology of these diseases.

  1. Clinical and CT characteristics of surgically resected lung adenocarcinomas harboring ALK rearrangements or EGFR mutations

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Hua [Department of Radiology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin (China); Schabath, Matthew B. [Department of Cancer Epidemiology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL (United States); Liu, Ying [Department of Radiology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin (China); Department of Cancer Imaging and Metabolism, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL (United States); Han, Ying [Department of Biotherapy, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin (China); Li, Qi [Department of Pathology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin (China); Gillies, Robert J. [Department of Cancer Imaging and Metabolism, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL (United States); Department of Radiology, H. Lee Moffitt Cancer Center and Research Institute, Tampa, FL (United States); Ye, Zhaoxiang, E-mail: yezhaoxiang@163.com [Department of Radiology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center of Cancer, Key Laboratory of Cancer Prevention and Therapy, Tianjin (China)

    2016-11-15

    Purpose: To determine if clinical and CT characteristics of surgically resected lung adenocarcinomas can distinguish those harboring ALK rearrangements from EGFR mutations. Materials and methods: Patients who had surgical resection and histologically confirmed lung adenocarcinoma were enrolled, including 41 patients with ALK rearrangements and 66 patients with EGFR mutations. Eighteen categorical and six quantitative CT characteristics were used to evaluate the tumors. Differences in clinical and CT characteristics between the two groups were investigated. Results: Age (P = 0.003), histological subtypes (P < 0.001), pathological stage (P = 0.007), and five CT characteristics, including size (P < 0.001), GGO (P = 0.001), bubble-like lucency (P = 0.048), lymphadenopathy (P = 0.001), and tumor shadow disappearance rate (P = 0.005) were significantly different between patients harboring ALK rearrangements compared to patients with EGFR mutations. When we compared histologic components, a solid pattern was more common (P = 0.009) in tumors with ALK rearrangements, and lepidic and acinar patterns were more common (P < 0.001 and P = 0.040, respectively) in those with EGFR mutations. Backward elimination analyses revealed that age (OR = 0.93; 95% CI 0.89–0.98), GGO (OR = 0.14; 95% CI 0.03–0.67), and lymphadenopathy (OR = 4.15; 95% CI 1.49–11.60) were significantly associated with ALK rearrangement status. Conclusion: Our analyses revealed that clinical and CT characteristics of lung adenocarcinomas harboring ALK rearrangements were significantly different, compared with those with EGFR mutations. These differences may be related to the molecular pathology of these diseases.

  2. Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins.

    Science.gov (United States)

    Zdżalik, Daria; Domańska, Anna; Prorok, Paulina; Kosicki, Konrad; van den Born, Erwin; Falnes, Pål Ø; Rizzo, Carmelo J; Guengerich, F Peter; Tudek, Barbara

    2015-06-01

    AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N(2)-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. Copyright © 2015 Elsevier B

  3. The second-generation ALK inhibitor alectinib effectively induces apoptosis in human neuroblastoma cells and inhibits tumor growth in a TH-MYCN transgenic neuroblastoma mouse model.

    Science.gov (United States)

    Lu, Jiaxiong; Guan, Shan; Zhao, Yanling; Yu, Yang; Woodfield, Sarah E; Zhang, Huiyuan; Yang, Kristine L; Bieerkehazhi, Shayahati; Qi, Lin; Li, Xiaonan; Gu, Jerry; Xu, Xin; Jin, Jingling; Muscal, Jodi A; Yang, Tianshu; Xu, Guo-Tong; Yang, Jianhua

    2017-08-01

    Activating germline mutations of anaplastic lymphoma kinase (ALK) occur in most cases of hereditary neuroblastoma (NB) and the constitutively active kinase activity of ALK promotes cell proliferation and survival in NB. Therefore, ALK kinase is a potential therapeutic target for NB. In this study, we show that the novel ALK inhibitor alectinib effectively suppressed cell proliferation and induces apoptosis in NB cell lines with either wild-type ALK or mutated ALK (F1174L and D1091N) by blocking ALK-mediated PI3K/Akt/mTOR signaling. In addition, alectinib enhanced doxorubicin-induced cytotoxicity and apoptosis in NB cells. Furthermore, alectinib induced apoptosis in an orthotopic xenograft NB mouse model. Also, in the TH-MYCN transgenic mouse model, alectinib resulted in decreased tumor growth and prolonged survival time. These results indicate that alectinib may be a promising therapeutic agent for the treatment of NB. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Activity and three-dimensional distribution of toluene-degrading Pseudomonas putida in a multispecies biofilm assessed by quantitative in situ hybridization and scanning confocal laser microscopy

    DEFF Research Database (Denmark)

    Møller, Søren; Pedersen, Anne Rathmann; Poulsen, L.K.

    1996-01-01

    As a representative member of the toluene-degrading population in a biofilter for waste gas treatment, Pseudomonas putida was investigated with a 16S rRNA targeting probe, The three-dimensional distribution of P. putida was visualized in the biofilm matrix by scanning confocal laser microscopy...

  5. Simultaneous VENTANA IHC and RT-PCR testing of ALK status in Chinese non-small cell lung cancer patients and response to crizotinib.

    Science.gov (United States)

    Xu, Chun-Wei; Wang, Wen-Xian; Chen, Yan-Ping; Chen, Yu; Liu, Wei; Zhong, Li-Hua; Chen, Fang-Fang; Zhuang, Wu; Song, Zheng-Bo; Chen, Xiao-Hui; Huang, Yun-Jian; Guan, Yan-Fang; Yi, Xin; Lv, Tang-Feng; Zhu, Wei-Feng; Lu, Jian-Ping; Wang, Xiao-Jiang; Shi, Yi; Lin, Xian-Dong; Chen, Gang; Song, Yong

    2018-04-11

    ALK rearrangement-advanced NSCLC patients respond to crizotinib. ALK rearrangement is currently determined with RT-PCR. VENTANA IHC is a standard method to identify ALK protein overexpression in NSCLC; however, VENTANA IHC has rarely been used to determine the response to crizotinib in Chinese patients with NSCLC and ALK overexpression. To better clarify the clinical implication of VENTANA IHC to detect ALK rearrangements, we conducted this study to analyze VENTANA IHC and RT-PCR in a large cohort of Chinese patients with NSCLC undergoing screening for ALK rearrangements. A total of 1720 patients with NSCLC who had ALK rearrangements detected by VENTANA IHC and/or RT-PCR were included in this analysis. We compared the efficacy and survival of ALK-positive patients detected by VENTANA IHC and RT-PCR. We used NGS to identify patients in whom the two methods were inconsistent. Among 1720 patients, 187 (10.87%) were shown to be ALK-positive by VENTANA IHC and/or RT-PCR, and 66 received crizotinib treatment. We identified 10.27% (172/1674) of patients as ALK-positive by the VENTANA IHC method, and 12.73% (41/322) of patients had ALK rearrangements by the RT-PCR method. Twenty-nine of 276 (10.51%) ALK-positive patients were simultaneously analyzed using VENTANA IHC and RT-PCR. The overall response rates were 65.90% (29/44) by VENTANA IHC and 55.88% (19/34) by RT-PCR. The disease control rates were 86.36% (38/44) by VENTANA IHC and 76.47% (26/34) by RT-PCR. In contrast, the median progression-free survival for VENTANA IHC and RT-PCR was 8.5 and 9.2 months, respectively. The VENTANA IHC and RT-PCR results obtained for 6 of 17 ALK-positive patients were inconsistent based on NGS; specifically, 4 patients had EML4-ALK fusions, 2 patients had non EML4-ALK fusions, 1 patient had a KCL1-ALK fusion, and one patient had a FBXO36-ALK fusion. VENTANA IHC is a reliable and rapid screening tool used in routine pathologic laboratories for the identification of suitable candidates for

  6. Imaging Characteristics in ALK Fusion-Positive Lung Adenocarcinomas by Using HRCT

    Science.gov (United States)

    Okumura, Sakae; Kuroda, Hiroaki; Uehara, Hirofumi; Mun, Mingyon; Takeuchi, Kengo; Nakagawa, Ken

    2014-01-01

    Objectives: We aimed to identify high-resolution computed tomography (HRCT) features useful to distinguish the anaplastic lymphoma kinase gene (ALK) fusion-positive and negative lung adenocarcinomas. Methods: We included 236 surgically resected adenocarcinoma lesions, which included 27 consecutive ALK fusion-positive (AP) lesions, 115 epidermal growth factor receptor mutation-positive lesions, and 94 double-negative lesions. HRCT parameters including size, air bronchograms, pleural indentation, spiculation, and tumor disappearance rate (TDR) were compared. In addition, prevalence of small lesions (≤20 mm) and solid lesions (TDR ≤20%) were compared. Results: AP lesions were significantly smaller and had lower TDR (%) than ALK fusion-negative (AN) lesions (tumor diameter: 20.7 mm ± 14.1 mm vs. 27.4 mm ± 13.8 mm, respectively, p 20 mm (n = 7, 25.9%) showed a solid pattern. Among all small lesions, AP lesions had lower TDR and more frequent spiculation than AN lesions (p 20 mm lesions may be ALK fusion-negative. PMID:24899136

  7. A case of lung adenocarcinoma harboring EGFR mutation and EML4-ALK fusion gene

    International Nuclear Information System (INIS)

    Tanaka, Hisashi; Hayashi, Akihito; Morimoto, Takeshi; Taima, Kageaki; Tanaka, Yoshihito; Shimada, Michiko; Kurose, Akira; Takanashi, Shingo; Okumura, Ken

    2012-01-01

    Lung cancer is the leading cause of cancer-related death worldwide. Epidermal growth factor receptor (EGFR) - tyrosine kinase inhibitor (TKI) is used for the patients with EGFR-mutant lung cancer. Recently, phase III studies in the patients with EGFR-mutant demonstrated that EGFR-TKI monotherapy improved progression-free survival compared with platinum-doublet chemotherapy. The echinoderm microtubule-associated protein-like 4 (EML4) - anaplastic lymphoma kinase (ALK) fusion oncogene represents one of the newest molecular targets in non-small cell lung cancer (NSCLC). Patients who harbor EML4-ALK fusions have been associated with a lack of EGFR or KRAS mutations. We report a 39-year-old patient diagnosed as adenocarcinoma harboring EGFR mutation and EML4-ALK fusion gene. We treated this patient with erlotinib as the third line therapy, but no clinical benefit was obtained. We experienced a rare case with EGFR mutation and EML4-ALK. Any clinical benefit using EGFR-TKI was not obtained in our case. The therapeutic choice for the patients with more than one driver mutations is unclear. We needs further understanding of the lung cancer molecular biology and the biomarker infomation

  8. Economic Analysis of Alternative Strategies for Detection of ALK Rearrangements in Non Small Cell Lung Cancer.

    Science.gov (United States)

    Doshi, Shivang; Ray, David; Stein, Karen; Zhang, Jie; Koduru, Prasad; Fogt, Franz; Wellman, Axel; Wat, Ricky; Mathews, Charles

    2016-01-06

    Identification of alterations in ALK gene and development of ALK-directed therapies have increased the need for accurate and efficient detection methodologies. To date, research has focused on the concordance between the two most commonly used technologies, fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC). However, inter-test concordance reflects only one, albeit important, aspect of the diagnostic process; laboratories, hospitals, and payors must understand the cost and workflow of ALK rearrangement detection strategies. Through literature review combined with interviews of pathologists and laboratory directors in the U.S. and Europe, a cost-impact model was developed that compared four alternative testing strategies-IHC only, FISH only, IHC pre-screen followed by FISH confirmation, and parallel testing by both IHC and FISH. Interviews were focused on costs of reagents, consumables, equipment, and personnel. The resulting model showed that testing by IHC alone cost less ($90.07 in the U.S., $68.69 in Europe) than either independent or parallel testing by both FISH and IHC ($441.85 in the U.S. and $279.46 in Europe). The strategies differed in cost of execution, turnaround time, reimbursement, and number of positive results detected, suggesting that laboratories must weigh the costs and the clinical benefit of available ALK testing strategies.

  9. Economic Analysis of Alternative Strategies for Detection of ALK Rearrangements in Non Small Cell Lung Cancer

    Directory of Open Access Journals (Sweden)

    Shivang Doshi

    2016-01-01

    Full Text Available Identification of alterations in ALK gene and development of ALK-directed therapies have increased the need for accurate and efficient detection methodologies. To date, research has focused on the concordance between the two most commonly used technologies, fluorescent in situ hybridization (FISH and immunohistochemistry (IHC. However, inter-test concordance reflects only one, albeit important, aspect of the diagnostic process; laboratories, hospitals, and payors must understand the cost and workflow of ALK rearrangement detection strategies. Through literature review combined with interviews of pathologists and laboratory directors in the U.S. and Europe, a cost-impact model was developed that compared four alternative testing strategies—IHC only, FISH only, IHC pre-screen followed by FISH confirmation, and parallel testing by both IHC and FISH. Interviews were focused on costs of reagents, consumables, equipment, and personnel. The resulting model showed that testing by IHC alone cost less ($90.07 in the U.S., $68.69 in Europe than either independent or parallel testing by both FISH and IHC ($441.85 in the U.S. and $279.46 in Europe. The strategies differed in cost of execution, turnaround time, reimbursement, and number of positive results detected, suggesting that laboratories must weigh the costs and the clinical benefit of available ALK testing strategies.

  10. Meta-analysis of Neuroblastomas Reveals a Skewed ALK Mutation Spectrum in Tumors with MYCN Amplification

    NARCIS (Netherlands)

    de Brouwer, Sara; de Preter, Katleen; Kumps, Candy; Zabrocki, Piotr; Porcu, Michaël; Westerhout, Ellen M.; Lakeman, Arjan; Vandesompele, Jo; Hoebeeck, Jasmien; van Maerken, Tom; de Paepe, Anne; Laureys, Geneviève; Schulte, Johannes H.; Schramm, Alexander; van den Broecke, Caroline; Vermeulen, Joëlle; van Roy, Nadine; Beiske, Klaus; Renard, Marleen; Noguera, Rosa; Delattre, Olivier; Janoueix-Lerosey, Isabelle; Kogner, Per; Martinsson, Tommy; Nakagawara, Akira; Ohira, Miki; Caron, Huib N.; Eggert, Angelika; Cools, Jan; Versteeg, Rogier; Speleman, Frank

    2010-01-01

    Purpose: Activating mutations of the anaplastic lymphoma kinase (ALK) were recently described in neuroblastoma. We carried out a meta-analysis of 709 neuroblastoma tumors to determine their frequency and mutation spectrum in relation to genomic and clinical parameters, and studied the prognostic

  11. Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells

    Science.gov (United States)

    Kraehling, Jan R.; Chidlow, John H.; Rajagopal, Chitra; Sugiyama, Michael G.; Fowler, Joseph W.; Lee, Monica Y.; Zhang, Xinbo; Ramírez, Cristina M.; Park, Eon Joo; Tao, Bo; Chen, Keyang; Kuruvilla, Leena; Larriveé, Bruno; Folta-Stogniew, Ewa; Ola, Roxana; Rotllan, Noemi; Zhou, Wenping; Nagle, Michael W.; Herz, Joachim; Williams, Kevin Jon; Eichmann, Anne; Lee, Warren L.; Fernández-Hernando, Carlos; Sessa, William C.

    2016-01-01

    In humans and animals lacking functional LDL receptor (LDLR), LDL from plasma still readily traverses the endothelium. To identify the pathways of LDL uptake, a genome-wide RNAi screen was performed in endothelial cells and cross-referenced with GWAS-data sets. Here we show that the activin-like kinase 1 (ALK1) mediates LDL uptake into endothelial cells. ALK1 binds LDL with lower affinity than LDLR and saturates only at hypercholesterolemic concentrations. ALK1 mediates uptake of LDL into endothelial cells via an unusual endocytic pathway that diverts the ligand from lysosomal degradation and promotes LDL transcytosis. The endothelium-specific genetic ablation of Alk1 in Ldlr-KO animals leads to less LDL uptake into the aortic endothelium, showing its physiological role in endothelial lipoprotein metabolism. In summary, identification of pathways mediating LDLR-independent uptake of LDL may provide unique opportunities to block the initiation of LDL accumulation in the vessel wall or augment hepatic LDLR-dependent clearance of LDL. PMID:27869117

  12. An Unusual Case of Systemic Inflammatory Myofibroblastic Tumor with Successful Treatment with ALK-Inhibitor

    Directory of Open Access Journals (Sweden)

    Sanjivini V. Jacob

    2014-01-01

    Full Text Available Systemic inflammatory myofibroblastic tumor is an exceedingly rare entity. A 45-year-old Hispanic female presented with a 6-month history of left-sided thigh pain, low back pain, and generalized weakness. PET/CT scan revealed abnormal activity in the liver, adrenal gland, and pancreas. MRI of the abdomen demonstrated two 6-7 cm masses in the liver. MRI of the lumbar spine demonstrated lesions in the L2 to L4 spinous processes, paraspinal muscles, and subcutaneous tissues, as well as an 8 mm enhancing intradural lesion at T11, all thought to be metastatic disease. A biopsy of the liver showed portal tract expansion by a spindle cell proliferation rich in inflammation. Tumor cells showed immunoreactivity for smooth muscle actin and anaplastic lymphoma kinase 1 (ALK1. Tissue from the L5 vertebra showed a process histologically identical to that seen in the liver. FISH analysis of these lesions demonstrated an ALK (2p23 gene rearrangement. The patient was successfully treated with an ALK-inhibitor, Crizotinib, and is now in complete remission. We present the first reported case, to our knowledge, of inflammatory myofibroblastic tumor with systemic manifestations and ALK translocation. This case is a prime example of how personalized medicine has vastly improved patient care through the use of molecular-targeted therapy.

  13. ALK-FISH borderline cases in non-small cell lung cancer: Implications for diagnostics and clinical decision making.

    Science.gov (United States)

    von Laffert, Maximilian; Stenzinger, Albrecht; Hummel, Michael; Weichert, Wilko; Lenze, Dido; Warth, Arne; Penzel, Roland; Herbst, Hermann; Kellner, Udo; Jurmeister, Philipp; Schirmacher, Peter; Dietel, Manfred; Klauschen, Frederick

    2015-12-01

    Fluorescence in-situ hybridization (FISH) for the detection of ALK-rearrangements in non-small cell lung cancer (NSCLC) is based on at first sight clear cut-off criteria (≥15% of tumor cells) for split signals (SS) and single red signals (SRS). However, NSCLC with SS-counts around the cut-off may cause interpretation problems. Tissue microarrays containing 753 surgically resected NSCLCs were independently tested for ALK-alterations by FISH and immunohistochemistry (IHC). Our analysis focused on samples with SS/SRS in the range between 10% and 20% (ALK-FISH borderline group). To better understand the role of these samples in routine diagnostics, we performed statistical analyses to systematically estimate the probability of ALK-FISH-misclassification (false negative or positive) for different numbers of evaluated tumor cell nuclei (30, 50, 100, and 200). 94.3% (710/753) of the cases were classified as unequivocally (FISH-negative (93%; 700/753) or positive (1.3%; 10/753) and showed concordant IHC results. 5.7% (43/753) of the samples showed SS/SRS between 10% and 20% of the tumor cells. Out of these, 7% (3/43; ALK-FISH: 14%, 18% and 20%) were positive by ALK-IHC, while 93% (40/43) had no detectable expression of the ALK-protein. Statistical analysis showed that ALK-FISH misclassifications occur frequently for samples with rearrangements between 10% and 20% if ALK-characterization is based on a sharp cut-off point (15%). If results in this interval are defined as equivocal (borderline), statistical sampling-related ALK-FISH misclassifications will occur in less than 1% of the cases if 100 tumor cells are evaluated. While ALK status can be determined robustly for the majority of NSCLC by FISH our analysis showed that ∼6% of the cases belong to a borderline group for which ALK-FISH evaluation has only limited reliability due to statistical sampling effects. These cases should be considered equivocal and therapy decisions should include additional tests and clinical

  14. Pseudomonas putida response in membrane bioreactors under salicylic acid-induced stress conditions

    Energy Technology Data Exchange (ETDEWEB)

    Collado, Sergio; Rosas, Irene; González, Elena; Gutierrez-Lavin, Antonio; Diaz, Mario, E-mail: mariodiaz@uniovi.es

    2014-02-01

    Highlights: • MBR under feed-induced stress conditions: starvation and changing feeding conditions. • High capacity of MBR to withstand high variations in feed loads. • Slow biofilm formation under starvation conditions during the first days. • Observed growth of P. putida for substrate to microorganism ratio higher than 0.6 g/g. • Maximum specific growth rate and growth yield values of around 37.5 h{sup −1} and 0.5 g/g. - Abstract: Starvation and changing feeding conditions are frequently characteristics of wastewater treatment plants. They are typical causes of unsteady-state operation of biological systems and provoke cellular stress. The response of a membrane bioreactor functioning under feed-induced stress conditions is studied here. In order to simplify and considerably amplify the response to stress and to obtain a reference model, a pure culture of Pseudomonas putida was selected instead of an activated sludge and a sole substrate (salicylic acid) was employed. The system degraded salicylic acid at 100–1100 mg/L with a high level of efficiency, showed rapid acclimation without substrate or product inhibition phenomena and good stability in response to unsteady states caused by feed variations. Under starvation conditions, specific degradation rates of around 15 mg/g h were achieved during the adaptation of the biomass to the new conditions and no biofilm formation was observed during the first days of experimentation using an initial substrate to microorganisms ratio lower than 0.1. When substrate was added to the reactor as pulses resulting in rapidly changing concentrations, P. putida growth was observed only for substrate to microorganism ratios higher than 0.6, with a maximum Y{sub X/S} of 0.5 g/g. Biofilm development under changing feeding conditions was fast, biomass detachment only being significant for biomass concentrations on the membrane surface that were higher than 16 g/m{sup 2}.

  15. Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O2.

    Science.gov (United States)

    Ibrahim, Susan A; Crack, Jason C; Rolfe, Matthew D; Borrero-de Acuña, José Manuel; Thomson, Andrew J; Le Brun, Nick E; Schobert, Max; Stapleton, Melanie R; Green, Jeffrey

    2015-07-03

    The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Kinetics of mercury reduction by Serratia marcescens mercuric reductase expressed by pseudomonas putida strains

    Energy Technology Data Exchange (ETDEWEB)

    Schneider, M.; Deckwer, W.D. [GBF-Gesellschaft fuer Biotechnologische Forschung mbH, Abteilung TU-BCE, Mascheroder Weg 1, D-38124 Braunschweig (Germany)

    2005-10-01

    Mercury (Hg) resistance is widespread among microorganisms and is based on the intracellular transformation of Hg(II) to less toxic elemental Hg(0). The use of microbial consortia to demercurize polluted wastewater streams and environments has been demonstrated. To develop efficient and versatile microbial cleanup strategies requires detailed knowledge of transport and reaction rates. This study focuses on the kinetics of the key enzyme of the microbial transformation, e.g., the mercuric reductase (MerA) under conditions closely resembling the cell interior. To this end, previously constructed and characterized Pseudomonas putida strains expressing MerA from Serratia marcescens were applied. Of the P. putida strains considered in this study P. putida KT2442::mer73 constitutively expressing broad spectrum mercury resistance (merTPAB) yielded the highest mercuric reductase (MerA) activity directly after cell disruption. MerA in the raw extract was further purified (about 100 fold). Reduction rates were measured for various substrates (HgCl{sub 2}, Hg{sub 2}SO{sub 4}, Hg(NO{sub 3}){sub 2} and phenyl mercury acetate) up to high concentrations dependent on the purification grade. In all cases, a pronounced substrate inhibition was found. The kinetic constants determined for the cell raw extract are in agreement with those measured for intact cells. However, the rate data exhibit reduced affinity and inhibition with rising purification grade (specific activity). Therefore, the findings seemingly point to reactions preceding the catalytic reduction. Based on simplified assumptions, a kinetic model is suggested which reasonably describes the experimental findings and can advantageously be applied to the bioreactor design. (Abstract Copyright [2005], Wiley Periodicals, Inc.)

  17. Pseudomonas putida response in membrane bioreactors under salicylic acid-induced stress conditions

    International Nuclear Information System (INIS)

    Collado, Sergio; Rosas, Irene; González, Elena; Gutierrez-Lavin, Antonio; Diaz, Mario

    2014-01-01

    Highlights: • MBR under feed-induced stress conditions: starvation and changing feeding conditions. • High capacity of MBR to withstand high variations in feed loads. • Slow biofilm formation under starvation conditions during the first days. • Observed growth of P. putida for substrate to microorganism ratio higher than 0.6 g/g. • Maximum specific growth rate and growth yield values of around 37.5 h −1 and 0.5 g/g. - Abstract: Starvation and changing feeding conditions are frequently characteristics of wastewater treatment plants. They are typical causes of unsteady-state operation of biological systems and provoke cellular stress. The response of a membrane bioreactor functioning under feed-induced stress conditions is studied here. In order to simplify and considerably amplify the response to stress and to obtain a reference model, a pure culture of Pseudomonas putida was selected instead of an activated sludge and a sole substrate (salicylic acid) was employed. The system degraded salicylic acid at 100–1100 mg/L with a high level of efficiency, showed rapid acclimation without substrate or product inhibition phenomena and good stability in response to unsteady states caused by feed variations. Under starvation conditions, specific degradation rates of around 15 mg/g h were achieved during the adaptation of the biomass to the new conditions and no biofilm formation was observed during the first days of experimentation using an initial substrate to microorganisms ratio lower than 0.1. When substrate was added to the reactor as pulses resulting in rapidly changing concentrations, P. putida growth was observed only for substrate to microorganism ratios higher than 0.6, with a maximum Y X/S of 0.5 g/g. Biofilm development under changing feeding conditions was fast, biomass detachment only being significant for biomass concentrations on the membrane surface that were higher than 16 g/m 2

  18. Co-clinical quantitative tumor volume imaging in ALK-rearranged NSCLC treated with crizotinib

    Energy Technology Data Exchange (ETDEWEB)

    Nishino, Mizuki, E-mail: Mizuki_Nishino@DFCI.HARVARD.EDU [Department of Radiology, Brigham and Women’s Hospital, 450 Brookline Ave., Boston MA, 02215 (United States); Department of Imaging, Dana-Farber Cancer Institute, 450 Brookline Ave., Boston MA, 02215 (United States); Sacher, Adrian G.; Gandhi, Leena; Chen, Zhao; Akbay, Esra [Department of Medical Oncology and Department of Medicine Dana-Farber Cancer Institute and Brigham and Women’s Hospital 450 Brookline Ave., Boston MA, 02215 (United States); Fedorov, Andriy; Westin, Carl F.; Hatabu, Hiroto [Department of Radiology, Brigham and Women’s Hospital, 450 Brookline Ave., Boston MA, 02215 (United States); Johnson, Bruce E.; Hammerman, Peter; Wong, Kwok-kin [Department of Medical Oncology and Department of Medicine Dana-Farber Cancer Institute and Brigham and Women’s Hospital 450 Brookline Ave., Boston MA, 02215 (United States)

    2017-03-15

    Highlights: • Role of co-clinical studies in precision cancer medicine is increasingly recognized. • This study compared tumor volume in co-clinical trials of ALK-rearranged NSCLC. • Similarities and differences of tumor volume changes in mice and humans were noted. • The study provides insights to optimize murine co-clinical trial designs. - Abstract: Purpose: To evaluate and compare the volumetric tumor burden changes during crizotinib therapy in mice and human cohorts with ALK-rearranged non-small-cell lung cancer (NSCLC). Methods: Volumetric tumor burden was quantified on serial imaging studies in 8 bitransgenic mice with ALK-rearranged adenocarcinoma treated with crizotinib, and in 33 human subjects with ALK-rearranged NSCLC treated with crizotinib. The volumetric tumor burden changes and the time to maximal response were compared between mice and humans. Results: The median tumor volume decrease (%) at the maximal response was −40.4% (range: −79.5%–+11.7%) in mice, and −72.9% (range: −100%–+72%) in humans (Wilcoxon p = 0.03). The median time from the initiation of therapy to maximal response was 6 weeks in mice, and 15.7 weeks in humans. Overall volumetric response rate was 50% in mice and 97% in humans. Spider plots of tumor volume changes during therapy demonstrated durable responses in the human cohort, with a median time on therapy of 13.1 months. Conclusion: The present study described an initial attempt to evaluate quantitative tumor burden changes in co-clinical imaging studies of genomically-matched mice and human cohorts with ALK-rearranged NSCLC treated with crizotinib. Differences are noted in the degree of maximal volume response between the two cohorts in this well-established paradigm of targeted therapy, indicating a need for further studies to optimize co-clinical trial design and interpretation.

  19. Alkane Hydroxylase Gene (alkB Phylotype Composition and Diversity in Northern Gulf of Mexico Bacterioplankton

    Directory of Open Access Journals (Sweden)

    Conor Blake Smith

    2013-12-01

    Full Text Available Natural and anthropogenic activities introduce alkanes into marine systems where they are degraded by alkane hydroxylases expressed by phylogenetically diverse bacteria. Partial sequences for alkB, one of the structural genes of alkane hydroxylase, have been used to assess the composition of alkane-degrading communities, and to determine their responses to hydrocarbon inputs. We present here the first spatially extensive analysis of alkB in bacterioplankton of the northern Gulf of Mexico (nGoM, a region that experiences numerous hydrocarbon inputs. We have analyzed 401 partial alkB gene sequences amplified from genomic extracts collected during March 2010 from 17 water column samples that included surface waters and bathypelagic depths. Previous analyses of 16S rRNA gene sequences for these and related samples have shown that nGoM bacterial community composition and structure stratify strongly with depth, with distinctly different communities above and below 100 m. Although we hypothesized that alkB gene sequences would exhibit a similar pattern, PCA analyses of operational protein units (OPU indicated that community composition did not vary consistently with depth or other major physical-chemical variables. We observed 22 distinct OPUs, one of which was ubiquitous and accounted for 57% of all sequences. This OPU clustered with alkB sequences from known hydrocarbon oxidizers (e.g., Alcanivorax and Marinobacter. Some OPUs could not be associated with known alkane degraders, however, and perhaps represent novel hydrocarbon-oxidizing populations or genes. These results indicate that the capacity for alkane hydrolysis occurs widely in the nGoM, but that alkane degrader diversity varies substantially among sites and responds differently than bulk communities to physical-chemical variables.

  20. ALK Positive Lung Cancer: Clinical Profile, Practice and Outcomes in a Developing Country.

    Directory of Open Access Journals (Sweden)

    Vanita Noronha

    Full Text Available To evaluate the performance and treatment profile of advanced EML4-ALK positive Non-small cell lung cancer (NSCLC patients in a developing country with potentially restricted access to Crizotinib.A retrospective analysis of advanced ALK positive NSCLC patients who were treated from June 2012 to September 2015 was conducted. The primary goal was to evaluate outcomes of advanced ALK positive NSCLC in our practice and examine the logistic constraints in procuring Crizotinib.94 patients were available for analysis. 21 (22.3% patients were started on Crizotinib upfront, 60 (63.8% on chemotherapy, 10 (10.6% on Tyrosine kinase inhibitors (in view of poor PS and 3 (3.2% patients were offered best supportive care. Reasons for not starting Crizotinib upfront included symptomatic patients needing early initiation of therapy (23.3%, ALK not tested upfront (23.3% and financial constraints (21.9%. 69 patients (73.4% received Crizotinib at some stage during treatment. Dose interruptions (> 1 week with Crizotinib were seen in 20 patients (29%, with drug toxicity being the commonest reason (85%. Median Progression free survival (PFS on first line therapy for the entire cohort was 10 months, with a significant difference between patients receiving Crizotinib and those who did not ever receive Crizotinib (10 months vs. 2 months, p = 0.028. Median Overall Survival (OS was not reached for the entire cohort, with 1 year survival being 81.2%. Patients with an ECOG Performance Status (PS of >2 had a significantly reduced PFS compared to patients with PS < = 2 (1.5 months vs. 11 months, p< 0.001. 47 patients with financial constraints (68.1% received Crizotinib completely free via various extramural support schemes.A majority of our ALK positive NSCLC patients were exposed to Crizotinib through the help of various support mechanisms and these patients had similar outcomes to that reported from previously published literature.

  1. DHPLC-based mutation analysis of ENG and ALK-1 genes in HHT Italian population.

    Science.gov (United States)

    Lenato, Gennaro M; Lastella, Patrizia; Di Giacomo, Marilena C; Resta, Nicoletta; Suppressa, Patrizia; Pasculli, Giovanna; Sabbà, Carlo; Guanti, Ginevra

    2006-02-01

    Hereditary haemorrhagic telangiectasia (HHT or Rendu-Osler-Weber syndrome) is an autosomal dominant disorder characterized by localized angiodysplasia due to mutations in endoglin, ALK-1 gene, and a still unidentified locus. The lack of highly recurrent mutations, locus heterogeneity, and the presence of mutations in almost all coding exons of the two genes makes the screening for mutations time-consuming and costly. In the present study, we developed a DHPLC-based protocol for mutation detection in ALK1 and ENG genes through retrospective analysis of known sequence variants, 20 causative mutations and 11 polymorphisms, and a prospective analysis on 47 probands with unknown mutation. Overall DHPLC analysis identified the causative mutation in 61 out 66 DNA samples (92.4%). We found 31 different mutations in the ALK1 gene, of which 15 are novel, and 20, of which 12 are novel, in the ENG gene, thus providing for the first time the mutational spectrum in a cohort of Italian HHT patients. In addition, we characterized the splicing pattern of ALK1 gene in lymphoblastoid cells, both in normal controls and in two individuals carrying a mutation in the non-invariant -3 position of the acceptor splice site upstream exon 6 (c.626-3C>G). Functional essay demonstrated the existence, also in normal individuals, of a small proportion of ALK1 alternative splicing, due to exon 5 skipping, and the presence of further aberrant splicing isoforms in the individuals carrying the c.626-3C>G mutation. 2006 Wiley-Liss, Inc.

  2. Co-clinical quantitative tumor volume imaging in ALK-rearranged NSCLC treated with crizotinib

    International Nuclear Information System (INIS)

    Nishino, Mizuki; Sacher, Adrian G.; Gandhi, Leena; Chen, Zhao; Akbay, Esra; Fedorov, Andriy; Westin, Carl F.; Hatabu, Hiroto; Johnson, Bruce E.; Hammerman, Peter; Wong, Kwok-kin

    2017-01-01

    Highlights: • Role of co-clinical studies in precision cancer medicine is increasingly recognized. • This study compared tumor volume in co-clinical trials of ALK-rearranged NSCLC. • Similarities and differences of tumor volume changes in mice and humans were noted. • The study provides insights to optimize murine co-clinical trial designs. - Abstract: Purpose: To evaluate and compare the volumetric tumor burden changes during crizotinib therapy in mice and human cohorts with ALK-rearranged non-small-cell lung cancer (NSCLC). Methods: Volumetric tumor burden was quantified on serial imaging studies in 8 bitransgenic mice with ALK-rearranged adenocarcinoma treated with crizotinib, and in 33 human subjects with ALK-rearranged NSCLC treated with crizotinib. The volumetric tumor burden changes and the time to maximal response were compared between mice and humans. Results: The median tumor volume decrease (%) at the maximal response was −40.4% (range: −79.5%–+11.7%) in mice, and −72.9% (range: −100%–+72%) in humans (Wilcoxon p = 0.03). The median time from the initiation of therapy to maximal response was 6 weeks in mice, and 15.7 weeks in humans. Overall volumetric response rate was 50% in mice and 97% in humans. Spider plots of tumor volume changes during therapy demonstrated durable responses in the human cohort, with a median time on therapy of 13.1 months. Conclusion: The present study described an initial attempt to evaluate quantitative tumor burden changes in co-clinical imaging studies of genomically-matched mice and human cohorts with ALK-rearranged NSCLC treated with crizotinib. Differences are noted in the degree of maximal volume response between the two cohorts in this well-established paradigm of targeted therapy, indicating a need for further studies to optimize co-clinical trial design and interpretation.

  3. Vanillin production using Escherichia coli cells over-expressing isoeugenol monooxygenase of Pseudomonas putida.

    Science.gov (United States)

    Yamada, Mamoru; Okada, Yukiyoshi; Yoshida, Toyokazu; Nagasawa, Toru

    2008-04-01

    The isoeugenol monooxygenase gene of Pseudomonas putida IE27 was inserted into an expression vector, pET21a, under the control of the T7 promoter. The recombinant plasmid was introduced into Escherichia coli BL21(DE3) cells, containing no vanillin-degrading activity. The transformed E. coli BL21(DE3) cells produced 28.3 g vanillin/l from 230 mM isoeugenol, with a molar conversion yield of 81% at 20 degrees C after 6 h. In the reaction system, no accumulation of undesired by-products, such as vanillic acid or acetaldehyde, was observed.

  4. Pyoverdine synthesis by the Mn(II-oxidizing bacterium Pseudomonas putida GB-1

    Directory of Open Access Journals (Sweden)

    Dorothy Lundquist Parker

    2014-05-01

    Full Text Available When iron-starved, the Mn(II-oxidizing bacteria Pseudomonas putida strains GB-1 and MnB1 produce pyoverdines (PVDGB-1 and PVDMnB1, siderophores that both influence iron uptake and inhibit manganese(II oxidation by these strains. To explore the properties and genetics of a PVD that can affect manganese oxidation, LC-MS/MS and various siderotyping techniques were used to identify the peptides of PVDGB-1 and PVDMnB1 as being (for both PVDs: chromophore-Asp-Lys-OHAsp-Ser-Gly-aThr-Lys-cOHOrn, resembling a structure previously reported for P. putida CFML 90-51, which does not oxidize Mn. All three strains also produced an azotobactin and a sulfonated PVD, each with the peptide sequence above, but with unknown regulatory or metabolic effects. Bioinformatic analysis of the sequenced genome of P. putida GB-1 suggested that a particular non-ribosomal peptide synthetase, coded by the operon PputGB1_4083-4086, could produce the peptide backbone of PVDGB-1. To verify this prediction, plasmid integration disruption of PputGB1_4083 was performed and the resulting mutant failed to produce detectable PVD. In silico analysis of the modules in PputGB1_4083-4086 predicted a peptide sequence of Asp-Lys-Asp-Ser-Ala-Thr-Lsy-Orn, which closely matches the peptide determined by MS/MS. To extend these studies to other organisms, various Mn(II-oxidizing and non-oxidizing isolates of P. putida, P. fluorescens, P. marincola, P. fluorescens-syringae group, P. mendocina-resinovorans group and P. stutzerii group were screened for PVD synthesis. The PVD producers (12 out of 16 tested strains were siderotyped and placed into four sets of differing PVD structures, some corresponding to previously characterized PVDs and some to novel PVDs. These results combined with previous studies suggested that the presence of OHAsp or the flexibility of the pyoverdine polypeptide may enable efficient binding of Mn(III.

  5. Production of Polyhydroxyalkanoates from Sludge Palm Oil Using Pseudomonas putida S12.

    Science.gov (United States)

    Kang, Du-Kyeong; Lee, Cho-Ryong; Lee, Sun Hee; Bae, Jung-Hoon; Park, Young-Kwon; Rhee, Young Ha; Sung, Bong Hyun; Sohn, Jung-Hoon

    2017-05-28

    Polyhydroxyalkanoates (PHAs) are biodegradable plastics produced by bacteria, but their use in diverse applications is prohibited by high production costs. To reduce these costs, the conversion by Pseudomonas strains of P HAs from crude s ludge p alm oil ( SPO) a s an inexpensive renewable raw material was tested. Pseudomonas putida S12 was found to produce the highest yield (~41%) of elastomeric medium-chain-length (MCL)-PHAs from SPO. The MCL-PHA characteristics were analyzed by gas-chromatography/mass spectrometry, gel permeation chromatography, and differential scanning calorimetry. These findings may contribute to more widespread use of PHAs by reducing PHA production costs.

  6. A case report of acute pediatric bacterial meningitis due to the rare isolate, Pseudomonas putida

    Institute of Scientific and Technical Information of China (English)

    Grishma V. Kulkarni

    2016-01-01

    Acute bacterial meningitis (ABM) is the medical emergency which warrants an early diagnosis and an aggressive therapy. Despite the availability of the potent newer antibiotics, the mortality caused by ABM and its complications remain high in India, ranging from 16% to 32%. The aim of this case report is to present the rare isolation ofPseudomonas putida from cerebrospinal lfuid sample. Besides this, the author also emphasizes the importance of correctly identifying the organism and thus the selection of the most accurate antibiotic from the susceptibility proifle to allow for early recovery and to improve the patient outcome and survival.

  7. Pyoverdine synthesis by the Mn(II)-oxidizing bacterium Pseudomonas putida GB-1

    Science.gov (United States)

    Parker, Dorothy L.; Lee, Sung-Woo; Geszvain, Kati; Davis, Richard E.; Gruffaz, Christelle; Meyer, Jean-Marie; Torpey, Justin W.; Tebo, Bradley M.

    2014-01-01

    When iron-starved, the Mn(II)-oxidizing bacteria Pseudomonas putida strains GB-1 and MnB1 produce pyoverdines (PVDGB-1 and PVDMnB1), siderophores that both influence iron uptake and inhibit manganese(II) oxidation by these strains. To explore the properties and genetics of a PVD that can affect manganese oxidation, LC-MS/MS, and various siderotyping techniques were used to identify the peptides of PVDGB-1 and PVDMnB1 as being (for both PVDs): chromophore-Asp-Lys-OHAsp-Ser-Gly-aThr-Lys-cOHOrn, resembling a structure previously reported for P. putida CFML 90-51, which does not oxidize Mn. All three strains also produced an azotobactin and a sulfonated PVD, each with the peptide sequence above, but with unknown regulatory or metabolic effects. Bioinformatic analysis of the sequenced genome of P. putida GB-1 suggested that a particular non-ribosomal peptide synthetase (NRPS), coded by the operon PputGB1_4083-4086, could produce the peptide backbone of PVDGB-1. To verify this prediction, plasmid integration disruption of PputGB1_4083 was performed and the resulting mutant failed to produce detectable PVD. In silico analysis of the modules in PputGB1_4083-4086 predicted a peptide sequence of Asp-Lys-Asp-Ser-Ala-Thr-Lsy-Orn, which closely matches the peptide determined by MS/MS. To extend these studies to other organisms, various Mn(II)-oxidizing and non-oxidizing isolates of P. putida, P. fluorescens, P. marincola, P. fluorescens-syringae group, P. mendocina-resinovorans group, and P. stutzerii group were screened for PVD synthesis. The PVD producers (12 out of 16 tested strains) were siderotyped and placed into four sets of differing PVD structures, some corresponding to previously characterized PVDs and some to novel PVDs. These results combined with previous studies suggested that the presence of OHAsp or the flexibility of the pyoverdine polypeptide may enable efficient binding of Mn(III). PMID:24847318

  8. Biosorption of heavy metals and uranium by starfish and Pseudomonas putida

    International Nuclear Information System (INIS)

    Choi, Jaeyoung; Lee, Ju Young; Yang, Jung-Seok

    2009-01-01

    Biosorption of heavy metals and uranium from contaminated wastewaters may represent an innovative purification process. This study investigates the removal ability of unit mass of Pseudomonas putida and starfish for lead, cadmium, and uranium by quantifying the adsorption capacity. The adsorption of heavy metals and uranium by the samples was influenced by pH, and increased with increasing Pb, Cd, and U concentrations. Dead cells adsorbed the largest quantity of all heavy metals than live cells and starfish. The adsorption capacity followed the order: U(VI) > Pb > Cd. The results also suggest that bacterial membrane cells can be used successfully in the treatment of high strength metal-contaminated wastewaters

  9. Regulation of phenylacetic acid uptake is sigma54 dependent in Pseudomonas putida CA-3.

    LENUS (Irish Health Repository)

    O' Leary, Niall D

    2011-10-13

    Abstract Background Styrene is a toxic and potentially carcinogenic alkenylbenzene used extensively in the polymer processing industry. Significant quantities of contaminated liquid waste are generated annually as a consequence. However, styrene is not a true xenobiotic and microbial pathways for its aerobic assimilation, via an intermediate, phenylacetic acid, have been identified in a diverse range of environmental isolates. The potential for microbial bioremediation of styrene waste has received considerable research attention over the last number of years. As a result the structure, organisation and encoded function of the genes responsible for styrene and phenylacetic acid sensing, uptake and catabolism have been elucidated. However, a limited understanding persists in relation to host specific regulatory molecules which may impart additional control over these pathways. In this study the styrene degrader Pseudomonas putida CA-3 was subjected to random mini-Tn5 mutagenesis and mutants screened for altered styrene\\/phenylacetic acid utilisation profiles potentially linked to non-catabolon encoded regulatory influences. Results One mutant, D7, capable of growth on styrene, but not on phenylacetic acid, harboured a Tn5 insertion in the rpoN gene encoding σ54. Complementation of the D7 mutant with the wild type rpoN gene restored the ability of this strain to utilise phenylacetic acid as a sole carbon source. Subsequent RT-PCR analyses revealed that a phenylacetate permease, PaaL, was expressed in wild type P. putida CA-3 cells utilising styrene or phenylacetic acid, but could not be detected in the disrupted D7 mutant. Expression of plasmid borne paaL in mutant D7 was found to fully restore the phenylacetic acid utilisation capacity of the strain to wild type levels. Bioinformatic analysis of the paaL promoter from P. putida CA-3 revealed two σ54 consensus binding sites in a non-archetypal configuration, with the transcriptional start site being resolved by

  10. Biosorption of heavy metals and uranium by starfish and Pseudomonas putida.

    Science.gov (United States)

    Choi, Jaeyoung; Lee, Ju Young; Yang, Jung-Seok

    2009-01-15

    Biosorption of heavy metals and uranium from contaminated wastewaters may represent an innovative purification process. This study investigates the removal ability of unit mass of Pseudomonas putida and starfish for lead, cadmium, and uranium by quantifying the adsorption capacity. The adsorption of heavy metals and uranium by the samples was influenced by pH, and increased with increasing Pb, Cd, and U concentrations. Dead cells adsorbed the largest quantity of all heavy metals than live cells and starfish. The adsorption capacity followed the order: U(VI)>Pb>Cd. The results also suggest that bacterial membrane cells can be used successfully in the treatment of high strength metal-contaminated wastewaters.

  11. Chromogenic in situ hybridization is a reliable assay for detection of ALK rearrangements in adenocarcinomas of the lung.

    Science.gov (United States)

    Schildhaus, Hans-Ulrich; Deml, Karl-Friedrich; Schmitz, Katja; Meiboom, Maren; Binot, Elke; Hauke, Sven; Merkelbach-Bruse, Sabine; Büttner, Reinhard

    2013-11-01

    Reliable detection of anaplastic lymphoma kinase (ALK) rearrangements is a prerequisite for personalized treatment of lung cancer patients, as ALK rearrangements represent a predictive biomarker for the therapy with specific tyrosine kinase inhibitors. Currently, fluorescent in situ hybridization (FISH) is considered to be the standard method for assessing formalin-fixed and paraffin-embedded tissue for ALK inversions and translocations. However, FISH requires a specialized equipment, the signals fade rapidly and it is difficult to detect overall morphology and tumor heterogeneity. Chromogenic in situ hybridization (CISH) has been successfully introduced as an alternative test for the detection of several genetic aberrations. This study validates a newly developed ALK CISH assay by comparing FISH and CISH signal patterns in lung cancer samples with and without ALK rearrangements. One hundred adenocarcinomas of the lung were included in this study, among them 17 with known ALK rearrangement. FISH and CISH were carried out and evaluated according to the manufacturers' recommendations. For both assays, tumors were considered positive if ≥15% of tumor cells showed either isolated 3' signals or break-apart patterns or a combination of both. A subset of tumors was exemplarily examined by using a novel EML4 (echinoderm microtubule-associated protein-like 4) CISH probe. Red, green and fusion CISH signals were clearcut and different signal patterns were easily recognized. The percentage of aberrant tumor cells was statistically highly correlated (PCISH. On the basis of 86 samples that were evaluable by ALK CISH, we found a 100% sensitivity and 100% specificity of this assay. Furthermore, EML4 rearrangements could be recognized by CISH. CISH is a highly reliable, sensitive and specific method for the detection of ALK gene rearrangements in pulmonary adenocarcinomas. Our results suggest that CISH might serve as a suitable alternative to FISH, which is the current gold

  12. ALK5 inhibition maintains islet endothelial cell survival but does not enhance islet graft revascularisation or function.

    Science.gov (United States)

    King, A J F; Clarkin, C E; Austin, A L F; Ajram, L; Dhunna, J K; Jamil, M O; Ditta, S I; Ibrahim, S; Raza, Z; Jones, P M

    2015-01-01

    Islet transplantation is a potential treatment for Type 1 diabetes but long term graft function is suboptimal. The rich supply of intraislet endothelial cells diminishes rapidly after islet isolation and culture, which affects the revascularisation rate of islets after transplantation. The ALK5 pathway inhibits endothelial cell proliferation and thus inhibiting ALK5 is a potential target for improving endothelial cell survival. The aim of the study was to establish whether ALK5 inhibition prevents the loss of intraislet endothelial cells during islet culture and thus improves the functional survival of transplanted islets by enhancing their subsequent revascularisation after implantation. Islets were cultured for 48 h in the absence or presence of 2 different ALK inhibitors: SB-431542 or A-83-01. Their vascular density after culture was analysed using immunohistochemistry. Islets pre-cultured with the ALK5 inhibitors were implanted into streptozotocin-diabetic mice for either 3 or 7 days and blood glucose concentrations were monitored and vascular densities of the grafts were analysed. Islets cultured with ALK5 inhibitors had higher vascular densities than control-cultured islets. Three days after implantation, endothelial cell numbers in islet grafts were minimal, irrespective of treatment during culture. Seven days after implantation, endothelial cells were evident within the islet grafts but there was no difference between control-cultured islets and islets pre-treated with an ALK5 inhibitor. Blood glucose concentrations were no different between the treatment groups. In conclusion, inhibition of ALK5 improved intraislet endothelial cell numbers after islet culture, but this effect was lost in the early post-transplantation period. © Georg Thieme Verlag KG Stuttgart · New York.

  13. Structural and mutational analysis of Escherichia coli AlkB provides insight into substrate specificity and DNA damage searching.

    Directory of Open Access Journals (Sweden)

    Paul J Holland

    Full Text Available BACKGROUND: In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG iron(II dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA and 3-methylcytosine (3-meC lesions, but it also repairs 1-methylguanine (1-meG and 3-methylthymine (3-meT at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. METHODOLOGY/PRINCIPAL FINDINGS: We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. CONCLUSIONS/SIGNIFICANCE: A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a "searching" mode and "repair" mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.

  14. A Pseudomonas putida strain genetically engineered for 1,2,3-trichloropropane bioremediation.

    Science.gov (United States)

    Samin, Ghufrana; Pavlova, Martina; Arif, M Irfan; Postema, Christiaan P; Damborsky, Jiri; Janssen, Dick B

    2014-09-01

    1,2,3-Trichloropropane (TCP) is a toxic compound that is recalcitrant to biodegradation in the environment. Attempts to isolate TCP-degrading organisms using enrichment cultivation have failed. A potential biodegradation pathway starts with hydrolytic dehalogenation to 2,3-dichloro-1-propanol (DCP), followed by oxidative metabolism. To obtain a practically applicable TCP-degrading organism, we introduced an engineered haloalkane dehalogenase with improved TCP degradation activity into the DCP-degrading bacterium Pseudomonas putida MC4. For this purpose, the dehalogenase gene (dhaA31) was cloned behind the constitutive dhlA promoter and was introduced into the genome of strain MC4 using a transposon delivery system. The transposon-located antibiotic resistance marker was subsequently removed using a resolvase step. Growth of the resulting engineered bacterium, P. putida MC4-5222, on TCP was indeed observed, and all organic chlorine was released as chloride. A packed-bed reactor with immobilized cells of strain MC4-5222 degraded >95% of influent TCP (0.33 mM) under continuous-flow conditions, with stoichiometric release of inorganic chloride. The results demonstrate the successful use of a laboratory-evolved dehalogenase and genetic engineering to produce an effective, plasmid-free, and stable whole-cell biocatalyst for the aerobic bioremediation of a recalcitrant chlorinated hydrocarbon. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  15. Metabolic engineering to expand the substrate spectrum of Pseudomonas putida toward sucrose.

    Science.gov (United States)

    Löwe, Hannes; Schmauder, Lukas; Hobmeier, Karina; Kremling, Andreas; Pflüger-Grau, Katharina

    2017-08-01

    Sucrose is an important disaccharide used as a substrate in many industrial applications. It is a major component of molasses, a cheap by-product of the sugar industry. Unfortunately, not all industrially relevant organisms, among them Pseudomonas putida, are capable of metabolizing sucrose. We chose a metabolic engineering approach to circumvent this blockage and equip P. putida with the activities necessary to consume sucrose. Therefore, we constructed a pair of broad-host range mini-transposons (pSST - sucrose splitting transposon), carrying either cscA, encoding an invertase able to split sucrose into glucose and fructose, or additionally cscB, encoding a sucrose permease. Introduction of cscA was sufficient to convey sucrose consumption and the additional presence of cscB had no further effect, though the sucrose permease was built and localized to the membrane. Sucrose was split extracellularly by the activity of the invertase CscA leaking out of the cell. The transposons were also used to confer sucrose consumption to Cupriavidus necator. Interestingly, in this strain, CscB acted as a glucose transporter, such that C. necator also gained the ability to grow on glucose. Thus, the pSST transposons are functional tools to extend the substrate spectrum of Gram-negative bacterial strains toward sucrose. © 2017 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

  16. Structural characterization of pyoverdines produced by Pseudomonas putida KT2440 and Pseudomonas taiwanensis VLB120.

    Science.gov (United States)

    Baune, Matthias; Qi, Yulin; Scholz, Karen; Volmer, Dietrich A; Hayen, Heiko

    2017-08-01

    The previously unknown sequences of several pyoverdines (PVD) produced by a biotechnologically-relevant bacterium, namely, Pseudomonas taiwanensis VLB120, were characterized by high performance liquid chromatography (HPLC)-high resolution mass spectrometry (HRMS). The same structural characterization scheme was checked before by analysis of Pseudomonas sp. putida KT2440 samples with known PVDs. A new sample preparation strategy based on solid-phase extraction was developed, requiring significantly reduced sample material as compared to existing methods. Chromatographic separation was performed using hydrophilic interaction liquid chromatography with gradient elution. Interestingly, no signals for apoPVDs were detected in these analyses, only the corresponding aluminum(III) and iron(III) complexes were seen. The chromatographic separation readily enabled separation of PVD complexes according to their individual structures. HPLC-HRMS and complementary fragmentation data from collision-induced dissociation and electron capture dissociation enabled the structural characterization of the investigated pyoverdines. In Pseudomonas sp. putida KT2240 samples, the known pyoverdines G4R and G4R A were readily confirmed. No PVDs have been previously described for Pseudomonas sp. taiwanensis VLB120. In our study, we identified three new PVDs, which only differed in their acyl side chains (succinic acid, succinic amide and malic acid). Peptide sequencing by MS/MS provided the sequence Orn-Asp-OHAsn-Thr-AcOHOrn-Ser-cOHOrn. Of particular interest is the presence of OHAsn, which has not been reported as PVD constituent before.

  17. Novel AroA from Pseudomonas putida Confers Tobacco Plant with High Tolerance to Glyphosate

    Science.gov (United States)

    Yan, Hai-Qin; Chang, Su-Hua; Tian, Zhe-Xian; Zhang, Le; Sun, Yi-Cheng; Li, Yan; Wang, Jing; Wang, Yi-Ping

    2011-01-01

    Glyphosate is a non-selective broad-spectrum herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, also designated as AroA), a key enzyme in the aromatic amino acid biosynthesis pathway in microorganisms and plants. Previously, we reported that a novel AroA (PpAroA1) from Pseudomonas putida had high tolerance to glyphosate, with little homology to class I or class II glyphosate-tolerant AroA. In this study, the coding sequence of PpAroA1 was optimized for tobacco. For maturation of the enzyme in chloroplast, a chloroplast transit peptide coding sequence was fused in frame with the optimized aroA gene (PparoA1optimized) at the 5′ end. The PparoA1optimized gene was introduced into the tobacco (Nicotiana tabacum L. cv. W38) genome via Agrobacterium-mediated transformation. The transformed explants were first screened in shoot induction medium containing kanamycin. Then glyphosate tolerance was assayed in putative transgenic plants and its T1 progeny. Our results show that the PpAroA1 from Pseudomonas putida can efficiently confer tobacco plants with high glyphosate tolerance. Transgenic tobacco overexpressing the PparoA1optimized gene exhibit high tolerance to glyphosate, which suggest that the novel PpAroA1 is a new and good candidate applied in transgenic crops with glyphosate tolerance in future. PMID:21611121

  18. Characterization of cell lysis in Pseudomonas putida induced upon expression of heterologous killing genes

    DEFF Research Database (Denmark)

    Ronchel, M.C.; Molina, L.; Witte, A.

    1998-01-01

    Active biological containment systems are based on the controlled expression of killing genes. These systems are of interest for the Pseudomonadaceae because of the potential applications of these microbes as bioremediation agents and biopesticides, The physiological effects that lead to cell dea...... protein was the killing agent. In both cases, cell death occurred as a result of impaired respiration, altered membrane permeability, and the release of some cytoplasmic contents to the extracellular medium.......Active biological containment systems are based on the controlled expression of killing genes. These systems are of interest for the Pseudomonadaceae because of the potential applications of these microbes as bioremediation agents and biopesticides, The physiological effects that lead to cell death......, respectively. Expression of the killing genes is controlled by the LacI protein, whose expression is initiated from the XylS-dependent Pm promoter. Under induced conditions, killing of P. putida CMC12 cells mediated by phi X174 lysis protein E was faster than that observed for P. putida CMC4, for which the Gef...

  19. Metabolic engineering of Pseudomonas putida KT2440 for the production of para-hydroxy benzoic acid

    Directory of Open Access Journals (Sweden)

    Shiqin Yu

    2016-11-01

    Full Text Available para-hydroxy benzoic acid (PHBA is the key component for preparing parabens, a common preservatives in food, drugs and personal care products, as well as high performance bioplastics such as liquid crystal polymers (LCP. Pseudomonas putida KT2440 was engineered to produce PHBA from glucose via the shikimate pathway intermediate chorismate. To obtain the PHBA production strain, chorismate lyase UbiC from Escherichia coli and a feedback resistant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase encoded by gene aroGD146N were overexpressed individually and simultaneously. In addition, genes related to product degradation (pobA or competing for the precursor chorismate (pheA and trpE were deleted from the genome. To further improve PHBA production, the glucose metabolism repressor hexR was knocked out in order to increase erythrose-4- phosphate and NAPH supply. The best strain achieved a maximum titre of 1.73 g L-1 and a carbon yield of 18.1 % (C-mol C-mol-1 in a non-optimized fed-batch fermentation. This is to date the highest PHBA concentration produced by P. putida using a chorismate lyase.

  20. The Bistable Behaviour of Pseudomonas putida KT2440 during PHA Depolymerization under Carbon Limitation

    Directory of Open Access Journals (Sweden)

    Stephanie Karmann

    2017-06-01

    Full Text Available Poly(hydroxyalkanoates (PHAs are bacterial polyesters offering a biodegradable alternative to petrochemical plastics. The intracellular formation and degradation of PHAs is a dynamic process that strongly depends on the availability of carbon and other nutrients. Carbon excess and nitrogen limitation are considered to favor PHA accumulation, whereas carbon limitation triggers PHA depolymerization when all other essential nutrients are present in excess. We studied the population dynamics of Pseudomonas putida KT2440 at the single cell level during different physiological conditions, favoring first PHA polymerization during growth on octanoic acid, and then PHA depolymerization during carbon limitation. PHAs accumulate intracellularly in granules, and were proposed to separate preferentially together with nucleic acids, leading to two daughter cells containing approximately equal amounts of PHA. However, we could show that such P. putida KT2440 cells show bistable behavior when exposed to carbon limitation, and separate into two subpopulations: one with high and one with low PHA. This suggests an asymmetric PHA distribution during cell division under carbon limitation, which has a significant influence on our understanding of PHA mobilization.

  1. Degradation of waste waters from olive oil mills by Yarrowia lipolytica ATCC 20255 and Pseudomonas putida

    Energy Technology Data Exchange (ETDEWEB)

    De Felice, B.; Pontecorvo, G.; Carfagna, M. [Univ. of Naples, Caserta (Italy). Inst. of Biology

    1997-12-31

    Waste water from olive oil processing may cause severe pollution in the Mediterranean area, since they have a high level of chemical oxygen demand (COD) (100-200 g/l) and contain other organic and inorganic compounds. In all olive oil producing countries, the reduction of pollution in olive oil mill waste waters at reasonable costs and using techniques suitable for most industrial applications is an unsolved problem. For this paper, the yeast Yarrowia lipolytica ATCC 20255 was grown on waste waters from an olive oil mill in a 3.5 l fermenter under batch culture conditions. The results showed that the yeast was capable of reducing the COD value by 80% in 24 h. In this way, a useful biomass of 22.45 g/l as single cell protein (SCP) and enzyme lipase were produced. During this process, most of the organic and inorganic substances were consumed, only aromatic pollutants were still present in the fermentation effluents. Therefore, we used a phenol degrader, namely Pseudomonas putida, to reduce phenolic compounds in the fermentation effluents after removing Yarrowia lipolytica cells. P. putida was effective in reducing phenols in only 12 h. (orig.)

  2. Bioaugmentation of a 4-chloronitrobenzene contaminated soil with Pseudomonas putida ZWL73

    Energy Technology Data Exchange (ETDEWEB)

    Guilan, Niu [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Graduate School, Chinese Academy of Sciences, Beijing 100049 (China); Junjie, Zhang [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Shuo, Zhao [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Graduate School, Chinese Academy of Sciences, Beijing 100049 (China); Hong, Liu [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Boon, Nico [Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, B-9000 Gent (Belgium); Zhou Ningyi [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China)], E-mail: n.zhou@pentium.whiov.ac.cn

    2009-03-15

    The strain Pseudomonas putida ZWL73, which metabolizes 4-chloronitrobenzene (4CNB) by a partial-reductive pathway, was inoculated into lab-scale 4CNB-contaminated soil for bioaugmentation purposes in this study. The degradation of 4CNB was clearly stimulated, as indicated with the gradual accumulation of ammonium and chloride. Simultaneously, the diversity and quantity of cultivable heterotrophic bacteria decreased due to 4CNB contamination, while the quantity of 4CNB-resistant bacteria increased. During the bioaugmentation, denaturing gradient gel electrophoresis analysis showed the changes of diversity in dominant populations of intrinsic soil microbiota. The results showed that Alphaproteobacteria and Betaproteobacteria were not distinctly affected, but Actinobacteria were apparently stimulated. In addition, an interesting dynamic within Acidobacteria was observed, as well as an influence on ammonia-oxidizing bacteria population. These combined findings demonstrate that the removal of 4CNB in soils by inoculating strain ZWL73 is feasible, and that specific populations in soils rapidly changed in response to 4CNB contamination and subsequent bioaugmentation. - Pseudomonas putida ZWL73 can accelerate 4CNB removal in lab-scale soils, causing dynamic changes within intrinsic Actinobacteria and Acidobacteria.

  3. Bioaugmentation of a 4-chloronitrobenzene contaminated soil with Pseudomonas putida ZWL73

    International Nuclear Information System (INIS)

    Niu Guilan; Zhang Junjie; Zhao Shuo; Liu Hong; Boon, Nico; Zhou Ningyi

    2009-01-01

    The strain Pseudomonas putida ZWL73, which metabolizes 4-chloronitrobenzene (4CNB) by a partial-reductive pathway, was inoculated into lab-scale 4CNB-contaminated soil for bioaugmentation purposes in this study. The degradation of 4CNB was clearly stimulated, as indicated with the gradual accumulation of ammonium and chloride. Simultaneously, the diversity and quantity of cultivable heterotrophic bacteria decreased due to 4CNB contamination, while the quantity of 4CNB-resistant bacteria increased. During the bioaugmentation, denaturing gradient gel electrophoresis analysis showed the changes of diversity in dominant populations of intrinsic soil microbiota. The results showed that Alphaproteobacteria and Betaproteobacteria were not distinctly affected, but Actinobacteria were apparently stimulated. In addition, an interesting dynamic within Acidobacteria was observed, as well as an influence on ammonia-oxidizing bacteria population. These combined findings demonstrate that the removal of 4CNB in soils by inoculating strain ZWL73 is feasible, and that specific populations in soils rapidly changed in response to 4CNB contamination and subsequent bioaugmentation. - Pseudomonas putida ZWL73 can accelerate 4CNB removal in lab-scale soils, causing dynamic changes within intrinsic Actinobacteria and Acidobacteria

  4. Receptor ligand-triggered resistance to alectinib and its circumvention by Hsp90 inhibition in EML4-ALK lung cancer cells.

    Science.gov (United States)

    Tanimoto, Azusa; Yamada, Tadaaki; Nanjo, Shigeki; Takeuchi, Shinji; Ebi, Hiromichi; Kita, Kenji; Matsumoto, Kunio; Yano, Seiji

    2014-07-15

    Alectinib is a new generation ALK inhibitor with activity against the gatekeeper L1196M mutation that showed remarkable activity in a phase I/II study with echinoderm microtubule associated protein-like 4 (EML4)--anaplastic lymphoma kinase (ALK) non-small cell lung cancer (NSCLC) patients. However, alectinib resistance may eventually develop. Here, we found that EGFR ligands and HGF, a ligand of the MET receptor, activate EGFR and MET, respectively, as alternative pathways, and thereby induce resistance to alectinib. Additionally, the heat shock protein 90 (Hsp90) inhibitor suppressed protein expression of ALK, MET, EGFR, and AKT, and thereby induced apoptosis in EML4-ALK NSCLC cells, even in the presence of EGFR ligands or HGF. These results suggest that Hsp90 inhibitors may overcome ligand-triggered resistance to new generation ALK inhibitors and may result in more successful treatment of NSCLC patients with EML4-ALK.

  5. Comprehensive proteome analysis of the response of Pseudomonas putida KT2440 to the flavor compound vanillin.

    Science.gov (United States)

    Simon, Oliver; Klaiber, Iris; Huber, Armin; Pfannstiel, Jens

    2014-09-23

    Understanding of the molecular response of bacteria to precursors, products and environmental conditions applied in bioconversions is essential for optimizing whole-cell biocatalysis. To investigate the molecular response of the potential biocatalyst Pseudomonas putida KT2440 to the flavor compound vanillin we applied complementary gel- and LC-MS-based quantitative proteomics approaches. Our comprehensive proteomics survey included cytoplasmic and membrane proteins and led to the identification and quantification of 1614 proteins, corresponding to 30% of the total KT2440 proteome. 662 proteins were altered in abundance during growth on vanillin as sole carbon source as compared to growth on glucose. The proteome response entailed an increased abundance of enzymes involved in vanillin degradation, significant changes in central energy metabolism and an activation of solvent tolerance mechanisms. With respect to vanillin metabolism, particularly enzymes belonging to the β-ketoadipate pathway including a transcriptional regulator and porins specific for vanillin uptake increased in abundance. However, catabolism of vanillin was not dependent on vanillin dehydrogenase (Vdh), as shown by quantitative proteome analysis of a Vdh-deficient KT2440 mutant (GN235). Other aldehyde dehydrogenases that were significantly increased in abundance in response to vanillin may replace Vdh and thus may represent interesting targets for improving vanillin production in P. putida KT2440. The high demand for the flavor compound vanillin by the food and fragrance industry makes natural vanillin from vanilla pods a scarce and expensive resource rendering its biotechnological production economically attractive. Pseudomonas bacteria are metabolically very versatile and accept a broad range of hydrocarbons as carbon source making them suitable candidates for bioconversion processes. This work describes the impact of vanillin on the metabolism of the reference strain P. putida KT2440 on a

  6. Toward a better understanding of the interaction between TGF-β family members and their ALK receptors

    KAUST Repository

    Romano, Valentina; Raimondo, Domenico; Calvanese, Luisa; D’ Auria, Gabriella; Tramontano, Anna; Falcigno, Lucia

    2012-01-01

    Transforming growth factor-beta (TGF-β) proteins are a family of structurally related extracellular proteins that trigger their signaling functions through interaction with the extracellular domains of their cognate serine/threonine kinase receptors. The specificity of TGF-β/receptor binding is complex and gives rise to multiple functional roles. Additionally, it is not completely understood at the atomic level. Here, we use the most reliable computational methods currently available to study systems involving activin-like kinase (ALK) receptors ALK4 and ALK7 and their multiple TGF-β ligands. We built models for all these proteins and their complexes for which experimental structures are not available. By analyzing the surfaces of interaction in six different TGF-β/ALK complexes we could infer which are the structural distinctive features of the ligand-receptor binding mode. Furthermore, this study allowed us to rationalize why binding of the growth factors GDF3 and Nodal to the ALK4 receptor requires the Cripto co-factor, whilst binding to the ALK7 receptor does not. © Springer-Verlag 2012.

  7. L1198F Mutation Resensitizes Crizotinib to ALK by Altering the Conformation of Inhibitor and ATP Binding Sites

    Directory of Open Access Journals (Sweden)

    Jian Li

    2017-02-01

    Full Text Available The efficacy of anaplastic lymphoma kinase (ALK positive non-small-cell lung cancer (NSCLC treatment with small molecule inhibitors is greatly challenged by acquired resistance. A recent study reported the newest generation inhibitor resistant mutation L1198F led to the resensitization to crizotinib, which is the first Food and Drug Administration (FDA approved drug for the treatment of ALK-positive NSCLC. It is of great importance to understand how this extremely rare event occurred for the purpose of overcoming the acquired resistance of such inhibitors. In this study, we exploited molecular dynamics (MD simulation to dissect the molecular mechanisms. Our MD results revealed that L1198F mutation of ALK resulted in the conformational change at the inhibitor site and altered the binding affinity of ALK to crizotinib and lorlatinib. L1198F mutation also affected the autoactivation of ALK as supported by the identification of His1124 and Tyr1278 as critical amino acids involved in ATP binding and phosphorylation. Our findings are valuable for designing more specific and potent inhibitors for the treatment of ALK-positive NSCLC and other types of cancer.

  8. Detection of EML4-ALK in lung adenocarcinoma using pleural effusion with FISH, IHC, and RT-PCR methods.

    Directory of Open Access Journals (Sweden)

    Leilei Liu

    Full Text Available Anaplastic lymphoma kinase (ALK and echinoderm microtubule-associated protein-like 4 (EML4 gene rearrangements occur in approximately 5% of non-small-cell lung cancers (NSCLC, leading to the overexpression of anaplastic lymphoma kinase and predicting a response to the targeted inhibitor, crizotinib. Malignant pleural effusion occurs in most patients with advanced lung cancer, especially adenocarcinoma, and tissue samples are not always available from these patients. We attempted to clarify the feasibility of detecting the EML4-ALK fusion gene in pleural effusion cells using different methods. We obtained 66 samples of pleural effusion from NSCLC patients. The pleural effusion fluid was centrifuged, and the cellular components obtained were formalin fixed and paraffin embedded. The EML4-ALK fusion gene status was determined with fluorescent in situ hybridization (FISH, reverse transcription-polymerase chain reaction (RT-PCR, and immunohistochemistry (IHC. EML4-ALK was detected in three of 66 patient samples (4.5% with RT-PCR. When the RT-PCR data were used as the standard, one false positive and one false negative samples were identified with IHC; and one false negative sample was identified with FISH. These results suggest that a block of pleural effusion cells can be used to detect the EML4-ALK fusion gene. IHC had good sensitivity, but low specificity. FISH had low sensitivity, but high specificity. RT-PCR is a good candidate method for detecting EML4-ALK in blocks of pleural effusion cells from lung cancer patients.

  9. Toward a better understanding of the interaction between TGF-β family members and their ALK receptors

    KAUST Repository

    Romano, Valentina

    2012-02-22

    Transforming growth factor-beta (TGF-β) proteins are a family of structurally related extracellular proteins that trigger their signaling functions through interaction with the extracellular domains of their cognate serine/threonine kinase receptors. The specificity of TGF-β/receptor binding is complex and gives rise to multiple functional roles. Additionally, it is not completely understood at the atomic level. Here, we use the most reliable computational methods currently available to study systems involving activin-like kinase (ALK) receptors ALK4 and ALK7 and their multiple TGF-β ligands. We built models for all these proteins and their complexes for which experimental structures are not available. By analyzing the surfaces of interaction in six different TGF-β/ALK complexes we could infer which are the structural distinctive features of the ligand-receptor binding mode. Furthermore, this study allowed us to rationalize why binding of the growth factors GDF3 and Nodal to the ALK4 receptor requires the Cripto co-factor, whilst binding to the ALK7 receptor does not. © Springer-Verlag 2012.

  10. Bioluminescent Bioreporter Pseudomonas putida TVA8 as a Detector of Water Pollution. Operational Conditions and Selectivity of Free Cells Sensor

    Czech Academy of Sciences Publication Activity Database

    Kuncová, Gabriela; Pazlarová, J.; Hlavatá, Alena; Ripp, S.; Sayler, G.S.

    2011-01-01

    Roč. 11, č. 3 (2011), s. 882-887 ISSN 1470-160X R&D Projects: GA MŠk ME 893 Institutional research plan: CEZ:AV0Z40720504 Keywords : whole-cell biosensor * bioluminiscence * pseudomonas putida TVA8 Subject RIV: EI - Biotechnology ; Bionics Impact factor: 2.695, year: 2011

  11. Effect of Genetically Modified Pseudomonas putida WCS358r on the Fungal Rhizosphere Microflora of Field-Grown Wheat

    NARCIS (Netherlands)

    Glandorf, D.C.M.; Verheggen, Patrick; Jansen, Timo; Jorritsma, J.-W.; Smit, Eric; Leeflang, Paula; Wernars, Karel; Thomashow, L.S.; Laureijs, Eric; Thomas-Oates, J.E.; Bakker, P.A.H.M.; Loon, L.C. van

    2001-01-01

    We released genetically modified Pseudomonas putida WCS358r into the rhizospheres of wheat plants. The two genetically modified derivatives, genetically modified microorganism (GMM) 2 and GMM 8, carried the phz biosynthetic gene locus of strain P. fluorescens 2-79 and constitutively produced the

  12. Engineering Pseudomonas putida KT2440 for simultaneous degradation of organophosphates and pyrethroids and its application in bioremediation of soil.

    Science.gov (United States)

    Zuo, Zhenqiang; Gong, Ting; Che, You; Liu, Ruihua; Xu, Ping; Jiang, Hong; Qiao, Chuanling; Song, Cunjiang; Yang, Chao

    2015-06-01

    Agricultural soils are usually co-contaminated with organophosphate (OP) and pyrethroid pesticides. To develop a stable and marker-free Pseudomonas putida for co-expression of two pesticide-degrading enzymes, we constructed a suicide plasmid with expression cassettes containing a constitutive promoter J23119, an OP-degrading gene (mpd), a pyrethroid-hydrolyzing carboxylesterase gene (pytH) that utilizes the upp gene as a counter-selectable marker for upp-deficient P. putida. By introduction of suicide plasmid and two-step homologous recombination, both mpd and pytH genes were integrated into the chromosome of a robust soil bacterium P. putida KT2440 and no selection marker was left on chromosome. Functional expression of mpd and pytH in P. putida KT2440 was demonstrated by Western blot analysis and enzyme activity assays. Degradation experiments with liquid cultures showed that the mixed pesticides including methyl parathion, fenitrothion, chlorpyrifos, permethrin, fenpropathrin, and cypermethrin (0.2 mM each) were degraded completely within 48 h. The inoculation of engineered strain (10(6) cells/g) to soils treated with the above mixed pesticides resulted in a higher degradation rate than in noninoculated soils. All six pesticides could be degraded completely within 15 days in fumigated and nonfumigated soils with inoculation. Theses results highlight the potential of the engineered strain to be used for in situ bioremediation of soils co-contaminated with OP and pyrethroid pesticides.

  13. P-glycoprotein confers acquired resistance to 17-DMAG in lung cancers with an ALK rearrangement

    International Nuclear Information System (INIS)

    Kim, Hee Joung; Lee, Kye Young; Kim, Young Whan; Choi, Yun Jung; Lee, Jung-Eun; Choi, Chang Min; Baek, In-Jeoung; Rho, Jin Kyung; Lee, Jae Cheol

    2015-01-01

    Because anaplastic lymphoma kinase (ALK) is dependent on Hsp90 for protein stability, Hsp90 inhibitors are effective in controlling growth of lung cancer cells with ALK rearrangement. We investigated the mechanism of acquired resistance to 17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), a geldanamycin analogue Hsp90 inhibitor, in H3122 and H2228 non-small cell lung cancer cell lines with ALK rearrangement. Resistant cell lines (H3122/DR-1, H3122/DR-2 and H2228/DR) were established by repeated exposure to increasing concentrations of 17-DMAG. Mechanisms for resistance by either NAD(P)H/quinone oxidoreductase 1 (NQO1), previously known as a factor related to 17-DMAG resistance, or P-glycoprotein (P-gp; ABCB1/MDR1) were queried using RT-PCR, western blot analysis, chemical inhibitors, the MTT cell proliferation/survival assay, and cellular efflux of rhodamine 123. The resistant cells showed no cross-resistance to AUY922 or ALK inhibitors, suggesting that ALK dependency persists in cells with acquired resistance to 17-DMAG. Although expression of NQO1 was decreased in H3122/DR-1 and H3122/DR-2, NQO1 inhibition by dicumarol did not affect the response of parental cells (H2228 and H3122) to 17-DMAG. Interestingly, all resistant cells showed the induction of P-gp at the protein and RNA levels, which was associated with an increased efflux of the P-gp substrate rhodamine 123 (Rho123). Transfection with siRNA directed against P-gp or treatment with verapamil, an inhibitor of P-gp, restored the sensitivity to the drug in all cells with acquired resistance to 17-DMAG. Furthermore, we also observed that the growth-inhibitory effect of 17-DMAG was decreased in A549/PR and H460/PR cells generated to over-express P-gp by long-term exposure to paclitaxel, and these cells recovered their sensitivity to 17-DMAG through the inhibition of P-gp. P-gp over-expression is a possible mechanism of acquired resistance to 17-DMAG in cells with ALK rearrangement. The online

  14. Development of a high efficiency integration system and promoter library for rapid modification of Pseudomonas putida KT2440

    Energy Technology Data Exchange (ETDEWEB)

    Elmore, Joshua R. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Furches, Anna [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Wolff, Gara N. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Gorday, Kent [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Guss, Adam M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division

    2017-04-15

    Pseudomonas putida strains are highly robust bacteria known for their ability to efficiently utilize a variety of carbon sources, including aliphatic and aromatic hydrocarbons. Recently, P. putida has been engineered to valorize the lignin stream of a lignocellulosic biomass pretreatment process. Nonetheless, when compared to platform organisms such as Escherichia coli, the toolkit for engineering P. putida is underdeveloped. Heterologous gene expression in particular is problematic. Plasmid instability and copy number variance provide challenges for replicative plasmids, while use of homologous recombination for insertion of DNA into the chromosome is slow and laborious. Furthermore, heterologous expression efforts to date typically rely on overexpression of exogenous pathways using a handful of poorly characterized promoters. In order to improve the P. putida toolkit, we developed a rapid genome integration system using the site-specific recombinase from bacteriophage Bxb1 to enable rapid, high efficiency integration of DNA into the P. putida chromosome. We also developed a library of synthetic promoters with various UP elements, -35 sequences, and -10 sequences, as well as different ribosomal binding sites. We tested these promoters using a fluorescent reporter gene, mNeonGreen, to characterize the strength of each promoter, and identified UP-element-promoter-ribosomal binding sites combinations capable of driving a ~150-fold range of protein expression levels. One additional integrating vector was developed that confers more robust kanamycin resistance when integrated at single copy into the chromosome. This genome integration and reporter systems are extensible for testing other genetic parts, such as examining terminator strength, and will allow rapid integration of heterologous pathways for metabolic engineering.

  15. Accurate identification of ALK positive lung carcinoma patients: novel FDA-cleared automated fluorescence in situ hybridization scanning system and ultrasensitive immunohistochemistry.

    Directory of Open Access Journals (Sweden)

    Esther Conde

    Full Text Available BACKGROUND: Based on the excellent results of the clinical trials with ALK-inhibitors, the importance of accurately identifying ALK positive lung cancer has never been greater. However, there are increasing number of recent publications addressing discordances between FISH and IHC. The controversy is further fuelled by the different regulatory approvals. This situation prompted us to investigate two ALK IHC antibodies (using a novel ultrasensitive detection-amplification kit and an automated ALK FISH scanning system (FDA-cleared in a series of non-small cell lung cancer tumor samples. METHODS: Forty-seven ALK FISH-positive and 56 ALK FISH-negative NSCLC samples were studied. All specimens were screened for ALK expression by two IHC antibodies (clone 5A4 from Novocastra and clone D5F3 from Ventana and for ALK rearrangement by FISH (Vysis ALK FISH break-apart kit, which was automatically captured and scored by using Bioview's automated scanning system. RESULTS: All positive cases with the IHC antibodies were FISH-positive. There was only one IHC-negative case with both antibodies which showed a FISH-positive result. The overall sensitivity and specificity of the IHC in comparison with FISH were 98% and 100%, respectively. CONCLUSIONS: The specificity of these ultrasensitive IHC assays may obviate the need for FISH confirmation in positive IHC cases. However, the likelihood of false negative IHC results strengthens the case for FISH testing, at least in some situations.

  16. Effects of changes in intracellular iron pool on AlkB-dependent and AlkB-independent mechanisms protecting E.coli cells against mutagenic action of alkylating agent

    Energy Technology Data Exchange (ETDEWEB)

    Sikora, Anna; Maciejewska, Agnieszka M.; Poznański, Jarosław; Pilżys, Tomasz; Marcinkowski, Michał; Dylewska, Małgorzata; Piwowarski, Jan [Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw (Poland); Jakubczak, Wioletta; Pawlak, Katarzyna [Department of Analytical Chemistry, Faculty of Chemistry, Warsaw University of Technology, Warsaw (Poland); Grzesiuk, Elżbieta, E-mail: elag@ibb.waw.pl [Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw (Poland)

    2015-08-15

    Highlights: • The intracellular free iron in E.coli hemH appear to be double that in wt strain. • Increased Fe(II) and AlkB concentrations result in decreased MMS-induced mutations. • Dealkylation of dNTPs takes place in the presence of Fe(II) and not requires AlkB. - Abstract: An Escherichia coli hemH mutant accumulates protoporphyrin IX, causing photosensitivity of cells to visible light. Here, we have shown that intracellular free iron in hemH mutants is double that observed in hemH{sup +} strain. The aim of this study was to recognize the influence of this increased free iron concentration on AlkB-directed repair of alkylated DNA by analyzing survival and argE3 → Arg{sup +} reversion induction after λ > 320 nm light irradiation and MMS-treatment in E. coli AB1157 hemH and alkB mutants. E.coli AlkB dioxygenase constitutes a direct single-protein repair system using non-hem Fe(II) and cofactors 2-oxoglutarate (2OG) and oxygen (O{sub 2}) to initiate oxidative dealkylation of DNA/RNA bases. We have established that the frequency of MMS-induced Arg{sup +} revertants in AB1157 alkB{sup +}hemH{sup –}/pMW1 strain was 40 and 26% reduced comparing to the alkB{sup +}hemH{sup –} and alkB{sup +}hemH{sup +}/pMW1, respectively. It is noteworthy that the effect was observed only when bacteria were irradiated with λ > 320 nm light prior MMS-treatment. This finding indicates efficient repair of alkylated DNA in photosensibilized cells in the presence of higher free iron pool and AlkB concentrations. Interestingly, a 31% decrease in the level of Arg{sup +} reversion was observed in irradiated and MMS-treated hemH{sup –}alkB{sup –} cells comparing to the hemH{sup +}alkB{sup –} strain. Also, the level of Arg{sup +} revertants in the irradiated and MMS treated hemH{sup –} alkB{sup –} mutant was significantly lower (by 34%) in comparison to the same strain but MMS-treated only. These indicate AlkB-independent repair involving Fe ions and reactive oxygen

  17. ALK-negative anaplastic large cell lymphoma mimicking a soft tissue sarcoma

    Directory of Open Access Journals (Sweden)

    Rachel Hudacko

    2011-01-01

    Full Text Available Anaplastic lymphoma kinase protein (ALK-negative anaplastic large cell lymphoma (ALCL has a vast morphologic spectrum and may mimic many other types of malignancies both cytologically and histologically. There are only a few published case reports/series describing the cytomorphologic features of ALCL on fine-needle aspiration (FNA biopsy specimens. We describe a case of ALK-negative ALCL mimicking a high-grade soft tissue sarcoma of the thigh in a 62-year-old man. The characteristic morphologic findings on FNA and core biopsy along with the immunophenotypic profile are described and reviewed. The diagnosis of ALCL on FNA biopsy may be difficult, but can be done successfully with the use of ancillary tests. Therefore, it must be considered in the differential diagnosis of lesions with pleomorphism, anaplasia, and wreath-like or horseshoe-shaped nuclei to ensure that adequate material is obtained for ancillary studies.

  18. Development of a flagellin surface display expression system in a moderate thermophile, Bacillus halodurans Alk36

    CSIR Research Space (South Africa)

    Crampton, Michael C

    2007-06-01

    Full Text Available BIOTECHNOLOGY The development of a flagellin surface display expression system in a moderate thermophile, Bacillus halodurans Alk36 Michael Crampton & Eldie Berger & Sharon Reid & Maureen Louw Received: 3 October 2006 /Revised: 29 January 2007 /Accepted... techniques Plasmid DNA was isolated using a Plasmid Midi Kit (Qiagen). Restriction enzymes were used as specified by the manufacturer (Fermentas and Roche Diagnostics). All mini-preps were done using Perfectprep Plasmid Mini Kit (Eppendorf). All DNA...

  19. A Genetically Engineered Mouse Model of Neuroblastoma Driven by Mutated ALK and MYCN

    Science.gov (United States)

    2014-09-01

    super-enhancer driven transcriptional programs in MYCN- amplified cells. George RE. Advances in Neuroblastoma Research Conference, Cologne, Germany ...crizotinib in ALK-mutated cells The above results suggested that deregulated MYCN could contribute to the sustained upregulation of mTORC1 activity in...incomplete inhibition of mTORC1 limits the activity of crizotinib in NB cells that express both ALKF1174L and deregulated MYCN. This interpretation is

  20. Decolorization of industrial synthetic dyes using engineered Pseudomonas putida cells with surface-immobilized bacterial laccase

    Directory of Open Access Journals (Sweden)

    Wang Wei

    2012-06-01

    Full Text Available Abstract Background Microbial laccases are highly useful in textile effluent dye biodegradation. However, the bioavailability of cellularly expressed or purified laccases in continuous operations is usually limited by mass transfer impediment or enzyme regeneration difficulty. Therefore, this study develops a regenerable bacterial surface-displaying system for industrial synthetic dye decolorization, and evaluates its effects on independent and continuous operations. Results A bacterial laccase (WlacD was engineered onto the cell surface of the solvent-tolerant bacterium Pseudomonas putida to construct a whole-cell biocatalyst. Ice nucleation protein (InaQ anchor was employed, and the ability of 1 to 3 tandemly aligned N-terminal repeats to direct WlacD display were compared. Immobilized WlacD was determined to be surface-displayed in functional form using Western blot analysis, immunofluorescence microscopy, flow cytometry, and whole-cell enzymatic activity assay. Engineered P. putida cells were then applied to decolorize the anthraquinone dye Acid Green (AG 25 and diazo-dye Acid Red (AR 18. The results showed that decolorization of both dyes is Cu2+- and mediator-independent, with an optimum temperature of 35°C and pH of 3.0, and can be stably performed across a temperature range of 15°C to 45°C. A high activity toward AG25 (1 g/l with relative decolorization values of 91.2% (3 h and 97.1% (18 h, as well as high activity to AR18 (1 g/l by 80.5% (3 h and 89.0% (18 h, was recorded. The engineered system exhibited a comparably high activity compared with those of separate dyes in a continuous three-round shake-flask decolorization of AG25/AR18 mixed dye (each 1 g/l. No significant decline in decolorization efficacy was noted during first two-rounds but reaction equilibriums were elongated, and the residual laccase activity eventually decreased to low levels. However, the decolorizing capacity of the system was easily retrieved

  1. Global Transcriptional Responses to Osmotic, Oxidative, and Imipenem Stress Conditions in Pseudomonas putida.

    Science.gov (United States)

    Bojanovič, Klara; D'Arrigo, Isotta; Long, Katherine S

    2017-04-01

    Bacteria cope with and adapt to stress by modulating gene expression in response to specific environmental cues. In this study, the transcriptional response of Pseudomonas putida KT2440 to osmotic, oxidative, and imipenem stress conditions at two time points was investigated via identification of differentially expressed mRNAs and small RNAs (sRNAs). A total of 440 sRNA transcripts were detected, of which 10% correspond to previously annotated sRNAs, 40% to novel intergenic transcripts, and 50% to novel transcripts antisense to annotated genes. Each stress elicits a unique response as far as the extent and dynamics of the transcriptional changes. Nearly 200 protein-encoding genes exhibited significant changes in all stress types, implicating their participation in a general stress response. Almost half of the sRNA transcripts were differentially expressed under at least one condition, suggesting possible functional roles in the cellular response to stress conditions. The data show a larger fraction of differentially expressed sRNAs than of mRNAs with >5-fold expression changes. The work provides detailed insights into the mechanisms through which P. putida responds to different stress conditions and increases understanding of bacterial adaptation in natural and industrial settings. IMPORTANCE This study maps the complete transcriptional response of P. putida KT2440 to osmotic, oxidative, and imipenem stress conditions at short and long exposure times. Over 400 sRNA transcripts, consisting of both intergenic and antisense transcripts, were detected, increasing the number of identified sRNA transcripts in the strain by a factor of 10. Unique responses to each type of stress are documented, including both the extent and dynamics of the gene expression changes. The work adds rich detail to previous knowledge of stress response mechanisms due to the depth of the RNA sequencing data. Almost half of the sRNAs exhibit significant expression changes under at least one

  2. ALK-positive anaplastic large cell lymphoma with soft tissue involvement in a young woman

    Directory of Open Access Journals (Sweden)

    Gao KH

    2016-07-01

    Full Text Available Kehai Gao, Hongtao Li, Caihong Huang, Huazhuang Li, Jun Fang, Chen Tian Department of Orthopaedics, Yidu Central Hospital, Shandong, People’s Republic of China Introduction: Anaplastic large cell lymphoma (ALCL is a type of non-Hodgkin lymphoma that has strong expression of CD30. ALCL can sometimes involve the bone marrow, and in advanced stages, it can produce destructive extranodal lesions. But anaplastic large cell lymphoma kinase (ALK+ ALCL with soft tissue involvement is very rare.Case report: A 35-year-old woman presented with waist pain for over 1 month. The biopsy of soft tissue lesions showed that these cells were positive for ALK-1, CD30, TIA-1, GranzymeB, CD4, CD8, and Ki67 (90%+ and negative for CD3, CD5, CD20, CD10, cytokeratin (CK, TdT, HMB-45, epithelial membrane antigen (EMA, and pan-CK, which identified ALCL. After six cycles of Hyper-CVAD/MA regimen, she achieved partial remission. Three months later, she died due to disease progression.Conclusion: This case illustrates the unusual presentation of ALCL in soft tissue with a bad response to chemotherapy. Because of the tendency for rapid progression, ALCL in young adults with extranodal lesions are often treated with high-grade chemotherapy, such as Hyper-CVAD/MA. Keywords: anaplastic large cell lymphoma, ALK+, soft tissue involvement, Hyper-CVAD/MA

  3. Evaluating the efficiency of two phase partitioning stirred tank bio-reactor for treating xylene vapors from the airstreamthrough a bed of Pseudomonas Putida

    Directory of Open Access Journals (Sweden)

    F. Golbabaei

    2015-04-01

    Conclusion: Overall, the results of the present research revealed that the application of two phase stirred tank bioreactors (TPPBs containing pure strains of Pseudomonas putida was successful for treatment of air streams with xylene.

  4. Chromogenic in situ hybridisation (CISH) is a powerful method to detect ALK-positive non-small cell lung carcinomas.

    Science.gov (United States)

    Wagner, F; Streubel, A; Roth, A; Stephan-Falkenau, S; Mairinger, T

    2014-05-01

    We assessed the potential of a chromogenic in situ hybridisation (CISH) assay in comparison with quantitative reverse transcription (RT)-PCR (qPCR) to detect anaplastic lymphoma kinase (ALK) break apart-positive lung carcinomas. Dual-colour CISH using a break apart probe for the ALK gene on 2p23 was performed with 181 formalin-fixed, paraffin-embedded tissue and agar block sections from 175 cases of non-small cell lung carcinomas (NSCLC). Stained slides were analysed with a standard bright-field microscope at 1000× magnification by counting signals from 60 non-overlapping nuclei from three different tumour areas. Samples with ≥15% of positive nuclei were judged as ALK break apart-positive. All samples were simultaneously analysed by qPCR for EML4-ALK to validate CISH results, and positive samples were subject to Sanger sequencing. CISH was successful with 173 of 181 hybridised samples (96%), and seven ALK break apart-positive cases were detected. CISH signals were specific and distinct for both colours. All positive cases were confirmed by qPCR and Sanger sequencing, and concordance between CISH and qPCR was 100%. Nearly all samples (9/10) which failed by qPCR were accessible to CISH analysis. CISH is a very reliable, convenient and inexpensive method to detect ALK-positive NSCLC. CISH success rate is comparably high as with qPCR, and it detects all ALK break apart events in a single assay. It is of special value when RNA quality is poor, or when small biopsies with a very limited amount of tumour cells have to be analysed.

  5. Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain.

    Science.gov (United States)

    Richards, Mark W; O'Regan, Laura; Roth, Daniel; Montgomery, Jessica M; Straube, Anne; Fry, Andrew M; Bayliss, Richard

    2015-05-01

    Proteins of the echinoderm microtubule (MT)-associated protein (EMAP)-like (EML) family contribute to formation of the mitotic spindle and interphase MT network. EML1-4 consist of Trp-Asp 40 (WD40) repeats and an N-terminal region containing a putative coiled-coil. Recurrent gene rearrangements in non-small cell lung cancer (NSCLC) fuse EML4 to anaplastic lymphoma kinase (ALK) causing expression of several oncogenic fusion variants. The fusions have constitutive ALK activity due to self-association through the EML4 coiled-coil. We have determined crystal structures of the coiled-coils from EML2 and EML4, which describe the structural basis of both EML self-association and oncogenic EML4-ALK activation. The structures reveal a trimeric oligomerization state directed by a conserved pattern of hydrophobic residues and salt bridges. We show that the trimerization domain (TD) of EML1 is necessary and sufficient for self-association. The TD is also essential for MT binding; however, this property requires an adjacent basic region. These observations prompted us to investigate MT association of EML4-ALK and EML1-ABL1 (Abelson 1) fusions in which variable portions of the EML component are present. Uniquely, EML4-ALK variant 3, which includes the TD and basic region of EML4 but none of the WD40 repeats, was localized to MTs, both when expressed recombinantly and when expressed in a patient-derived NSCLC cell line (H2228). This raises the question of whether the mislocalization of ALK activity to MTs might influence downstream signalling and malignant properties of cells. Furthermore, the structure of EML4 TD may enable the development of protein-protein interaction inhibitors targeting the trimerization interface, providing a possible avenue towards therapeutic intervention in EML4-ALK NSCLC.

  6. Malignant pleural effusion cell blocks are substitutes for tissue in EML4-ALK rearrangement detection in patients with advanced non-small-cell lung cancer.

    Science.gov (United States)

    Zhong, J; Li, X; Bai, H; Zhao, J; Wang, Z; Duan, J; An, T; Wu, M; Wang, Y; Wang, S; Wang, J

    2016-12-01

    To evaluate the feasibility of malignant pleural effusions (MPE) as surrogate samples for the detection of echinoderm microtubule-associated protein-like4 (EML4)-anaplastic lymphoma kinase (ALK) and to investigate the prognostic and predictive value of EML4-ALK in MPE of non-small-cell lung cancer (NSCLC). One hundred and nine NSCLC patients were retrospectively analysed. EML4-ALK was identified using paraffin-embedded tumour cells in MPE samples by immunohistochemistry (IHC, Ventana) and confirmed by fluorescence using in situ hybridisation (FISH) and qRT-PCR. The EGFR mutation was determined by MPE, using denaturing high-performance liquid chromatography (DHPLC). A total of 5 out of 109 (4.58%) patients were identified as EML4-ALK rearrangement in MPE by IHC.; In addition to two metachronous samples, the consistency of MPE and tissue for EML4-ALK detection was 100% (21/21), and the sensitivity and specificity were 100% (2/2) and 100% (19/19), respectively. EML4-ALK rearrangement cases were confirmed by FISH and qRT-PCR; the sensitivity were both 100% (2/2) when compared with tissue, and it was 60% (3/5) and 100% (5/5), respectively, when compared with MPE by IHC. The overall response rate (ORR) was 100% (2/2) for patients with EML4-ALK in MPE. Moreover, the PFS of these patients appeared to be prolonged in chemotherapy (9.27 versus 6.53 and versus 4.67 months, P = 0.122), compared with the EGFR mutation and the EGFR/ALK double negative group, respectively. EML4-ALK rearrangement detection in malignant pleural effusions is a complementary method for EML4-ALK detection. VETANA and qRT-PCR are more appropriate for MPE detection. EML4-ALK rearrangement in pleural effusions has a predictive value for treatment. © 2016 John Wiley & Sons Ltd.

  7. The Identification and Validation of Novel Small Proteins in Pseudomonas Putida KT-2440

    DEFF Research Database (Denmark)

    Yang, Xiaochen; Long, Katherine

    2014-01-01

    and activities and may lead to the discovery of novel antimicrobial agents. Our project focuses on the identification, validation and characterization of novel s-­‐proteins in the bacterium Pseudomonas putida KT-­2440. As there is virtually no information on s-­‐proteins in pseudomonads, the first step......, total protein samples are prepared, fractionated, and analyzed with mass spectrometry (MS/MS). The MS/MS data are compared to a custom database containing >80000 putative sORF sequences to identify candidates for validation. A total of 56 and 22 putative sORFs were obtained from MS/MS data...... and bioinformatics prediction, respectively, where there is no overlap between the putative sORFs obtained from the two approaches. The sequences encoding the putative sORFs will be integrated onto the Tn7 site on the chromosome as well as on a plasmid expression vector for validation....

  8. Pseudomonas putida KT2440 as a host for biochemicals production

    DEFF Research Database (Denmark)

    Calero Valdayo, Patricia

    The extensive use of cell factories for production of biochemicals is still facing a number of challenges to become economically competitive. One of the problems that need to be overcome is the toxicity of certain chemical products, which prevents yields high enough for their implementation...... in industry.This thesis aims at contributing to developing and characterizing tools for the use of alternative hosts organisms with high tolerance towards toxic compounds, such as Pseudomonas putida. The thesis also focuses on identifying target compounds that may be relevant to produce in this strain...... such as p-coumaric acid, and the mechanisms responsible for such natural tolerance. Moreover, the tolerance towards the highly toxic compound p-hydroxystyrene was improved as an alternative strategy to deal with the effect on growth during the production of such compounds. Expanding the toolbox...

  9. Silver nanotoxicity using a light-emitting biosensor Pseudomonas putida isolated from a wastewater treatment plant.

    Science.gov (United States)

    Dams, R I; Biswas, A; Olesiejuk, A; Fernandes, T; Christofi, N

    2011-11-15

    The effect of silver ions, nano- and micro-particles on a luminescent biosensor bacterium Pseudomonas putida originally isolated from activated sludge was assessed. The bacterium carrying a stable chromosomal copy of the lux operon (luxCDABE) was able to detect toxicity of ionic and particulate silver over short term incubations ranging from 30 to 240 min. The IC(50) values obtained at different time intervals showed that highest toxicity (lowest IC(50)) was obtained after 90 min incubation for all toxicants and this is considered the optimum incubation for testing. The data show that ionic silver is the most toxic followed by nanosilver particles with microsilver particles being least toxic. Release of nanomaterials is likely to have an effect on the activated sludge process as indicated by the study using a common sludge bacterium involved in biodegradation of organic wastes. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Novel polyhydroxyalkanoate copolymers produced in Pseudomonas putida by metagenomic polyhydroxyalkanoate synthases.

    Science.gov (United States)

    Cheng, Jiujun; Charles, Trevor C

    2016-09-01

    Bacterially produced biodegradable polyhydroxyalkanoates (PHAs) with versatile properties can be achieved using different PHA synthases (PhaCs). This work aims to expand the diversity of known PhaCs via functional metagenomics and demonstrates the use of these novel enzymes in PHA production. Complementation of a PHA synthesis-deficient Pseudomonas putida strain with a soil metagenomic cosmid library retrieved 27 clones expressing either class I, class II, or unclassified PHA synthases, and many did not have close sequence matches to known PhaCs. The composition of PHA produced by these clones was dependent on both the supplied growth substrates and the nature of the PHA synthase, with various combinations of short-chain-length (SCL) and medium-chain-length (MCL) PHA. These data demonstrate the ability to isolate diverse genes for PHA synthesis by functional metagenomics and their use for the production of a variety of PHA polymer and copolymer mixtures.

  11. Clinical and Pathologic Findings of Spitz Nevi and Atypical Spitz Tumors with ALK Fusions

    Science.gov (United States)

    Busam, Klaus J; Kutzner, Heinz; Cerroni, Lorenzo; Wiesner, Thomas

    2016-01-01

    Spitz tumors represent a group of melanocytic neoplasms that typically affects young individuals. Microscopically the lesions are composed of cytologically distinct spindle and epithelioid melanocytes, with a range in the architectural display or the cells, their nuclear features, and secondary epidermal or stromal changes. Recently, kinase fusions have been documented in a subset of Spitz tumors, but there is limited information on the clinical and pathologic features associated with those lesions. Here, we report a series of 17 patients (9 male, 8 female) with spitzoid neoplasms showing ALK fusions (5 Spitz nevi and 12 atypical Spitz tumors). The patients’ ages ranged from 2 years to 35 years (mean = 17; median = 16). Most lesions were located on the lower extremities and presented clinically as polypoid nodules. All tumors were compound melanocytic proliferations with a predominant intradermal growth. Tumor thickness ranged from 1.1 to 6 mm (mean = 2.9 mm; median = 2.5 mm). The most characteristic histopathologic feature of the tumors (seen in all but two lesions) was a plexiform dermal growth of intersecting fascicles of fusiform melanocytes. All but two tumors were amelanotic. All tumors were strongly immunoreactive for ALK. The ALK rearrangements were confirmed in all cases by fluorescence in situ hybridization (FISH) and the fusion partner was determined by quantitative polymerase chain reaction as TPM3 (tropomyosin 3) in 11 cases and DCTN1 (dynactin 1) in 6 cases. None of the eight tumors, which were analyzed by FISH for copy number changes of 6p, 6q, 9p, or 11q met criteria for melanoma. Two patients underwent a sentinel lymph node biopsy, and in both cases melanocytes nests were found in the subcapsular sinus of the node. Array comparative genomic hybridization of these two tumors revealed no chromosomal gains or losses. In conclusion, our study revealed that Spitz nevi/tumors with ALK rearrangement show a characteristic plexiform morphology and that

  12. Global Genome Comparative Analysis Reveals Insights of Resistome and Life-Style Adaptation of Pseudomonas putida Strain T2-2 in Oral Cavity

    Directory of Open Access Journals (Sweden)

    Xin Yue Chan

    2014-01-01

    Full Text Available Most Pseudomonas putida strains are environmental microorganisms exhibiting a wide range of metabolic capability but certain strains have been reported as rare opportunistic pathogens and some emerged as multidrug resistant P. putida. This study aimed to assess the drug resistance profile of, via whole genome analysis, P. putida strain T2-2 isolated from oral cavity. At the same time, we also compared the nonenvironmental strain with environmentally isolated P. putida. In silico comparative genome analysis with available reference strains of P. putida shows that T2-2 has lesser gene counts on carbohydrate and aromatic compounds metabolisms, which suggested its little versatility. The detection of its edd gene also suggested T2-2’s catabolism of glucose via ED pathway instead of EMP pathway. On the other hand, its drug resistance profile was observed via in silico gene prediction and most of the genes found were in agreement with drug-susceptibility testing in laboratory by automated VITEK 2. In addition, the finding of putative genes of multidrug resistance efflux pump and ATP-binding cassette transporters in this strain suggests a multidrug resistant phenotype. In summary, it is believed that multiple metabolic characteristics and drug resistance in P. putida strain T2-2 helped in its survival in human oral cavity.

  13. Solubilization and mineralization of polycyclic aromatic hydrocarbons by Pseudomonas putida in the presence of surfactant

    International Nuclear Information System (INIS)

    Doong Rueyan; Lei Wengang

    2003-01-01

    The solubilization and mineralization of polycyclic aromatic hydrocarbons (PAHs) in a soil system amended with different surfactants was examined. Mineralization experiments were conducted with the addition of [ 14 C]pyrene. An inoculum of the PAH-degrading microorganism, Pseudomonas putida, was investigated for its sensitivity towards four non-ionic and one anionic surfactants with different polyoxyethylene (POE) chain lengths. The addition of surfactant was found to enhance the bioavailability of naphthalene, phenanthrene and pyrene with efficiencies ranging from 21.1 to 60.6%, 33.3 to 62.8% and 26.8 to 70.9%, respectively. The enhanced efficiency followed the order of Brij 30, Triton X-100, Tween 80, and Brij 35, which is correlated with the polyoxyethylene chain of the surfactants. Brij 35 and Tween 80 inhibited the growth of P. putida. However, microorganisms can utilize Triton X-100 and Brij 30 as the sole carbon and energy sources at concentrations above CMC values. In the aqueous system without the addition of surfactants, microorganisms could mineralize [ 14 C]pyrene to 14 CO 2 which corresponds to 28% of mineralization. The addition of surfactants decreased the mineralization rate of pyrene. Also, the fraction of the micellar-phase pyrene that can be directly biodegraded decreased as the concentration of micelle increases. However, the mineralization rate can be enhanced by the amendment of Brij 30 when soil was applied to the cultures. This suggests that biodegradable surfactants can be applicable for increasing the bioavailability and mineralization of PAHs in soil systems

  14. Degradation of phenol and TCE using suspended and chitosan-bead immobilized Pseudomonas putida.

    Science.gov (United States)

    Chen, Yan-Min; Lin, Tsair-Fuh; Huang, Chih; Lin, Jui-Che; Hsieh, Feng-Ming

    2007-09-30

    The degradability of phenol and trichloroethene (TCE) by Pseudomonas putida BCRC 14349 in both suspended culture and immobilized culture systems are investigated. Chitosan beads at a size of about 1-2mm were employed to encapsulate the P. putida cells, becoming an immobilized culture system. The phenol concentration was controlled at 100 mg/L, and that of TCE was studied from 0.2 to 20 mg/L. The pH, between 6.7 and 10, did not affect the degradation of either phenol or TCE in the suspended culture system. However, it was found to be an important factor in the immobilized culture system in which the only significant degradation was observed at pH >8. This may be linked to the surface properties of the chitosan beads and its influence on the activity of the bacteria. The transfer yield of TCE on a phenol basis was almost the same for the suspended and immobilized cultures (0.032 mg TCE/mg phenol), except that these yields occurred at different TCE concentrations. The transfer yield at a higher TCE concentration for the immobilized system suggested that the cells immobilized in carriers can be protected from harsh environmental conditions. For kinetic rate interpretation, the Monod equation was employed to describe the degradation rates of phenol, while the Haldane's equation was used for TCE degradation. Based on the kinetic parameters obtained from the two equations, the rate for the immobilized culture systems was only about 1/6 to that of the suspended culture system for phenol degradation, and was about 1/2 for TCE degradation. The slower kinetics observed for the immobilized culture systems was probably due to the slow diffusion of substrate molecules into the beads. However, compared with the suspended cultures, the immobilized cultures may tolerate a higher TCE concentration as much less inhibition was observed and the transfer yield occurred at a higher TCE concentration.

  15. Rapid generation of recombinant Pseudomonas putida secondary metabolite producers using yTREX

    Directory of Open Access Journals (Sweden)

    Andreas Domröse

    2017-12-01

    Full Text Available Microbial secondary metabolites represent a rich source of valuable compounds with a variety of applications in medicine or agriculture. Effective exploitation of this wealth of chemicals requires the functional expression of the respective biosynthetic genes in amenable heterologous hosts. We have previously established the TREX system which facilitates the transfer, integration and expression of biosynthetic gene clusters in various bacterial hosts. Here, we describe the yTREX system, a new tool adapted for one-step yeast recombinational cloning of gene clusters. We show that with yTREX, Pseudomonas putida secondary metabolite production strains can rapidly be constructed by random targeting of chromosomal promoters by Tn5 transposition. Feasibility of this approach was corroborated by prodigiosin production after yTREX cloning, transfer and expression of the respective biosynthesis genes from Serratia marcescens. Furthermore, the applicability of the system for effective pathway rerouting by gene cluster adaptation was demonstrated using the violacein biosynthesis gene cluster from Chromobacterium violaceum, producing pathway metabolites violacein, deoxyviolacein, prodeoxyviolacein, and deoxychromoviridans. Clones producing both prodigiosin and violaceins could be readily identified among clones obtained after random chromosomal integration by their strong color-phenotype. Finally, the addition of a promoter-less reporter gene enabled facile detection also of phenazine-producing clones after transfer of the respective phenazine-1-carboxylic acid biosynthesis genes from Pseudomonas aeruginosa. All compounds accumulated to substantial titers in the mg range. We thus corroborate here the suitability of P. putida for the biosynthesis of diverse natural products, and demonstrate that the yTREX system effectively enables the rapid generation of secondary metabolite producing bacteria by activation of heterologous gene clusters, applicable for

  16. The Regulation of para-Nitrophenol Degradation in Pseudomonas putida DLL-E4.

    Directory of Open Access Journals (Sweden)

    Qiongzhen Chen

    Full Text Available Pseudomonas putida DLL-E4 can efficiently degrade para-nitrophenol and its intermediate metabolite hydroquinone. The regulation of para-nitrophenol degradation was studied, and PNP induced a global change in the transcriptome of P. putida DLL-E4. When grown on PNP, the wild-type strain exhibited significant downregulation of 2912 genes and upregulation of 845 genes, whereas 2927 genes were downregulated and 891 genes upregulated in a pnpR-deleted strain. Genes related to two non-coding RNAs (ins1 and ins2, para-nitrophenol metabolism, the tricarboxylic acid cycle, the outer membrane porin OprB, glucose dehydrogenase Gcd, and carbon catabolite repression were significantly upregulated when cells were grown on para-nitrophenol plus glucose. pnpA, pnpR, pnpC1C2DECX1X2, and pnpR1 are key genes in para-nitrophenol degradation, whereas pnpAb and pnpC1bC2bDbEbCbX1bX2b have lost the ability to degrade para-nitrophenol. Multiple components including transcriptional regulators and other unknown factors regulate para-nitrophenol degradation, and the transcriptional regulation of para-nitrophenol degradation is complex. Glucose utilization was enhanced at early stages of para-nitrophenol supplementation. However, it was inhibited after the total consumption of para-nitrophenol. The addition of glucose led to a significant enhancement in para-nitrophenol degradation and up-regulation in the expression of genes involved in para-nitrophenol degradation and carbon catabolite repression (CCR. It seemed that para-nitrophenol degradation can be regulated by CCR, and relief of CCR might contribute to enhanced para-nitrophenol degradation. In brief, the regulation of para-nitrophenol degradation seems to be controlled by multiple factors and requires further study.

  17. Fructose 1-phosphate is the preferred effector of the metabolic regulator Cra of Pseudomonas putida.

    Science.gov (United States)

    Chavarría, Max; Santiago, César; Platero, Raúl; Krell, Tino; Casasnovas, José M; de Lorenzo, Víctor

    2011-03-18

    The catabolite repressor/activator (Cra) protein is a global sensor and regulator of carbon fluxes through the central metabolic pathways of gram-negative bacteria. To examine the nature of the effector (or effectors) that signal such fluxes to the protein of Pseudomonas putida, the Cra factor of this soil microorganism has been purified and characterized and its three-dimensional structure determined. Analytical ultracentrifugation, gel filtration, and mobility shift assays showed that the effector-free Cra is a dimer that binds an operator DNA sequence in the promoter region of the fruBKA cluster. Furthermore, fructose 1-phosphate (F1P) was found to most efficiently dissociate the Cra-DNA complex. Thermodynamic parameters of the F1P-Cra-DNA interaction calculated by isothermal titration calorimetry revealed that the factor associates tightly to the DNA sequence 5'-TTAAACGTTTCA-3' (K(D) = 26.3 ± 3.1 nM) and that F1P binds the protein with an apparent stoichiometry of 1.06 ± 0.06 molecules per Cra monomer and a K(D) of 209 ± 20 nM. Other possible effectors, like fructose 1,6-bisphosphate, did not display a significant affinity for the regulator under the assay conditions. Moreover, the structure of Cra and its co-crystal with F1P at a 2-Å resolution revealed that F1P fits optimally the geometry of the effector pocket. Our results thus single out F1P as the preferred metabolic effector of the Cra protein of P. putida.

  18. Fructose 1-Phosphate Is the Preferred Effector of the Metabolic Regulator Cra of Pseudomonas putida*

    Science.gov (United States)

    Chavarría, Max; Santiago, César; Platero, Raúl; Krell, Tino; Casasnovas, José M.; de Lorenzo, Víctor

    2011-01-01

    The catabolite repressor/activator (Cra) protein is a global sensor and regulator of carbon fluxes through the central metabolic pathways of Gram-negative bacteria. To examine the nature of the effector (or effectors) that signal such fluxes to the protein of Pseudomonas putida, the Cra factor of this soil microorganism has been purified and characterized and its three-dimensional structure determined. Analytical ultracentrifugation, gel filtration, and mobility shift assays showed that the effector-free Cra is a dimer that binds an operator DNA sequence in the promoter region of the fruBKA cluster. Furthermore, fructose 1-phosphate (F1P) was found to most efficiently dissociate the Cra-DNA complex. Thermodynamic parameters of the F1P-Cra-DNA interaction calculated by isothermal titration calorimetry revealed that the factor associates tightly to the DNA sequence 5′-TTAAACGTTTCA-3′ (KD = 26.3 ± 3.1 nm) and that F1P binds the protein with an apparent stoichiometry of 1.06 ± 0.06 molecules per Cra monomer and a KD of 209 ± 20 nm. Other possible effectors, like fructose 1,6-bisphosphate, did not display a significant affinity for the regulator under the assay conditions. Moreover, the structure of Cra and its co-crystal with F1P at a 2-Å resolution revealed that F1P fits optimally the geometry of the effector pocket. Our results thus single out F1P as the preferred metabolic effector of the Cra protein of P. putida. PMID:21239488

  19. Potential of Pseudomonas putida PCI2 for the Protection of Tomato Plants Against Fungal Pathogens.

    Science.gov (United States)

    Pastor, Nicolás; Masciarelli, Oscar; Fischer, Sonia; Luna, Virginia; Rovera, Marisa

    2016-09-01

    Tomato is one of the most economically attractive vegetable crops due to its high yields. Diseases cause significant losses in tomato production worldwide. We carried out Polymerase Chain Reaction studies to detect the presence of genes encoding antifungal compounds in the DNA of Pseudomonas putida strain PCI2. We also used liquid chromatography-electrospray tandem mass spectrometry to detect and quantify the production of compounds that increase the resistance of plants to diseases from culture supernatants of PCI2. In addition, we investigated the presence of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase in PCI2. Finally, PCI2 was used for inoculation of tomato seeds to study its potential biocontrol activity against Fusarium oxysporum MR193. The obtained results showed that no fragments for the encoding genes of hydrogen cyanide, pyoluteorin, 2,4-diacetylphloroglucinol, pyrrolnitrin, or phenazine-1-carboxylic acid were amplified from the DNA of PCI2. On the other hand, PCI2 produced salicylic acid and jasmonic acid in Luria-Bertani medium and grew in a culture medium containing ACC as the sole nitrogen source. We observed a reduction in disease incidence from 53.33 % in the pathogen control to 30 % in tomato plants pre-inoculated with PCI2 as well as increases in shoot and root dry weights in inoculated plants, as compared to the pathogenicity control. This study suggests that inoculation of tomato seeds with P. putida PCI2 increases the resistance of plants to root rot caused by F. oxysporum and that PCI2 produces compounds that may be involved at different levels in increasing such resistance. Thus, PCI2 could represent a non-contaminating management strategy potentially applicable in vegetable crops such as tomato.

  20. Competition triggers plasmid-mediated enhancement of substrate utilisation in Pseudomonas putida.

    Directory of Open Access Journals (Sweden)

    Hiren Joshi

    2009-06-01

    Full Text Available Competition between species plays a central role in the activity and structure of communities. Stable co-existence of diverse organisms in communities is thought to be fostered by individual tradeoffs and optimization of competitive strategies along resource gradients. Outside the laboratory, microbes exist as multispecies consortia, continuously interacting with one another and the environment. Survival and proliferation of a particular species is governed by its competitive fitness. Therefore, bacteria must be able to continuously sense their immediate environs for presence of competitors and prevailing conditions. Here we present results of our investigations on a novel competition sensing mechanism in the rhizosphere-inhabiting Pseudomonas putida KT2440, harbouring gfpmut3b-modified Kan(R TOL plasmid. We monitored benzyl alcohol (BA degradation rate, along with GFP expression profiling in mono species and dual species cultures. Interestingly, enhanced plasmid expression (monitored using GFP expression and consequent BA degradation were observed in dual species consortia, irrespective of whether the competitor was a BA degrader (Pseudomonas aeruginosa or a non-degrader (E. coli. Attempts at elucidation of the mechanistic aspects of induction indicated the role of physical interaction, but not of any diffusible compounds emanating from the competitors. This contention is supported by the observation that greater induction took place in presence of increasing number of competitors. Inert microspheres mimicking competitor cell size and concentration did not elicit any significant induction, further suggesting the role of physical cell-cell interaction. Furthermore, it was also established that cell wall compromised competitor had minimal induction capability. We conclude that P. putida harbouring pWW0 experience a competitive stress when grown as dual-species consortium, irrespective of the counterpart being BA degrader or not. The immediate

  1. Anaplastic lymphoma kinase (ALK) gene rearrangements in radiation-related human papillary thyroid carcinoma after the Chernobyl accident.

    Science.gov (United States)

    Arndt, Annette; Steinestel, Konrad; Rump, Alexis; Sroya, Manveer; Bogdanova, Tetiana; Kovgan, Leonila; Port, Matthias; Abend, Michael; Eder, Stefan

    2018-04-06

    Childhood radiation exposure has been associated with increased papillary thyroid carcinoma (PTC) risk. The role of anaplastic lymphoma kinase (ALK) gene rearrangements in radiation-related PTC remains unclear, but STRN-ALK fusions have recently been detected in PTCs from radiation exposed persons after Chernobyl using targeted next-generation sequencing and RNA-seq. We investigated ALK and RET gene rearrangements as well as known driver point mutations in PTC tumours from 77 radiation-exposed patients (mean age at surgery 22.4 years) and PTC tumours from 19 non-exposed individuals after the Chernobyl accident. ALK rearrangements were detected by fluorescence in situ hybridisation (FISH) and confirmed with immunohistochemistry (IHC); point mutations in the BRAF and RAS genes were detected by DNA pyrosequencing. Among the 77 tumours from exposed persons, we identified 7 ALK rearrangements and none in the unexposed group. When combining ALK and RET rearrangements, we found 24 in the exposed (31.2%) compared to two (10.5%) in the unexposed group. Odds ratios increased significantly in a dose-dependent manner up to 6.2 (95%CI: 1.1, 34.7; p = 0.039) at Iodine-131 thyroid doses >500 mGy. In total, 27 cases carried point mutations of BRAF or RAS genes, yet logistic regression analysis failed to identify significant dose association. To our knowledge we are the first to describe ALK rearrangements in post-Chernobyl PTC samples using routine methods such as FISH and IHC. Our findings further support the hypothesis that gene rearrangements, but not oncogenic driver mutations, are associated with ionizing radiation-related tumour risk. IHC may represent an effective method for ALK-screening in PTCs with known radiation aetiology, which is of clinical value since oncogenic ALK activation might represent a valuable target for small molecule inhibitors. © 2018 The Authors The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and

  2. The emerging role of ALK inhibitors in the treatment of advanced non-small cell lung cancer.

    Science.gov (United States)

    Galetta, Domenico; Rossi, Antonio; Pisconti, Salvatore; Colucci, Giuseppe

    2012-04-01

    Most NSCLC patients are diagnosed in the advanced stage of the disease. Recently, chemotherapeutic agents have reached a plateau of effectiveness. Increased understanding of cancer biology has revealed several potential therapeutic strategies that have led to marketing of new biologic agents. The echinoderm microtubule-associated protein like-4-anaplastic lymphoma kinase (EML4-ALK) fusion oncogene represents one of the newest molecular targets in NSCLC, identifying a subset of NSCLC patients characterized by distinct clinicopathological features. The available results concerning ALK inhibitors for the treatment of advanced NSCLC patients. An electronic search was used to retrieve the articles addressing this topic. In a pivotal Phase I clinical trial, crizotinib (PF-02341066), a small-molecule ALK inhibitor, demonstrated impressive antitumor activity in the majority of NSCLC patients with ALK fusions. Phase III randomized trials investigating crizotinib in this subgroup of patients are ongoing. If the results from these large international trials confirm the efficacy of crizotinib in the subset of patients, the next few years could see the treatment of advanced NSCLC patients with ALK fusions. Specific inhibitors would realize the so called personalized medicine in subsets of this disease.

  3. The Relevance of External Quality Assessment for Molecular Testing for ALK Positive Non-Small Cell Lung Cancer : Results from Two Pilot Rounds Show Room for Optimization

    NARCIS (Netherlands)

    Tembuyser, Lien; Tack, Veronique; Zwaenepoel, Karen; Pauwels, Patrick; Miller, Keith; Bubendorf, Lukas; Kerr, Keith; Schuuring, Ed; Thunnissen, Erik; Dequeker, Elisabeth M. C.

    2014-01-01

    Background and Purpose: Molecular profiling should be performed on all advanced non-small cell lung cancer with non-squamous histology to allow treatment selection. Currently, this should include EGFR mutation testing and testing for ALK rearrangements. ROS1 is another emerging target. ALK

  4. Oncogenic kinase NPM/ALK induces through STAT3 expression of immunosuppressive protein CD274 (PD-L1, B7-H1)

    DEFF Research Database (Denmark)

    Marzec, Michal; Zhang, Qian; Goradia, Ami

    2008-01-01

    The mechanisms of malignant cell transformation caused by the oncogenic, chimeric nucleophosmin (NPM)/anaplastic lymphoma kinase (ALK) remain only partially understood, with most of the previous studies focusing mainly on the impact of NPM/ALK on cell survival and proliferation. Here we report th...

  5. CHARACTERIZATION OF THE ALKANE-INDUCIBLE CYTOCHROME P450 (P450ALK) GENE FROM THE YEAST CANDIDA TROPICALIS: IDENTIFICATION OF A NEW P450 FAMILY

    Science.gov (United States)

    The P450alk gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. Structural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures ...

  6. A kinetic model for growth and biosynthesis of medium-chain-length poly-(3-hydroxyalkanoates in Pseudomonas putida

    Directory of Open Access Journals (Sweden)

    M. S. M. Annuar

    2008-06-01

    Full Text Available A kinetic model is presented giving a mathematical description of batch culture of Pseudomonas putida PGA1 grown using saponified palm kernel oil as carbon source and ammonium as the limiting nutrient. The growth of the micro-organism is well-described using Tessier-type model which takes into account the inhibitory effect of ammonium at high concentrations. The ammonium consumption rate by the cells is related in proportion to the rate of growth. The intracellular production of medium-chain-length poly-(3-hydroxyalkanoates (PHA MCL by P. putida PGA1 cells is reasonably modeled by the modified Luedeking-Piret kinetics, which incorporate a function of product synthesis inhibition (or reduction by ammonium above a threshold level.

  7. Pseudomonas putida KT2442 as a platform for the biosynthesis of polyhydroxyalkanoates with adjustable monomer contents and compositions

    DEFF Research Database (Denmark)

    Tripathi, Lakshmi; Wu, Lin-Ping; Dechuan, Meng

    2013-01-01

    of P(3HHx-co-3HD) consisting of 3-hydroxyhexanoate (3HHx) and 3-hydroxydecanoate (3HD), the monomer fraction of 3HHx ranged from 16 mol% to 63 mol%. The comonomer compositions were easily regulated by varying the fatty acid concentrations. P. putida KTQQ20 produced a novel diblock copolymer P3HHx-b-P(3......6), random copolymers of P(3HB-co-3HHx) consisting of 3-hydroxybutyrate (3HB), 3-hydroxyhexanoate (3HHx), were accumulated with 3HHx content ranged from 19 mol% to 75 mol%. While recombinant P. putida KTQQ20 grown on mixtures of sodium hexanoate and decanoic acid (C6:C10), produced random copolymers......HD-co-3HDD) consisting of 49 mol% P3HHx and 51 mol% P(3HD-co-3HDD) [35.25 mol% 3HDD (3-hydroxydodecanoate)], which was characterized by (13)C NMR, HMBC NMR, DSC, GPC and universal testing machine....

  8. Nitrogen Removal Characteristics of Pseudomonas putida Y-9 Capable of Heterotrophic Nitrification and Aerobic Denitrification at Low Temperature

    Directory of Open Access Journals (Sweden)

    Yi Xu

    2017-01-01

    Full Text Available The cold-adapted bacterium Pseudomonas putida Y-9 was investigated and exhibited excellent capability for nitrogen removal at 15°C. The strain capable of heterotrophic nitrification and aerobic denitrification could efficiently remove ammonium, nitrate, and nitrite at an average removal rate of 2.85 mg, 1.60 mg, and 1.83 mg NL−1 h−1, respectively. Strain Y-9 performed nitrification in preference to denitrification when ammonium and nitrate or ammonium and nitrite coexisted in the solution. Meantime, the presence of nitrate had no effect on the ammonium removal rate of strain Y-9, and yet the presence of high concentration of nitrite would inhibit the cell growth and decrease the nitrification rate. The experimental results indicate that P. putida Y-9 has potential application for the treatment of wastewater containing high concentrations of ammonium along with its oxidation products at low temperature.

  9. From dirt to industrial applications: Pseudomonas putida as a Synthetic Biology chassis for hosting harsh biochemical reactions.

    Science.gov (United States)

    Nikel, Pablo I; Chavarría, Max; Danchin, Antoine; de Lorenzo, Víctor

    2016-10-01

    The soil bacterium Pseudomonas putida is endowed with a central carbon metabolic network capable of fulfilling high demands of reducing power. This situation arises from a unique metabolic architecture that encompasses the partial recycling of triose phosphates to hexose phosphates-the so-called EDEMP cycle. In this article, the value of P. putida as a bacterial chassis of choice for contemporary, industrially-oriented metabolic engineering is addressed. The biochemical properties that make this bacterium adequate for hosting biotransformations involving redox reactions as well as toxic compounds and intermediates are discussed. Finally, novel developments and open questions in the continuous quest for an optimal microbial cell factory are presented at the light of current and future needs in the area of biocatalysis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  10. Effect of Crc and Hfq proteins on the transcription, processing, and stability of the Pseudomonas putida CrcZ sRNA.

    Science.gov (United States)

    Hernández-Arranz, Sofía; Sánchez-Hevia, Dione; Rojo, Fernando; Moreno, Renata

    2016-12-01

    In Pseudomonas putida, the Hfq and Crc proteins regulate the expression of many genes in response to nutritional and environmental cues, by binding to mRNAs that bear specific target motifs and inhibiting their translation. The effect of these two proteins is antagonized by the CrcZ and CrcY small RNAs (sRNAs), the levels of which vary greatly according to growth conditions. The crcZ and crcY genes are transcribed from promoters PcrcZ and PcrcY, respectively, a process that relies on the CbrB transcriptional activator and the RpoN σ factor. Here we show that crcZ can also be transcribed from the promoter of the immediate upstream gene, cbrB, a weak constitutive promoter. The cbrB-crcZ transcript was processed to render a sRNA very similar in size to the CrcZ produced from promoter PcrcZ The processed sRNA, termed CrcZ*, was able to antagonize Hfq/Crc because, when provided in trans, it relieved the deregulated Hfq/Crc-dependent hyperrepressing phenotype of a ΔcrcZΔcrcY strain. CrcZ* may help in attaining basal levels of CrcZ/CrcZ* that are sufficient to protect the cell from an excessive Hfq/Crc-dependent repression. Since a functional sRNA can be produced from PcrcZ, an inducible strong promoter, or by cleavage of the cbrB-crcZ mRNA, crcZ can be considered a 3'-untranslated region of the cbrB-crcZ mRNA. In the absence of Hfq, the processed form of CrcZ was not observed. In addition, we show that Crc and Hfq increase CrcZ stability, which supports the idea that these proteins can form a complex with CrcZ and protect it from degradation by RNases. © 2016 Hernández-Arranz et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  11. Metabolism of chlorofluorocarbons and polybrominated compounds by Pseudomonas putida G786(pHG-2) via an engineered metabolic pathway.

    Science.gov (United States)

    Hur, H G; Sadowsky, M J; Wackett, L P

    1994-11-01

    The recombinant bacterium Pseudomonas putida G786(pHG-2) metabolizes pentachloroethane to glyoxylate and carbon dioxide, using cytochrome P-450CAM and toluene dioxygenase to catalyze consecutive reductive and oxidative dehalogenation reactions (L.P. Wackett, M.J. Sadowsky, L.N. Newman, H.-G. Hur, and S. Li, Nature [London] 368:627-629, 1994). The present study investigated metabolism of brominated and chlorofluorocarbon compounds by the recombinant strain. Under anaerobic conditions, P. putida G786(pHG-2) reduced 1,1,2,2-tetrabromoethane, 1,2-dibromo-1,2-dichloroethane, and 1,1,1,2-tetrachloro-2,2-difluoroethane to products bearing fewer halogen substituents. Under aerobic conditions, P. putida G786(pHG-2) oxidized cis- and trans-1,2-dibromoethenes, 1,1-dichloro-2,2-difluoroethene, and 1,2-dichloro-1-fluoroethene. Several compounds were metabolized by sequential reductive and oxidative reactions via the constructed metabolic pathway. For example, 1,1,2,2-tetrabromoethane was reduced by cytochrome P-450CAM to 1,2-dibromoethenes, which were subsequently oxidized by toluene dioxygenase. The same pathway metabolized 1,1,1,2-tetrachloro-2,2-difluoroethane to oxalic acid as one of the final products. The results obtained in this study indicate that P. putida G786(pHG-2) metabolizes polyfluorinated, chlorinated, and brominated compounds and further demonstrates the value of using a knowledge of catabolic enzymes and recombinant DNA technology to construct useful metabolic pathways.

  12. Metabolism of chlorofluorocarbons and polybrominated compounds by Pseudomonas putida G786(pHG-2) via an engineered metabolic pathway.

    OpenAIRE

    Hur, H G; Sadowsky, M J; Wackett, L P

    1994-01-01

    The recombinant bacterium Pseudomonas putida G786(pHG-2) metabolizes pentachloroethane to glyoxylate and carbon dioxide, using cytochrome P-450CAM and toluene dioxygenase to catalyze consecutive reductive and oxidative dehalogenation reactions (L.P. Wackett, M.J. Sadowsky, L.N. Newman, H.-G. Hur, and S. Li, Nature [London] 368:627-629, 1994). The present study investigated metabolism of brominated and chlorofluorocarbon compounds by the recombinant strain. Under anaerobic conditions, P. putid...

  13. Combinatorial metabolic engineering of Pseudomonas putida KT2440 for efficient mineralization of 1,2,3-trichloropropane.

    Science.gov (United States)

    Gong, Ting; Xu, Xiaoqing; Che, You; Liu, Ruihua; Gao, Weixia; Zhao, Fengjie; Yu, Huilei; Liang, Jingnan; Xu, Ping; Song, Cunjiang; Yang, Chao

    2017-08-01

    An industrial waste, 1,2,3-trichloropropane (TCP), is toxic and extremely recalcitrant to biodegradation. To date, no natural TCP degraders able to mineralize TCP aerobically have been isolated. In this work, we engineered a biosafety Pseudomonas putida strain KT2440 for aerobic mineralization of TCP by implantation of a synthetic biodegradation pathway into the chromosome and further improved TCP mineralization using combinatorial engineering strategies. Initially, a synthetic pathway composed of haloalkane dehalogenase, haloalcohol dehalogenase and epoxide hydrolase was functionally assembled for the conversion of TCP into glycerol in P. putida KT2440. Then, the growth lag-phase of using glycerol as a growth precursor was eliminated by deleting the glpR gene, significantly enhancing the flux of carbon through the pathway. Subsequently, we improved the oxygen sequestering capacity of this strain through the heterologous expression of Vitreoscilla hemoglobin, which makes this strain able to mineralize TCP under oxygen-limited conditions. Lastly, we further improved intracellular energy charge (ATP/ADP ratio) and reducing power (NADPH/NADP + ratio) by deleting flagella-related genes in the genome of P. putida KT2440. The resulting strain (named KTU-TGVF) could efficiently utilize TCP as the sole source of carbon for growth. Degradation studies in a bioreactor highlight the value of this engineered strain for TCP bioremediation.

  14. Evaluation of Zosteric Acid for Mitigating Biofilm Formation of Pseudomonas putida Isolated from a Membrane Bioreactor System

    Directory of Open Access Journals (Sweden)

    Andrea Polo

    2014-05-01

    Full Text Available This study provides data to define an efficient biocide-free strategy based on zosteric acid to counteract biofilm formation on the membranes of submerged bioreactor system plants. 16S rRNA gene phylogenetic analysis showed that gammaproteobacteria was the prevalent taxa on fouled membranes of an Italian wastewater plant. Pseudomonas was the prevalent genus among the cultivable membrane-fouler bacteria and Pseudomonas putida was selected as the target microorganism to test the efficacy of the antifoulant. Zosteric acid was not a source of carbon and energy for P. putida cells and, at 200 mg/L, it caused a reduction of bacterial coverage by 80%. Biofilm experiments confirmed the compound caused a significant decrease in biomass (−97% and thickness (−50%, and it induced a migration activity of the peritrichous flagellated P. putida over the polycarbonate surface not amenable to a biofilm phenotype. The low octanol-water partitioning coefficient and the high water solubility suggested a low bioaccumulation potential and the water compartment as its main environmental recipient and capacitor. Preliminary ecotoxicological tests did not highlight direct toxicity effects toward Daphnia magna. For green algae Pseudokirchneriella subcapitata an effect was observed at concentrations above 100 mg/L with a significant growth of protozoa that may be connected to a concurrent algal growth inhibition.

  15. Evaluation of medium-chain-length polyhydroxyalkanoate production by Pseudomonas putida LS46 using biodiesel by-product streams.

    Science.gov (United States)

    Fu, Jilagamazhi; Sharma, Umesh; Sparling, Richard; Cicek, Nazim; Levin, David B

    2014-07-01

    Medium-chain-length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46 was analyzed in shake-flask-based batch reactions, using pure chemical-grade glycerol (PG), biodiesel-derived "waste" glycerol (WG), and biodiesel-derived "waste" free fatty acids (WFA). Cell growth, substrate consumption, mcl-PHA accumulation within the cells, and the monomer composition of the synthesized biopolymers were monitored. The patterns of mcl-PHA synthesis in P. putida LS46 cells grown on PG and WG were similar but differed from that of cells grown with WFA. Polymer accumulation in glycerol-based cultures was stimulated by nitrogen limitation and plateaued after 48 h in both PG and WG cultures, with a total accumulation of 17.9% cell dry mass and 16.3% cell dry mass, respectively. In contrast, mcl-PHA synthesis was independent of nitrogen concentration in P. putida LS46 cells cultured with WFA, which accumulated to 29% cell dry mass. In all cases, the mcl-PHAs synthesized consisted primarily of 3-hydroxyoctanoate (C(8)) and 3-hydroxydecanoate (C(10)). WG and WFA supported similar or greater cell growth and mcl-PHA accumulation than PG under the experimental conditions used. These results suggest that biodiesel by-product streams could be used as low-cost carbon sources for sustainable mcl-PHA production.

  16. Determination of copper binding in Pseudomonas putida CZ1 by chemical modifications and X-ray absorption spectroscopy.

    Science.gov (United States)

    Chen, XinCai; Shi, JiYan; Chen, YingXu; Xu, XiangHua; Chen, LiTao; Wang, Hui; Hu, TianDou

    2007-03-01

    Previously performed studies have shown that Pseudomonas putida CZ1 biomass can bind an appreciable amount of Cu(II) and Zn(II) ions from aqueous solutions. The mechanisms of Cu- and Zn-binding by P. putida CZ1 were ascertained by chemical modifications of the biomass followed by Fourier transform infrared and X-ray absorption spectroscopic analyses of the living or nonliving cells. A dramatic decrease in Cu(II)- and Zn(II)-binding resulted after acidic methanol esterification of the nonliving cells, indicating that carboxyl functional groups play an important role in the binding of metal to the biomaterial. X-ray absorption spectroscopy was used to determine the speciation of Cu ions bound by living and nonliving cells, as well as to elucidate which functional groups were involved in binding of the Cu ions. The X-ray absorption near-edge structure spectra analysis showed that the majority of the Cu was bound in both samples as Cu(II). The fitting results of Cu K-edge extended X-ray absorption fine structure spectra showed that N/O ligands dominated in living and nonliving cells. Therefore, by combining different techniques, our results indicate that carboxyl functional groups are the major ligands responsible for the metal binding in P. putida CZ1.

  17. Protein as chemical cue: non-nutritional growth enhancement by exogenous protein in Pseudomonas putida KT2440.

    Directory of Open Access Journals (Sweden)

    Hiren Joshi

    Full Text Available Research pertaining to microbe-microbe and microbe-plant interactions has been largely limited to small molecules like quorum sensing chemicals. However, a few recent reports have indicated the role of complex molecules like proteins and polysaccharides in microbial communication. Here we demonstrate that exogenous proteins present in culture media can considerably accelerate the growth of Pseudomonas putida KT2440, even when such proteins are not internalized by the cells. The growth enhancement is observed when the exogenous protein is not used as a source of carbon or nitrogen. The data show non-specific nature of the protein inducing growth; growth enhancement was observed irrespective of the protein type. It is shown that growth enhancement is mediated via increased siderophore secretion in response to the exogenous protein, leading to better iron uptake. We highlight the ecological significance of the observation and hypothesize that exogenous proteins serve as chemical cues in the case of P.putida and are perceived as indicator of the presence of competitors in the environment. It is argued that enhanced siderophore secretion in response to exogenous protein helps P.putida establish numerical superiority over competitors by way of enhanced iron assimilation and quicker utilization of aromatic substrates.

  18. Rearranged EML4-ALK fusion transcripts sequester in circulating blood platelets and enable blood-based crizotinib response monitoring in non-small-cell lung cancer

    Science.gov (United States)

    Nilsson, R. Jonas A.; Karachaliou, Niki; Berenguer, Jordi; Gimenez-Capitan, Ana; Schellen, Pepijn; Teixido, Cristina; Tannous, Jihane; Kuiper, Justine L.; Drees, Esther; Grabowska, Magda; van Keulen, Marte; Heideman, Danielle A.M.; Thunnissen, Erik; Dingemans, Anne-Marie C.; Viteri, Santiago; Tannous, Bakhos A.; Drozdowskyj, Ana; Rosell, Rafael; Smit, Egbert F.; Wurdinger, Thomas

    2016-01-01

    Purpose: Non-small-cell lung cancers harboring EML4-ALK rearrangements are sensitive to crizotinib. However, despite initial response, most patients will eventually relapse, and monitoring EML4-ALK rearrangements over the course of treatment may help identify these patients. However, challenges associated with serial tumor biopsies have highlighted the need for blood-based assays for the monitoring of biomarkers. Platelets can sequester RNA released by tumor cells and are thus an attractive source for the non-invasive assessment of biomarkers. Methods: EML4-ALK rearrangements were analyzed by RT-PCR in platelets and plasma isolated from blood obtained from 77 patients with non-small-cell lung cancer, 38 of whom had EML4-ALK-rearranged tumors. In a subset of 29 patients with EML4-ALK-rearranged tumors who were treated with crizotinib, EML4-ALK rearrangements in platelets were correlated with progression-free and overall survival. Results: RT-PCR demonstrated 65% sensitivity and 100% specificity for the detection of EML4-ALK rearrangements in platelets. In the subset of 29 patients treated with crizotinib, progression-free survival was 3.7 months for patients with EML4-ALK+ platelets and 16 months for those with EML4-ALK− platelets (hazard ratio, 3.5; P = 0.02). Monitoring of EML4-ALK rearrangements in the platelets of one patient over a period of 30 months revealed crizotinib resistance two months prior to radiographic disease progression. Conclusions: Platelets are a valuable source for the non-invasive detection of EML4-ALK rearrangements and may prove useful for predicting and monitoring outcome to crizotinib, thereby improving clinical decisions based on radiographic imaging alone. PMID:26544515

  19. Characterization of a marine-isolated mercury-resistant Pseudomonas putida strain SP1 and its potential application in marine mercury reduction

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Weiwei; Chen, Lingxin; Liu, Dongyan [Chinese Academy of Sciences, Yantai, SD (China). Yantai Inst. of Coastal Zone Research (YICCAS); Chinese Academy of Sciences, Yantai, SD (China). Shandong Provincial Key Lab. of Coastal Zone Environmental Processes

    2012-02-15

    The Pseudomonas putida strain SP1 was isolated from marine environment and was found to be resistant to 280 {mu}M HgCl{sub 2}. SP1 was also highly resistant to other metals, including CdCl{sub 2}, CoCl{sub 2}, CrCl{sub 3}, CuCl{sub 2}, PbCl{sub 2}, and ZnSO{sub 4}, and the antibiotics ampicillin (Ap), kanamycin (Kn), chloramphenicol (Cm), and tetracycline (Tc). mer operon, possessed by most mercury-resistant bacteria, and other diverse types of resistant determinants were all located on the bacterial chromosome. Cold vapor atomic absorption spectrometry and a volatilization test indicated that the isolated P. putida SP1 was able to volatilize almost 100% of the total mercury it was exposed to and could potentially be used for bioremediation in marine environments. The optimal pH for the growth of P. putida SP1 in the presence of HgCl{sub 2} and the removal of HgCl{sub 2} by P. putida SP1 was between 8.0 and 9.0, whereas the optimal pH for the expression of merA, the mercuric reductase enzyme in mer operon that reduces reactive Hg{sup 2+} to volatile and relatively inert monoatomic Hg{sup 0} vapor, was around 5.0. LD50 of P. putida SP1 to flounder and turbot was 1.5 x 10{sup 9} CFU. Biofilm developed by P. putida SP1 was 1- to 3-fold lower than biofilm developed by an aquatic pathogen Pseudomonas fluorescens TSS. The results of this study indicate that P. putida SP1 is a low virulence strain that can potentially be applied in the bioremediation of HgCl{sub 2} contamination over a broad range of pH. (orig.)

  20. Chloroacetaldehyde-induced mutagenesis in Escherichia coli: The role of AlkB protein in repair of 3,N4-ethenocytosine and 3,N4-α-hydroxyethanocytosine

    International Nuclear Information System (INIS)

    Maciejewska, Agnieszka M.; Ruszel, Karol P.; Nieminuszczy, Jadwiga; Lewicka, Joanna; Sokolowska, Beata; Grzesiuk, Elzbieta; Kusmierek, Jaroslaw T.

    2010-01-01

    Etheno (ε) adducts are formed in reaction of DNA bases with various environmental carcinogens and endogenously created products of lipid peroxidation. Chloroacetaldehyde (CAA), a metabolite of carcinogen vinyl chloride, is routinely used to generate ε-adducts. We studied the role of AlkB, along with AlkA and Mug proteins, all engaged in repair of ε-adducts, in CAA-induced mutagenesis. The test system used involved pIF102 and pIF104 plasmids bearing the lactose operon of CC102 or CC104 origin (Cupples and Miller (1989) ) which allowed to monitor Lac + revertants, the latter arose by GC → AT or GC → TA substitutions, respectively, as a result of modification of guanine and cytosine. The plasmids were CAA-damaged in vitro and replicated in Escherichia coli of various genetic backgrounds. To modify the levels of AlkA and AlkB proteins, mutagenesis was studied in E. coli cells induced or not in adaptive response. Formation of εC proceeds via a relatively stable intermediate, 3,N 4 -α-hydroxyethanocytosine (HEC), which allowed to compare repair of both adducts. The results indicate that all three genes, alkA, alkB and mug, are engaged in alleviation of CAA-induced mutagenesis. The frequency of mutation was higher in AlkA-, AlkB- and Mug-deficient strains in comparison to alkA + , alkB + , and mug + controls. Considering the levels of CAA-induced Lac + revertants in strains harboring the pIF plasmids and induced or not in adaptive response, we conclude that AlkB protein is engaged in the repair of εC and HEC in vivo. Using the modified TTCTT 5-mers as substrates, we confirmed in vitro that AlkB protein repairs εC and HEC although far less efficiently than the reference adduct 3-methylcytosine. The pH optimum for repair of HEC and εC is significantly different from that for 3-methylcytosine. We propose that the protonated form of adduct interact in active site of AlkB protein.

  1. Comparison of IHC, FISH and RT-PCR methods for detection of ALK rearrangements in 312 non-small cell lung cancer patients in Taiwan.

    Science.gov (United States)

    Wu, Yi-Cheng; Chang, Il-Chi; Wang, Chi-Liang; Chen, Tai-Di; Chen, Ya-Ting; Liu, Hui-Ping; Chu, Yen; Chiu, Yu-Ting; Wu, Tzu-Hua; Chou, Li-Hui; Chen, Yi-Rong; Huang, Shiu-Feng

    2013-01-01

    Recently Echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK) fusion gene has become an important biomarker for ALK tyrosine kinase inhibitor (crizotinib) treatment in NSCLC. However, the best detection method and the significance of EML4-ALK variant types remain uncertain. Reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in Situ hybridization (FISH) and Immunohistochemical (IHC) stain were performed on tumor tissues of 312 NSCLC patients for detection of ALK rearrangements. Mutation analyses for EGFR and KRAS genes were also performed. Thirteen of the 312 patients (4.17%) had ALK rearrangements detected by RT-PCR. If RT-PCR data was used as the gold standard, FISH tests had a low sensitivity (58.33%), but very good specificity (99.32%). IHC stain had better sensitivity (91.67%) than FISH, but lower specificity (79.52%), when the cut off was IHC2+. All of the 8 patients with high abundance of EML4-ALK positive cells in tumor tissues (assessed by the signal intensities of the RT-PCR product), were also have high expression of ALK protein (IHC3+), and positive for FISH, except one failed in FISH. Variants 3a+3b (4/5, 80%) of EML4-ALK fusion gene were more common to have high abundance of EML4-ALK positive cells in tumor tissues than variant 1 (1/3, 33.3%). Meta-analysis of the published data of 2273 NSCLC patients revealed that variant 3 (23/44, 52.3%) was the most common type in Chinese population, while variant 1 (28/37, 75.7%) was most common in Caucasian. Among the three detection methods, RT-PCR could detect not only the presence of EML4-ALK fusion gene and their variant types, but also the abundance of EML4-ALK positive cells in NSCLC tumor tissues. The latter two factors might affect the treatment response to anti-ALK inhibitor. Including RT-PCR as a diagnostic test for ALK inhibitor treatment in the prospective clinical trials is recommended.

  2. Comparison of IHC, FISH and RT-PCR methods for detection of ALK rearrangements in 312 non-small cell lung cancer patients in Taiwan.

    Directory of Open Access Journals (Sweden)

    Yi-Cheng Wu

    Full Text Available BACKGROUND: Recently Echinoderm microtubule-associated protein-like 4- anaplastic lymphoma kinase (EML4-ALK fusion gene has become an important biomarker for ALK tyrosine kinase inhibitor (crizotinib treatment in NSCLC. However, the best detection method and the significance of EML4-ALK variant types remain uncertain. METHODS: Reverse transcriptase-polymerase chain reaction (RT-PCR, fluorescence in Situ hybridization (FISH and Immunohistochemical (IHC stain were performed on tumor tissues of 312 NSCLC patients for detection of ALK rearrangements. Mutation analyses for EGFR and KRAS genes were also performed. RESULTS: Thirteen of the 312 patients (4.17% had ALK rearrangements detected by RT-PCR. If RT-PCR data was used as the gold standard, FISH tests had a low sensitivity (58.33%, but very good specificity (99.32%. IHC stain had better sensitivity (91.67% than FISH, but lower specificity (79.52%, when the cut off was IHC2+. All of the 8 patients with high abundance of EML4-ALK positive cells in tumor tissues (assessed by the signal intensities of the RT-PCR product, were also have high expression of ALK protein (IHC3+, and positive for FISH, except one failed in FISH. Variants 3a+3b (4/5, 80% of EML4-ALK fusion gene were more common to have high abundance of EML4-ALK positive cells in tumor tissues than variant 1 (1/3, 33.3%. Meta-analysis of the published data of 2273 NSCLC patients revealed that variant 3 (23/44, 52.3% was the most common type in Chinese population, while variant 1 (28/37, 75.7% was most common in Caucasian. CONCLUSIONS: Among the three detection methods, RT-PCR could detect not only the presence of EML4-ALK fusion gene and their variant types, but also the abundance of EML4-ALK positive cells in NSCLC tumor tissues. The latter two factors might affect the treatment response to anti-ALK inhibitor. Including RT-PCR as a diagnostic test for ALK inhibitor treatment in the prospective clinical trials is recommended.

  3. Lead Sorption from Aqueous Solutions by Pseudomonas putida (p168 and its Composites with Palygorskite and Sepiolite Clays

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    Marzieh tavanaei

    2017-02-01

    Full Text Available Introduction: Heavy metals contamination due to natural and anthropogenic sources is a global environmental concern. Lead (Pb is one of the very toxic heavy metals. Industrial production processes and their emissions, mining operation, smelting, combustion sources and solid waste incinerators are the primary sources of lead. This heavy metal has aberrant effects on the environment and living organisms. Hence, proper treatment of lead from soil and industrial wastewaters is very important. In order to remove toxic heavy metals from contaminated water systems, conventional methods such as chemical precipitation, coagulation, ion exchange, solvent extraction and filtration, evaporation and membrane methods are being used. These conventional methods generally have high costs and technical problems. Therefore, biosorption processes, in which microorganisms are used as sorbents, have been considered as economical and environmentally friendly options for removal of heavy metals from aqueous solution. Clay minerals are another group of sorbents used in removal of heavy metals from polluted environments. Furthermore, bacterial cells can be attached on clay mineral surfaces and form bacteria-mineral composites. These composites adsorb heavy metals and convert them into forms with low mobility and bioavailability. Pseudomonas putida is a unique microorganism with a high tendency to sorb and/or degrade certain environmental pollutants. Palygorskite and sepiolite are the fibrous clay minerals of arid and semiarid regions; their structures consist of ribbons and channels. These fibrous minerals have various applications in industry and the environment because of its large surface area and high adsorption capacity. The present study was conducted in order to determine the ability of Pseudomonas putida (P168, and its composites with palygorskite and sepiolite in lead sorption. Materials and Methods: The bacterial strain used in the present study was Pseudomonas

  4. Dependence of toxicity of silver nanoparticles on Pseudomonas putida biofilm structure.

    Science.gov (United States)

    Thuptimdang, Pumis; Limpiyakorn, Tawan; Khan, Eakalak

    2017-12-01

    Susceptibility of biofilms with different physical structures to silver nanoparticles (AgNPs) was studied. Biofilms of Pseudomonas putida KT2440 were formed in batch conditions under different carbon sources (glucose, glutamic acid, and citrate), glucose concentrations (5 and 50 mM), and incubation temperatures (25 and 30 °C). The biofilms were observed using confocal laser scanning microscopy for their physical characteristics (biomass amount, thickness, biomass volume, surface to volume ratio, and roughness coefficient). The biofilms forming under different growth conditions exhibited different physical structures. The biofilm thickness and the roughness coefficient were found negatively and positively correlated with the biofilm susceptibility to AgNPs, respectively. The effect of AgNPs on biofilms was low (1-log reduction of cell number) when the biofilms had high biomass amount, high thickness, high biomass volume, low surface to volume ratio, and low roughness coefficient. Furthermore, the extracellular polymeric substance (EPS) stripping process was applied to confirm the dependence of susceptibility to AgNPs on the structure of biofilm. After the EPS stripping process, the biofilms forming under different conditions showed reduction in thickness and biomass volume, and increases in surface to volume ratio and roughness coefficient, which led to more biofilm susceptibility to AgNPs. The results of this study suggest that controlling the growth conditions to alter the biofilm physical structure is a possible approach to reduce the impact of AgNPs on biofilms in engineered and natural systems. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Effect of silver nanoparticles on Pseudomonas putida biofilms at different stages of maturity

    Energy Technology Data Exchange (ETDEWEB)

    Thuptimdang, Pumis, E-mail: pumis.th@gmail.com [International Program in Hazardous Substance and Environmental Management, Graduate School, Chulalongkorn University, Bangkok 10330 (Thailand); Center of Excellence on Hazardous Substance Management, Bangkok 10330 (Thailand); Limpiyakorn, Tawan, E-mail: tawan.l@chula.ac.th [Center of Excellence on Hazardous Substance Management, Bangkok 10330 (Thailand); Department of Environmental Engineering, Chulalongkorn University, Bangkok 10330 (Thailand); Research Unit Control of Emerging Micropollutants in Environment, Chulalongkorn University, Bangkok 10330 (Thailand); McEvoy, John, E-mail: john.mcevoy@ndsu.edu [Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND 58108 (United States); Prüß, Birgit M., E-mail: birgit.pruess@ndsu.edu [Department of Veterinary and Microbiological Sciences, North Dakota State University, Fargo, ND 58108 (United States); Khan, Eakalak, E-mail: eakalak.khan@ndsu.edu [Department of Civil and Environmental Engineering, North Dakota State University, Fargo, ND 58108 (United States)

    2015-06-15

    Highlights: • Biofilm stages in static batch conditions were similar to dynamic conditions. • Expression of csgA gene increased earlier than alg8 gene in biofilm maturation. • AgNPs had higher effect on less mature biofilms. • Removal of extracellular polymeric substance made biofilms susceptible to AgNPs. - Abstract: This study determined the effect of silver nanoparticles (AgNPs) on Pseudomonas putida KT2440 biofilms at different stages of maturity. Three biofilm stages (1–3, representing early to late stages of development) were identified from bacterial adenosine triphosphate (ATP) activity under static (96-well plate) and dynamic conditions (Center for Disease Control and Prevention biofilm reactor). Extracellular polymeric substance (EPS) levels, measured using crystal violet and total carbohydrate assays, and expression of the EPS-associated genes, csgA and alg8, supported the conclusion that biofilms at later stages were older than those at earlier stages. More mature biofilms (stages 2 and 3) showed little to no reduction in ATP activity following exposure to AgNPs. In contrast, the same treatment reduced ATP activity by more than 90% in the less mature stage 1 biofilms. Regardless of maturity, biofilms with EPS stripped off were more susceptible to AgNPs than controls with intact EPS, demonstrating that EPS is critical for biofilm tolerance of AgNPs. The findings from this study show that stage of maturity is an important factor to consider when studying effect of AgNPs on biofilms.

  6. Effect of silver nanoparticles on Pseudomonas putida biofilms at different stages of maturity

    International Nuclear Information System (INIS)

    Thuptimdang, Pumis; Limpiyakorn, Tawan; McEvoy, John; Prüß, Birgit M.; Khan, Eakalak

    2015-01-01

    Highlights: • Biofilm stages in static batch conditions were similar to dynamic conditions. • Expression of csgA gene increased earlier than alg8 gene in biofilm maturation. • AgNPs had higher effect on less mature biofilms. • Removal of extracellular polymeric substance made biofilms susceptible to AgNPs. - Abstract: This study determined the effect of silver nanoparticles (AgNPs) on Pseudomonas putida KT2440 biofilms at different stages of maturity. Three biofilm stages (1–3, representing early to late stages of development) were identified from bacterial adenosine triphosphate (ATP) activity under static (96-well plate) and dynamic conditions (Center for Disease Control and Prevention biofilm reactor). Extracellular polymeric substance (EPS) levels, measured using crystal violet and total carbohydrate assays, and expression of the EPS-associated genes, csgA and alg8, supported the conclusion that biofilms at later stages were older than those at earlier stages. More mature biofilms (stages 2 and 3) showed little to no reduction in ATP activity following exposure to AgNPs. In contrast, the same treatment reduced ATP activity by more than 90% in the less mature stage 1 biofilms. Regardless of maturity, biofilms with EPS stripped off were more susceptible to AgNPs than controls with intact EPS, demonstrating that EPS is critical for biofilm tolerance of AgNPs. The findings from this study show that stage of maturity is an important factor to consider when studying effect of AgNPs on biofilms

  7. Effects of Pseudomonas putida and Glomusintraradices Inoculations on Morphological and Biochemical Traitsin Trigonellafoenum-graecum L.

    Directory of Open Access Journals (Sweden)

    simin irankhah

    2017-02-01

    Full Text Available Introduction: Fenugreek (Trigonellafoenum-graecum L. is a traditional medicinal plant belonging to the legume family Fabaceae. Diverse groups of microorganisms are symbiotic with Fenugreek roots system. This integration leads to significant increases in the development and production by increasing nitrogen fixation, phytohormones production, siderophores and phosphate solubilization. Plant growth-promoting bacteria increase plant growth byimproving nutrientuptake and phytohormones production. In addition, the beneficial effect of these bacteria could be due totheirinteractionwithArbuscularMycorrhizal fungi(VAM. Drought is one of the major limiting factors for crop production in many parts of the world including Iran. Symbiotic microorganisms can enhance plant tolerance to drought. This experiment was carried out to investigate the effect of Vesicular ArbuscularMycorrhiza (VAM and Plant Growth Promoting Rhizobacteria (PGPR on morphological and biochemical characteristics of Fenugreek in drought stress conditions. Materials and Methods: The experiment was carried out in completely random design with 3 replications.There were four treatments including inoculation with Pseudomonas putida, inoculation with Glomusintraradices, combined association of Pseudomonas putida and Glomusintraradices and untreated as a check under drought stress (40% of field capacity and non-stress conditions (80% of field capacity. In this experiment fiveseeds were sowninplastic pots. Before sowing, seeds were inoculated with microorganisms. In order to inoculation ofseed with Mycorrhizal fungi, for each kilogram of soil, 100 grams of powder containing 10 to 15 thousand spores of fungal soil (produced by the biotech company Toos was added to three centimeters of soil in the pot. For seed inoculation with Plant Growth Promoting Rhizobacteria, the growth curve of the bacteria was drawn at first and then the best time for the growth of bacteria was determined. The bacteria at

  8. Environmental stress speeds up DNA replication in Pseudomonas putida in chemostat cultivations.

    Science.gov (United States)

    Lieder, Sarah; Jahn, Michael; Koepff, Joachim; Müller, Susann; Takors, Ralf

    2016-01-01

    Cellular response to different types of stress is the hallmark of the cell's strategy for survival. How organisms adjust their cell cycle dynamics to compensate for changes in environmental conditions is an important unanswered question in bacterial physiology. A cell using binary fission for reproduction passes through three stages during its cell cycle: a stage from cell birth to initiation of replication, a DNA replication phase and a period of cell division. We present a detailed analysis of durations of cell cycle phases, investigating their dynamics under environmental stress conditions. Applying continuous steady state cultivations (chemostats), the DNA content of a Pseudomonas putida KT2440 population was quantified with flow cytometry at distinct growth rates. Data-driven modeling revealed that under stress conditions, such as oxygen deprivation, solvent exposure and decreased iron availability, DNA replication was accelerated correlated to the severity of the imposed stress (up to 1.9-fold). Cells maintained constant growth rates by balancing the shortened replication phase with extended cell cycle phases before and after replication. Transcriptome data underpin the transcriptional upregulation of crucial genes of the replication machinery. Hence adaption of DNA replication speed appears to be an important strategy to withstand environmental stress. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Functional analysis of aromatic biosynthetic pathways in Pseudomonas putida KT2440

    Science.gov (United States)

    Molina‐Henares, M. Antonia; García‐Salamanca, Adela; Molina‐Henares, A. Jesús; De La Torre, Jesús; Herrera, M. Carmen; Ramos, Juan L.; Duque, Estrella

    2009-01-01

    Summary Pseudomonas putida KT2440 is a non‐pathogenic prototrophic bacterium with high potential for biotechnological applications. Despite all that is known about this strain, the biosynthesis of essential chemicals has not been fully analysed and auxotroph mutants are scarce. We carried out massive mini‐Tn5 random mutagenesis and screened for auxotrophs that require aromatic amino acids. The biosynthesis of aromatic amino acids was analysed in detail including physical and transcriptional organization of genes, complementation assays and feeding experiments to establish pathway intermediates. There is a single pathway from chorismate leading to the biosynthesis of tryptophan, whereas the biosynthesis of phenylalanine and tyrosine is achieved through multiple convergent pathways. Genes for tryptophan biosynthesis are grouped in unlinked regions with the trpBA and trpGDE genes organized as operons and the trpI, trpE and trpF genes organized as single transcriptional units. The pheA and tyrA gene‐encoding multifunctional enzymes for phenylalanine and tyrosine biosynthesis are linked in the chromosome and form an operon with the serC gene involved in serine biosynthesis. The last step in the biosynthesis of these two amino acids requires an amino transferase activity for which multiple tyrB‐like genes are present in the host chromosome. PMID:21261884

  10. Daya Tahan Hidup Pseudomonas putida Strain Pf-20 dalam Beberapa Macam Inokulum

    Directory of Open Access Journals (Sweden)

    Yenny Wuryandari

    2004-07-01

    Full Text Available For any crop-protection agent, an efficient formulation is a necessity to translate laboratory activity into adequate field performance. There are particular challenges to be faced in formulation of biological control agents, because the active ingredient is a living organism that must be kept relatively immobile and inactive while in storage, but quickly resume its  normal metabolic processes once applied to the target site. The objective of the research was  to study survival of Pseudomonas putida strain Pf-20 in various formulations at the storage  time and germination. The twelve formulations include carriers, additives and concentration  of Pf-20. The efficacy of various formulation in maintaining the population of Pf-20 in storage was assessed. The research result showed that population of Pf 20  in the formulation  number seven was the highest, with the combination peat+talc, CMC+arginin and  concentration of Pf[20 10^10 CFU/ml. In peat+talc, CMC+arginin, Pf-20 10^10  CFU/ml based formulation the bacteria survived even up to 84 days of storage although the population declined. In all formulations, population of Pf-20 increased at the time of seed germination. At the time of seed germination, formulation number seven was the highest too.

  11. A protocatechuate biosensor for Pseudomonas putida KT2440 via promoter and protein evolution

    Directory of Open Access Journals (Sweden)

    Ramesh K. Jha

    2018-06-01

    Full Text Available Robust fluorescence-based biosensors are emerging as critical tools for high-throughput strain improvement in synthetic biology. Many biosensors are developed in model organisms where sophisticated synthetic biology tools are also well established. However, industrial biochemical production often employs microbes with phenotypes that are advantageous for a target process, and biosensors may fail to directly transition outside the host in which they are developed. In particular, losses in sensitivity and dynamic range of sensing often occur, limiting the application of a biosensor across hosts. Here we demonstrate the optimization of an Escherichia coli-based biosensor in a robust microbial strain for the catabolism of aromatic compounds, Pseudomonas putida KT2440, through a generalizable approach of modulating interactions at the protein-DNA interface in the promoter and the protein-protein dimer interface. The high-throughput biosensor optimization approach demonstrated here is readily applicable towards other allosteric regulators. Keywords: Whole cell biosensor, Aromatic catabolism, Transcription factor, PcaU, Shikimate

  12. NPM-ALK mediates phosphorylation of MSH2 at tyrosine 238, creating a functional deficiency in MSH2 and the loss of mismatch repair

    International Nuclear Information System (INIS)

    Bone, K M; Wang, P; Wu, F; Wu, C; Li, L; Bacani, J T; Andrew, S E; Lai, R

    2015-01-01

    The vast majority of anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ALCL) tumors express the characteristic oncogenic fusion protein NPM-ALK, which mediates tumorigenesis by exerting its constitutive tyrosine kinase activity on various substrates. We recently identified MSH2, a protein central to DNA mismatch repair (MMR), as a novel binding partner and phosphorylation substrate of NPM-ALK. Here, using liquid chromatography–mass spectrometry, we report for the first time that MSH2 is phosphorylated by NPM-ALK at a specific residue, tyrosine 238. Using GP293 cells transfected with NPM-ALK, we confirmed that the MSH2 Y238F mutant is not tyrosine phosphorylated. Furthermore, transfection of MSH2 Y238F into these cells substantially decreased the tyrosine phosphorylation of endogenous MSH2. Importantly, gene transfection of MSH2 Y238F abrogated the binding of NPM-ALK with endogenous MSH2, re-established the dimerization of MSH2:MSH6 and restored the sensitivity to DNA mismatch-inducing drugs, indicative of MMR return. Parallel findings were observed in two ALK+ALCL cell lines, Karpas 299 and SUP-M2. In addition, we found that enforced expression of MSH2 Y238F into ALK+ALCL cells alone was sufficient to induce spontaneous apoptosis. In conclusion, our findings have identified NPM-ALK-induced phosphorylation of MSH2 at Y238 as a crucial event in suppressing MMR. Our studies have provided novel insights into the mechanism by which oncogenic tyrosine kinases disrupt MMR

  13. Isolation of the alkane inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis

    Science.gov (United States)

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  14. CT radiogenomic characterization of EGFR, K-RAS, and ALK mutations in non-small cell lung cancer

    Energy Technology Data Exchange (ETDEWEB)

    Rizzo, Stefania; Rampinelli, Cristiano [European Institute of Oncology, Department of Radiology, Milan (Italy); Petrella, Francesco; Spaggiari, Lorenzo [European Institute of Oncology, Department of Thoracic Surgery, Milan (Italy); Buscarino, Valentina; De Maria, Federica [University of Milan, Department of Health Sciences, Milan (Italy); Raimondi, Sara [European Institute of Oncology, Department of Epidemiology and Biostatistics, Milan (Italy); Barberis, Massimo; Fumagalli, Caterina [European Institute of Oncology, Department of Pathology, Milan (Italy); Spitaleri, Gianluca; De Marinis, Filippo [European Institute of Oncology, Department of Thoracic Oncology, Milan (Italy); Bellomi, Massimo [European Institute of Oncology, Department of Radiology, Milan (Italy); University of Milan, Department of Health Sciences, Milan (Italy)

    2016-01-15

    To assess the association between CT features and EGFR, ALK, KRAS mutations in non-small cell lung cancer. Patients undergoing chest CT and testing for the above gene mutations were included. Qualitative evaluation of CTs included: lobe; lesion diameter; shape; margins; ground-glass opacity; density; cavitation; air bronchogram; pleural thickening; intratumoral necrosis; nodules in tumour lobe; nodules in non-tumour lobes; pleural retraction; location; calcifications; emphysema; fibrosis; pleural contact; pleural effusion. Statistical analysis was performed to assess association of features with each gene mutation. ROC curves for gene mutations were drawn; the corresponding area under the curve was calculated. P-values <0.05 were considered significant. Of 285 patients, 60/280 (21.43 %) were positive for EGFR mutation; 31/270 (11.48 %) for ALK rearrangement; 64/240 (26.67 %) for KRAS mutation. EGFR mutation was associated with air bronchogram, pleural retraction, females, non-smokers, small lesion size, and absence of fibrosis. ALK rearrangements were associated with age and pleural effusion. KRAS mutation was associated with round shape, nodules in non-tumour lobes, and smoking. This study disclosed associations between CT features and alterations of EGFR (air bronchogram, pleural retraction, small lesion size, absence of fibrosis), ALK (pleural effusion) and KRAS (round lesion shape, nodules in non-tumour lobes). (orig.)

  15. Real-life experience of ceritinib in crizotinib-pretreated ALK+ advanced non-small cell lung cancer patients

    Directory of Open Access Journals (Sweden)

    Jacques Cadranel

    2018-02-01

    Full Text Available Here we report our experience of ceritinib in crizotinib-pretreated patients with anaplastic lymphoma kinase (ALK positive (ALK+ non-small cell lung cancer (NSCLC in a French temporary authorisation for use (TAU study. The French TAU study included crizotinib-pretreated patients with advanced ALK+ or ROS proto-oncogene 1 positive (ROS1+ tumours. Patients received oral ceritinib (750 mg·day−1 as a starting dose and best tumour response (as evaluated by the investigator and safety were reported every 3 months. A total of 242 TAUs were granted from March 12, 2013 to August 05, 2015. Of the 242 patients, 228 had ALK+ NSCLC and 13 had ROS1+ NSCLC. The median age of ALK+ patients (n=214 was 58.5 years, 51.9% were female, 70.8% had an Eastern Cooperative Oncology Group (ECOG performance status (PS of 0–1 and 50.0% had brain metastases. Of the 149 efficacy evaluable ALK+ NSCLC patients, 5.4% had a complete response (CR, 47.0% had a partial response (PR and 22.8% had stable disease (SD. At September 05, 2015, the median duration of ceritinib treatment (n=182 was 3.9 months but 5.5 months for patients (n=71 with a follow-up of ≥12 months. Higher objective response rate (ORR was observed for patients with ECOG PS 0 to 1 (55.0% versus 42.4% and those receiving prior crizotinib for >5 months (51.6% versus 36.1%. Treatment-related adverse events (AEs were reported in 118 of 208 patients (56.7%, the most common being diarrhoea (22.1% and hepatic toxicity (19.7%. Ceritinib (750 mg·day−1 demonstrated efficacy similar efficacy to ASCEND-1, ASCEND-2 and phase 3 ASCEND-5 trials with manageable safety in crizotinib-pretreated patients with ALK+ NSCLC.

  16. Nitrogen contribution proportion from soil, fertilizer and pseudomonas putida like in sorghum plantation on South Sumatra's inceptisols; Proposi Sumbangan Nitrogen Oleh Tanah, Pupuk Dan Pseudomonas putida like Dalam Tanaman Sorghum Pada Inceptisol Sumatera Selatan

    Energy Technology Data Exchange (ETDEWEB)

    Kesumadewi, A A.I. [Departement Agriculture Udayana, Bali (Indonesia); Anas, Iswandi; Santosa, D A [Departement Agriculture, Bogor Agriculture Institute (Indonesia); Sisworo, Elsje L [Center for Application of Isotop and Radiation, National Nuclear Energy Agency, Jakarta (Indonesia)

    2000-02-23

    The proportion of N which absorbed either from natural source (soil), N-fertilizer or N sub.2-fixing microorganism on sorghum plantation in marginal alang-alang lands that group to inceptisol should be studied in securing the soil fertility. In this experiment, N contribution of those N sources was determined during vegetative stage of sorghum on three subgroup of south sumatera's inceptisols. A greenhouse experiment that arranged in randomized complete block design using 2 factorial split plot was carried out in IPB Bogor from May-December 1998. The observation was focused on partition of N contribution from the soil, N-fertilizer and Pseudomonas putida like(N sub.2-fixing microorganism) on total plant-content 4 and 8 weeks after plantations (WAP). N-partition from those N-sources was done based o A-value method. Sorghum was absorb larger proportion of N from soil(63,36%-48,83% on 4 WAP and 64,58 % on WAP) than from fertilizer and P.putida like on all of the soil subgroups. Soil-N absorption was highest on oxic dystropept. Pseudomonas putida like was not able to subtite soil-N and fertilizer-N in sufficient amount to vigorous plant growth particularly on typica humitropept and typic dystropept. The microorganism was only provide a small amount of N to sorghum on oxic dystropept (23,05-23,95 % on 4 WAP and 15,91-34,44 % on 8WAP). The increment of plant root surface area could be enhance in greater extent of plant-N absorption than N sub.2-fixation. Thus, plantation of marginal tolerant sorghum still need addition of N-fertilizer.

  17. Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries

    Directory of Open Access Journals (Sweden)

    Daniela Toro-Ascuy

    2016-11-01

    Full Text Available The human immunodeficiency virus type-1 (HIV-1 unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1, Staufen double-stranded RNA binding protein 1/2 (STAU1/2, or components of miRNA-induced silencing complex (miRISC and processing bodies (PBs. More recently, the HIV-1 unspliced mRNA was shown to contain N6-methyladenosine (m6A, allowing the recruitment of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2, an m6A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries.

  18. Multiple Modes of Nematode Control by Volatiles of Pseudomonas putida 1A00316 from Antarctic Soil against Meloidogyne incognita

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    Yile Zhai

    2018-02-01

    Full Text Available Pseudomonas putida 1A00316 isolated from Antarctic soil showed nematicidal potential for biological control of Meloidogyne incognita; however, little was known about whether strain 1A00316 could produce volatile organic compounds (VOCs, and if they had potential for use in biological control against M. incognita. In this study, VOCs produced by a culture filtrate of P. putida 1A00316 were evaluated by in vitro experiments in three-compartment Petri dishes and 96-well culture plates. Our results showed that M. incognita juveniles gradually reduced their movement within 24–48 h of incubation with mortality ranging from 6.49 to 86.19%, and mostly stopped action after 72 h. Moreover, egg hatching in culture filtrates of strain 1A00316 was much reduced compared to that in sterile distilled water or culture medium. Volatiles from P. putida 1A00316 analysis carried out by solid-phase micro-extraction gas chromatography–mass spectrometry (SPME-GC/MS included dimethyl-disulfide, 1-undecene, 2-nonanone, 2-octanone, (Z-hexen-1-ol acetate, 2-undecanone, and 1-(ethenyloxy-octadecane. Of these, dimethyl-disulfide, 2-nonanone, 2-octanone, (Z-hexen-1-ol acetate, and 2-undecanone had strong nematicidal activity against M. incognita J2 larvae by direct-contact in 96-well culture plates, and only 2-undecanone acted as a fumigant. In addition, the seven VOCs inhibited egg hatching of M. incognita both by direct-contact and by fumigation. All of the seven VOCs repelled M. incognita J2 juveniles in 2% water agar Petri plates. These results show that VOCs from strain 1A00316 act on different stages in the development of M. incognita via nematicidal, fumigant, and repellent activities and have potential for development as agents with multiple modes of control of root-knot nematodes.

  19. Nitrogen contribution proportion from soil, fertilizer and pseudomonas putida like in sorghum plantation on South Sumatra's inceptisols

    International Nuclear Information System (INIS)

    Kesumadewi, A.A.I.; Anas, Iswandi; Santosa, D.A.; Sisworo, Elsje L.

    2000-01-01

    The proportion of N which absorbed either from natural source (soil), N-fertilizer or N sub.2-fixing microorganism on sorghum plantation in marginal alang-alang lands that group to inceptisol should be studied in securing the soil fertility. In this experiment, N contribution of those N sources was determined during vegetative stage of sorghum on three subgroup of south sumatera's inceptisols. A greenhouse experiment that arranged in randomized complete block design using 2 factorial split plot was carried out in IPB Bogor from May-December 1998. The observation was focused on partition of N contribution from the soil, N-fertilizer and Pseudomonas putida like(N sub.2-fixing microorganism) on total plant-content 4 and 8 weeks after plantations (WAP). N-partition from those N-sources was done based o A-value method. Sorghum was absorb larger proportion of N from soil(63,36%-48,83% on 4 WAP and 64,58 % on WAP) than from fertilizer and P.putida like on all of the soil subgroups. Soil-N absorption was highest on oxic dystropept. Pseudomonas putida like was not able to subtite soil-N and fertilizer-N in sufficient amount to vigorous plant growth particularly on typica humitropept and typic dystropept. The microorganism was only provide a small amount of N to sorghum on oxic dystropept (23,05-23,95 % on 4 WAP and 15,91-34,44 % on 8WAP). The increment of plant root surface area could be enhance in greater extent of plant-N absorption than N sub.2-fixation. Thus, plantation of marginal tolerant sorghum still need addition of N-fertilizer

  20. Carbon Source-Dependent Inducible Metabolism of Veratryl Alcohol and Ferulic Acid in Pseudomonas putida CSV86

    Science.gov (United States)

    Mohan, Karishma

    2017-01-01

    ABSTRACT Pseudomonas putida CSV86 degrades lignin-derived metabolic intermediates, viz., veratryl alcohol, ferulic acid, vanillin, and vanillic acid, as the sole sources of carbon and energy. Strain CSV86 also degraded lignin sulfonate. Cell respiration, enzyme activity, biotransformation, and high-pressure liquid chromatography (HPLC) analyses suggest that veratryl alcohol and ferulic acid are metabolized to vanillic acid by two distinct carbon source-dependent inducible pathways. Vanillic acid was further metabolized to protocatechuic acid and entered the central carbon pathway via the β-ketoadipate route after ortho ring cleavage. Genes encoding putative enzymes involved in the degradation were found to be present at fer, ver, and van loci. The transcriptional analysis suggests a carbon source-dependent cotranscription of these loci, substantiating the metabolic studies. Biochemical and quantitative real-time (qRT)-PCR studies revealed the presence of two distinct O-demethylases, viz., VerAB and VanAB, involved in the oxidative demethylation of veratric acid and vanillic acid, respectively. This report describes the various steps involved in metabolizing lignin-derived aromatic compounds at the biochemical level and identifies the genes involved in degrading veratric acid and the arrangement of phenylpropanoid metabolic genes as three distinct inducible transcription units/operons. This study provides insight into the bacterial degradation of lignin-derived aromatics and the potential of P. putida CSV86 as a suitable candidate for producing valuable products. IMPORTANCE Pseudomonas putida CSV86 metabolizes lignin and its metabolic intermediates as a carbon source. Strain CSV86 displays a unique property of preferential utilization of aromatics, including for phenylpropanoids over glucose. This report unravels veratryl alcohol metabolism and genes encoding veratric acid O-demethylase, hitherto unknown in pseudomonads, thereby providing new insight into the

  1. Self-amplifying mRNA vaccines.

    Science.gov (United States)

    Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

    2015-01-01

    This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Truncated ALK derived from chromosomal translocation t(2;5)(p23;q35) binds to the SH3 domain of p85-PI3K.

    Science.gov (United States)

    Polgar, Doris; Leisser, Christina; Maier, Susanne; Strasser, Stephan; Rüger, Beate; Dettke, Markus; Khorchide, Maya; Simonitsch, Ingrid; Cerni, Christa; Krupitza, Georg

    2005-02-15

    The chromosomal translocation t(2;5)(p23;q35) is associated with "Anaplastic large cell lymphomas" (ALCL), a Non Hodgkin Lymphoma occurring in childhood. The fusion of the tyrosine kinase gene-ALK (anaplastic lymphoma kinase) on chromosome 2p23 to the NPM (nucleophosmin/B23) gene on chromosome 5q35 results in a 80 kDa chimeric protein, which activates the "survival" kinase PI3K. However, the binding mechanism between truncated ALK and PI3K is poorly understood. Therefore, we attempted to elucidate the molecular interaction between ALK and the regulatory p85 subunit of PI3K. Here we provide evidence that the truncated ALK homodimer binds to the SH3 domain of p85. This finding may be useful for the development of a new target-specific intervention.

  3. Catabolite-mediated mutations in alternate toluene degradative pathways in Pseudomonas putida.

    Science.gov (United States)

    Leddy, M B; Phipps, D W; Ridgway, H F

    1995-01-01

    Pseudomonas putida 54g grew on mineral salts with toluene and exhibited catechol-2,3-dioxygenase (C23O) activity, indicating a meta pathway. After 10 to 15 days on toluene, nondegrading (Tol-) variants approached nearly 10% of total CFU. Auxotrophs were not detected among variants, suggesting selective loss of catabolic function(s). Variant formation was substrate dependent, since Tol- cells were observed on neither ethylbenzene, glucose, nor peptone-based media nor when toluene catabolism was suppressed by glucose. Unlike wild-type cells, variants did not grow on gasoline, toluene, benzene, ethylbenzene, benzoate, or catechol, suggesting loss of meta pathway function. Catabolic and C23O activities were restored to variants via transfer of a 78-mDa TOL-like plasmid from a wild-type Tol+ donor. Tests for reversion of variants to Tol+ were uniformly negative, suggesting possible delection or excision of catabolic genes. Deletions were confirmed in some variants by failure to hybridize with a DNA probe specific for the xylE gene encoding C23O. Cells grown on benzoate remained Tol+ but were C23O- and contained a plasmid of reduced size or were plasmid free, suggesting an alternate chromosomal catabolic pathway, also defective in variants. Cells exposed to benzyl alcohol, the initial oxidation product of toluene, accumulated > 13% variants in 5 days, even when cell division was repressed by nitrogen deprivation to abrogate selection processes. No variants formed in identical ethylbenzene-exposed controls. The results suggest that benzyl alcohol mediates irreversible defects in both a plasmid-associated meta pathway and an alternate chromosomal pathway. PMID:7642499

  4. Conversion and assimilation of furfural and 5-(hydroxymethylfurfural by Pseudomonas putida KT2440

    Directory of Open Access Journals (Sweden)

    Michael T. Guarnieri

    2017-06-01

    Full Text Available The sugar dehydration products, furfural and 5-(hydroxymethylfurfural (HMF, are commonly formed during high-temperature processing of lignocellulose, most often in thermochemical pretreatment, liquefaction, or pyrolysis. Typically, these two aldehydes are considered major inhibitors in microbial conversion processes. Many microbes can convert these compounds to their less toxic, dead-end alcohol counterparts, furfuryl alcohol and 5-(hydroxymethylfurfuryl alcohol. Recently, the genes responsible for aerobic catabolism of furfural and HMF were discovered in Cupriavidus basilensis HMF14 to enable complete conversion of these compounds to the TCA cycle intermediate, 2-oxo-glutarate. In this work, we engineer the robust soil microbe, Pseudomonas putida KT2440, to utilize furfural and HMF as sole carbon and energy sources via complete genomic integration of the 12 kB hmf gene cluster previously reported from Burkholderia phytofirmans. The common intermediate, 2-furoic acid, is shown to be a bottleneck for both furfural and HMF metabolism. When cultured on biomass hydrolysate containing representative amounts of furfural and HMF from dilute-acid pretreatment, the engineered strain outperforms the wild type microbe in terms of reduced lag time and enhanced growth rates due to catabolism of furfural and HMF. Overall, this study demonstrates that an approach for biological conversion of furfural and HMF, relative to the typical production of dead-end alcohols, enables both enhanced carbon conversion and substantially improves tolerance to hydrolysate inhibitors. This approach should find general utility both in emerging aerobic processes for the production of fuels and chemicals from biomass-derived sugars and in the biological conversion of high-temperature biomass streams from liquefaction or pyrolysis where furfural and HMF are much more abundant than in biomass hydrolysates from pretreatment.

  5. Conversion and assimilation of furfural and 5-(hydroxymethyl)furfural by Pseudomonas putida KT2440.

    Science.gov (United States)

    Guarnieri, Michael T; Ann Franden, Mary; Johnson, Christopher W; Beckham, Gregg T

    2017-06-01

    The sugar dehydration products, furfural and 5-(hydroxymethyl)furfural (HMF), are commonly formed during high-temperature processing of lignocellulose, most often in thermochemical pretreatment, liquefaction, or pyrolysis. Typically, these two aldehydes are considered major inhibitors in microbial conversion processes. Many microbes can convert these compounds to their less toxic, dead-end alcohol counterparts, furfuryl alcohol and 5-(hydroxymethyl)furfuryl alcohol. Recently, the genes responsible for aerobic catabolism of furfural and HMF were discovered in Cupriavidus basilensis HMF14 to enable complete conversion of these compounds to the TCA cycle intermediate, 2-oxo-glutarate. In this work, we engineer the robust soil microbe, Pseudomonas putida KT2440, to utilize furfural and HMF as sole carbon and energy sources via complete genomic integration of the 12 kB hmf gene cluster previously reported from Burkholderia phytofirmans . The common intermediate, 2-furoic acid, is shown to be a bottleneck for both furfural and HMF metabolism. When cultured on biomass hydrolysate containing representative amounts of furfural and HMF from dilute-acid pretreatment, the engineered strain outperforms the wild type microbe in terms of reduced lag time and enhanced growth rates due to catabolism of furfural and HMF. Overall, this study demonstrates that an approach for biological conversion of furfural and HMF, relative to the typical production of dead-end alcohols, enables both enhanced carbon conversion and substantially improves tolerance to hydrolysate inhibitors. This approach should find general utility both in emerging aerobic processes for the production of fuels and chemicals from biomass-derived sugars and in the biological conversion of high-temperature biomass streams from liquefaction or pyrolysis where furfural and HMF are much more abundant than in biomass hydrolysates from pretreatment.

  6. Lethal and mutagenic properties of MMS-generated DNA lesions in Escherichia coli cells deficient in BER and AlkB-directed DNA repair.

    Science.gov (United States)

    Sikora, Anna; Mielecki, Damian; Chojnacka, Aleksandra; Nieminuszczy, Jadwiga; Wrzesinski, Michal; Grzesiuk, Elzbieta

    2010-03-01

    Methylmethane sulphonate (MMS), an S(N)2-type alkylating agent, generates DNA methylated bases exhibiting cytotoxic and mutagenic properties. Such damaged bases can be removed by a system of base excision repair (BER) and by oxidative DNA demethylation catalysed by AlkB protein. Here, we have shown that the lack of the BER system and functional AlkB dioxygenase results in (i) increased sensitivity to MMS, (ii) elevated level of spontaneous and MMS-induced mutations (measured by argE3 --> Arg(+) reversion) and (iii) induction of the SOS response shown by visualization of filamentous growth of bacteria. In the xth nth nfo strain additionally mutated in alkB gene, all these effects were extreme and led to 'error catastrophe', resulting from the presence of unrepaired apurinic/apyrimidinic (AP) sites and 1-methyladenine (1meA)/3-methylcytosine (3meC) lesions caused by deficiency in, respectively, BER and AlkB dioxygenase. The decreased level of MMS-induced Arg(+) revertants in the strains deficient in polymerase V (PolV) (bearing the deletion of the umuDC operon), and the increased frequency of these revertants in bacteria overproducing PolV (harbouring the pRW134 plasmid) indicate the involvement of PolV in the error-prone repair of 1meA/3meC and AP sites. Comparison of the sensitivity to MMS and the induction of Arg(+) revertants in the double nfo alkB and xth alkB, and the quadruple xth nth nfo alkB mutants showed that the more AP sites there are in DNA, the stronger the effect of the lack of AlkB protein. Since the sum of MMS-induced Arg(+) revertants in xth, nfo and nth xth nfo and alkB mutants is smaller than the frequency of these revertants in the BER(-) alkB(-) strain, we consider two possibilities: (i) the presence of AP sites in DNA results in relaxation of its structure that facilitates methylation and (ii) additional AP sites are formed in the BER(-) alkB(-) mutants.

  7. Imaging Characteristics of Driver Mutations in EGFR, KRAS, and ALK among Treatment-Naïve Patients with Advanced Lung Adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Jangchul Park

    Full Text Available This study aimed to identify the computed tomography characteristics of treatment-naïve patients with lung adenocarcinoma and known driver mutations in EGFR, KRAS, or ALK. Patients with advanced lung adenocarcinoma (stage IIIB-IV and known mutations in EGFR, KRAS, or ALK were assessed. The radiological findings for the main tumor and intra-thoracic status were retrospectively analyzed in each group, and the groups' characteristics were compared. We identified 265 treatment-naïve patients with non-small-cell carcinoma, who had EGFR mutations (n = 159, KRAS mutations (n = 55, or ALK rearrangements (n = 51. Among the three groups, we evaluated only patients with stage IIIB-IV lung adenocarcinoma who had EGFR mutations (n = 126, KRAS mutations (n = 35, or ALK rearrangements (n = 47. We found that ground-glass opacity at the main tumor was significantly more common among EGFR-positive patients, compared to ALK-positive patients (p = 0.009. Lymphadenopathy was significantly more common among ALK-positive patients, compared to EGFR-positive patients (p = 0.003. Extranodal invasion was significantly more common among ALK-positive patients, compared to EGFR-positive patients and KRAS-positive patients (p = 0.001 and p = 0.049, respectively. Lymphangitis was significantly more common among ALK-positive patients, compared to EGFR-positive patients (p = 0.049. Pleural effusion was significantly less common among KRAS-positive patients, compared to EGFR-positive patients and ALK-positive patients (p = 0.046 and p = 0.026, respectively. Lung metastases were significantly more common among EGFR-positive patients, compared to KRAS-positive patients and ALK-positive patients (p = 0.007 and p = 0.04, respectively. In conclusion, EGFR mutations were associated with ground-glass opacity, KRAS-positive tumors were generally solid and less likely to metastasize to the lung and pleura, and ALK-positive tumors tended to present with lymphadenopathy, extranodal

  8. Clinical significance of LUNX mRNA, CK19 mRNA, CEA mRNA expression in detecting micrometastasis from lung cancer

    International Nuclear Information System (INIS)

    Zhu Guangying; Liu Delin; Chen Jie

    2003-01-01

    Objective: To evaluate the sensitivity, specificity and clinical significance of CK19 mRNA, CEA mRNA and LUNX mRNA for detecting micrometastasis by sampling the peripheral blood and regional lymph nodes of lung cancer patients. Methods: Reverse transcriptase chain reaction (RT-PCR) was used to detect LUNX mRNA, CK19 mRNA, CEA mRNA for micrometastasis by sampling the peripheral blood of 48 lung cancer patients and 44 regional lymph nodes of such patients treated by curative resection. Peripheral blood of 30 patients with pulmonary benign lesions and 10 normal healthy volunteers and lymph nodes of 6 patients with benign pulmonary diseases served as control. Results: 1) LUNX mRNA, CK19 mRNA, CEA mRNA were expressed in all (35/35) lung cancer tissues. 2) In the peripheral blood from 48 lung cancer patients, 30 (62.5%) were positive for LUNX mRNA, 24 (50.0%) positive for CK19 mRNA and 32(66.7%) positive for CEA mRNA. The positive detection rates of micrometastasis in 44 lymph nodes from lung cancer patients were 36.4% (16 out of 44) for LUNX mRNA, 27.3% (12 out of 44) for CK19 mRNA and 40.9% (18 out of 44) for CEA mRNA. 3) In the 30 blood samples from patients with pulmonary benign diseases, 2 (6.7%) expressed CK19 mRNA, but none expressed LUNX mRNA or CEA mRNA. All the 3 molecular markers were negative in the 10 blood samples from healthy volunteers. In 11 lymph nodes from patients with pulmonary benign lesions, none was positive for any of the three markers. 4) In 44 regional lymph nodes from lung cancer patients, 6 (13.6%) were positive for metastasis by histopathological examination, with a positive rate significantly lower than that of the RT-PCR (P<0.05). 5) The micrometastatic positive rate in the peripheral blood of 40 non-small cell lung cancer (NSCLC) patients was significantly related to TNM stage (P=0.01). Conclusions: LUNX mRNA, CK19 MRNA, CEA mRNA are all appropriate target genes for the detection of micrometastasis from lung cancer. LUNX mRNA and CEA mRNA

  9. Systemic treatment in EGFR-ALK NSCLC patients: second line therapy and beyond

    International Nuclear Information System (INIS)

    Karachaliou, Niki; Rosell, Rafael

    2014-01-01

    Lung cancer is the most frequently diagnosed cancer and a leading cause of cancer mortality worldwide, with adenocarcinoma being the most common histological subtype. Deeper understanding of the pathobiology of non-small cell lung cancer (NSCLC) has led to the development of small molecules that target genetic mutations known to play critical roles in progression to metastatic disease and to influence response to targeted therapies. The principle goal of precision medicine is to define those patient populations most likely to respond to targeted therapies. However, the cancer genome landscape is composed of relatively few “mountains” [representing the most commonly mutated genes like KRAS, epidermal growth factor (EGFR), and anaplastic lymphoma kinase (ALK)] and a vast number of “hills” (representing low frequency but potentially actionable mutations). Low-frequency lesions that affect a druggable gene product allow a relatively small population of cancer patients for targeted therapy to be selected

  10. TFE3-positive renal cell carcinomas are not always Xp11 translocation carcinomas: Report of a case with a TPM3-ALK translocation.

    Science.gov (United States)

    Thorner, Paul Scott; Shago, Mary; Marrano, Paula; Shaikh, Furqan; Somers, Gino R

    2016-10-01

    Translocation-associated renal cell carcinoma (RCC) is a distinct subtype of RCC with gene rearrangements of the TFE3 or TFEB loci. The TFE3 gene is located at Xp11 and can fuse to a number of translocation partners, resulting in high nuclear expression of TFE3 protein. TFE3 immunostaining is often used as a surrogate marker for a TFE3 translocation. We report a case of an RCC that expressed TFE3 but showed only gain of TFE3 rather than a translocation. Moreover, this case had a t(1;2) translocation fusing ALK and TMP3, identical to that seen in inflammatory myofibroblastic tumour. There was resulting overexpression of ALK protein in a cytoplasmic and membranous pattern. The patient was not treated with chemotherapy but following regional nodal recurrence, an ALK inhibitor was added and the patient remains alive one year later. There are only rare reports of RCC with an ALK-TMP3 fusion, and these tumours can express TFE3 on some unknown basis not related to a TFE3 translocation. Any RCC positive for TFE3 and lacking a translocation should be tested for ALK expression and translocation. Recognition of this subtype of RCC will allow ALK inhibitor therapy to be added, in the hope of improving patient outcome. Copyright © 2016 Elsevier GmbH. All rights reserved.

  11. Discovery of 2,4-diarylaminopyrimidines bearing a resorcinol motif as novel ALK inhibitors to overcome the G1202R resistant mutation.

    Science.gov (United States)

    Geng, Kaijun; Xia, Zongjun; Ji, Yinchun; Zhang, Ruisi Ruthy; Sun, Deqiao; Ai, Jing; Song, Zilan; Geng, Meiyu; Zhang, Ao

    2018-01-20

    To address drug resistance caused by ALK kinase mutations, especially the most refractory and predominant mutation G1202R for the second-generation ALK inhibitor, a series of new diarylaminopyrimidine analogues were designed by incorporating a resorcinol moiety (A-ring) to interact the ALK kinase domain where the G1202R is located. Compound 12d turns out as the most potent with IC 50 values of 1.7, 3.5, and 1.8 nM against ALK wild type, gatekeeper mutant L1196M, and the G1202R mutant, respectively. More importantly, compound 12d has excellent inhibitory effects against the proliferation of BaF3 cells specifically expressing ALK wild type, gatekeeper L1196M, and the most challenging mutant G1202R, with IC 50 values all less than 1.5 nM. Collectively, compound 12d is worthy of further investigation as a new more potent third-generation ALK inhibitor to circumvent drug resistance of both the first-generation and the second-generation inhibitors. Copyright © 2017. Published by Elsevier Masson SAS.

  12. Micro-cost Analysis of ALK Rearrangement Testing by FISH to Determine Eligibility for Crizotinib Therapy in NSCLC: Implications for Cost Effectiveness of Testing and Treatment

    Science.gov (United States)

    Parker, David; Belaud-Rotureau, Marc-Antoine

    2014-01-01

    Break-apart fluorescence in situ hybridization (FISH) is the gold standard test for anaplastic lymphoma kinase (ALK) gene rearrangement. However, this methodology often is assumed to be expensive and potentially cost-prohibitive given the low prevalence of ALK-positive non-small cell lung cancer (NSCLC) cases. To more accurately estimate the cost of ALK testing by FISH, we developed a micro-cost model that accounts for all cost elements of the assay, including laboratory reagents, supplies, capital equipment, technical and pathologist labor, and the acquisition cost of the commercial test and associated reagent kits and controls. By applying a set of real-world base-case parameter values, we determined that the cost of a single ALK break-apart FISH test result is $278.01. Sensitivity analysis on the parameters of batch size, testing efficiency, and the cost of the commercial diagnostic testing products revealed that the cost per result is highly sensitive to batch size, but much less so to efficiency or product cost. This implies that ALK testing by FISH will be most cost effective when performed in high-volume centers. Our results indicate that testing cost may not be the primary determinant of crizotinib (Xalkori®) treatment cost effectiveness, and suggest that testing cost is an insufficient reason to limit the use of FISH testing for ALK rearrangement. PMID:25520569

  13. Micro-cost Analysis of ALK Rearrangement Testing by FISH to Determine Eligibility for Crizotinib Therapy in NSCLC: Implications for Cost Effectiveness of Testing and Treatment.

    Science.gov (United States)

    Parker, David; Belaud-Rotureau, Marc-Antoine

    2014-01-01

    Break-apart fluorescence in situ hybridization (FISH) is the gold standard test for anaplastic lymphoma kinase (ALK) gene rearrangement. However, this methodology often is assumed to be expensive and potentially cost-prohibitive given the low prevalence of ALK-positive non-small cell lung cancer (NSCLC) cases. To more accurately estimate the cost of ALK testing by FISH, we developed a micro-cost model that accounts for all cost elements of the assay, including laboratory reagents, supplies, capital equipment, technical and pathologist labor, and the acquisition cost of the commercial test and associated reagent kits and controls. By applying a set of real-world base-case parameter values, we determined that the cost of a single ALK break-apart FISH test result is $278.01. Sensitivity analysis on the parameters of batch size, testing efficiency, and the cost of the commercial diagnostic testing products revealed that the cost per result is highly sensitive to batch size, but much less so to efficiency or product cost. This implies that ALK testing by FISH will be most cost effective when performed in high-volume centers. Our results indicate that testing cost may not be the primary determinant of crizotinib (Xalkori(®)) treatment cost effectiveness, and suggest that testing cost is an insufficient reason to limit the use of FISH testing for ALK rearrangement.

  14. Key role of the endothelial TGF-β/ALK1/endoglin signaling pathway in humans and rodents pulmonary hypertension.

    Directory of Open Access Journals (Sweden)

    Benoît Gore

    Full Text Available Mutations affecting transforming growth factor-beta (TGF-β superfamily receptors, activin receptor-like kinase (ALK-1, and endoglin (ENG occur in patients with pulmonary arterial hypertension (PAH. To determine whether the TGF-β/ALK1/ENG pathway was involved in PAH, we investigated pulmonary TGF-β, ALK1, ALK5, and ENG expressions in human lung tissue and cultured pulmonary-artery smooth-muscle-cells (PA-SMCs and pulmonary endothelial cells (PECs from 14 patients with idiopathic PAH (iPAH and 15 controls. Seeing that ENG was highly expressed in PEC, we assessed the effects of TGF-β on Smad1/5/8 and Smad2/3 activation and on growth factor production by the cells. Finally, we studied the consequence of ENG deficiency on the chronic hypoxic-PH development by measuring right ventricular (RV systolic pressure (RVSP, RV hypertrophy, and pulmonary arteriolar remodeling in ENG-deficient (Eng+/- and wild-type (Eng+/+ mice. We also evaluated the pulmonary blood vessel density, macrophage infiltration, and cytokine expression in the lungs of the animals. Compared to controls, iPAH patients had higher serum and pulmonary TGF-β levels and increased ALK1 and ENG expressions in lung tissue, predominantly in PECs. Incubation of the cells with TGF-β led to Smad1/5/8 phosphorylation and to a production of FGF2, PDGFb and endothelin-inducing PA-SMC growth. Endoglin deficiency protected mice from hypoxic PH. As compared to wild-type, Eng+/- mice had a lower pulmonary vessel density, and no change in macrophage infiltration after exposure to chronic hypoxia despite the higher pulmonary expressions of interleukin-6 and monocyte chemoattractant protein-1. The TGF-β/ALK1/ENG signaling pathway plays a key role in iPAH and experimental hypoxic PH via a direct effect on PECs leading to production of growth factors and inflammatory cytokines involved in the pathogenesis of PAH.

  15. Effect of growth conditions on the biodegradation kinetics of toluene by P. putida 54G in a vapor phase bioreactor

    International Nuclear Information System (INIS)

    Mirpuri, R.; Jones, W.; Krieger, E.; McFeters, G.

    1994-01-01

    Biodegradation of volatile organic compounds such as petroleum hydrocarbons and xenobiotic agents in the vapor phase is a promising new concept in well-head and end-of-pipe treatment which may have wide application where in-situ approaches are not feasible. The microbial degradation of the volatile organics can be carried out in vapor phase bioreactors which contain inert packing materials. Scale-up of these reactors from a bench scale to a pilot plant can best be achieved by the use of a predictive model, the success of which depends on accurate estimates of parameters defined in the model such as biodegradation kinetic and stoichiometric coefficients. The phenomena of hydrocarbon stress and injury may also affect performance of a vapor phase bioreactor. Batch kinetic studies on the biodegradation of toluene by P. Putida 54G will be compared to those obtained from continuous culture studies for both suspended and biofilm cultures of the same microorganism. These results will be compared to the activity of the P. putida 54G biofilm in a vapor phase bioreactor to evaluate the impact of hydrocarbon stress and injury on biodegradative processes

  16. Genetic engineering of Pseudomonas putida KT2440 for rapid and high-yield production of vanillin from ferulic acid.

    Science.gov (United States)

    Graf, Nadja; Altenbuchner, Josef

    2014-01-01

    Vanillin is one of the most important flavoring agents used today. That is why many efforts have been made on biotechnological production from natural abundant substrates. In this work, the nonpathogenic Pseudomonas putida strain KT2440 was genetically optimized to convert ferulic acid to vanillin. Deletion of the vanillin dehydrogenase gene (vdh) was not sufficient to prevent vanillin degradation. Additional inactivation of a molybdate transporter, identified by transposon mutagenesis, led to a strain incapable to grow on vanillin as sole carbon source. The bioconversion was optimized by enhanced chromosomal expression of the structural genes for feruloyl-CoA synthetase (fcs) and enoyl-CoA hydratase/aldolase (ech) by introduction of the strong tac promoter system. Further genetic engineering led to high initial conversion rates and molar vanillin yields up to 86% within just 3 h accompanied with very low by-product levels. To our knowledge, this represents the highest productivity and molar vanillin yield gained with a Pseudomonas strain so far. Together with its high tolerance for ferulic acid, the developed, plasmid-free P. putida strain represents a promising candidate for the biotechnological production of vanillin.

  17. Systemic resistance and lipoxygenase-related defence response induced in tomato by Pseudomonas putida strain BTP1

    Directory of Open Access Journals (Sweden)

    Dommes Jacques

    2008-11-01

    Full Text Available Abstract Background Previous studies showed the ability of Pseudomonas putida strain BTP1 to promote induced systemic resistance (ISR in different host plants. Since ISR is long-lasting and not conducive for development of resistance of the targeted pathogen, this phenomenon can take part of disease control strategies. However, in spite of the numerous examples of ISR induced by PGPR in plants, only a few biochemical studies have associated the protective effect with specific host metabolic changes. Results In this study, we showed the protective effect of this bacterium in tomato against Botrytis cinerea. Following treatment by P. putida BTP1, analyses of acid-hydrolyzed leaf extracts showed an accumulation of antifungal material after pathogen infection. The fungitoxic compounds thus mainly accumulate as conjugates from which active aglycones may be liberated through the activity of hydrolytic enzymes. These results suggest that strain BTP1 can elicit systemic phytoalexin accumulation in tomato as one defence mechanism. On another hand, we have shown that key enzymes of the lipoxygenase pathway are stimulated in plants treated with the bacteria as compared with control plants. Interestingly, this stimulation is observed only after pathogen challenge in agreement with the priming concept almost invariably associated with the ISR phenomenon. Conclusion Through the demonstration of phytoalexin accumulation and LOX pathway stimulation in tomato, this work provides new insights into the diversity of defence mechanisms that are inducible by non-pathogenic bacteria in the context of ISR.

  18. Investigation of Halohydrins Degradation by Whole Cells and Cell-free Extract of Pseudomonas putida DSM 437: A Kinetic Approach

    Directory of Open Access Journals (Sweden)

    A. Konti

    2017-10-01

    Full Text Available The biodegradation of two halohydrins (1,3-dichloro-2-propanol and 3-chloro-1,2-propanediol by P. putida DSM 437 was investigated. Intact cells of previously acclimatized P. putida DSM 437 as well as cell-free extracts were used in order to study the degradation kinetics. When whole cells were used, a maximum biodegradation rate of 3-CPD (vmax = 1.28.10–5 mmol mg–1 DCW h–1 was determined, which was more than 4 times higher than that of 1,3-DCP. However, the affinity towards both halohydrins (Km was practically the same. When using cell-free extract, the apparent vmax and Km values for 1,3-DCP were estimated at 9.61.10–6 mmol mg–1 protein h–1 and 8.00 mM, respectively, while for 3-CPD the corresponding values were 2.42.10–5 mmol mg–1 protein h–1 and 9.07 mM. GC-MS analysis of cell-free extracts samples spiked with 1,3-DCP revealed the presence of 3-CPD and glycerol, intermediates of 1,3-DCP degradation pathway. 3-CPD degradation was strongly inhibited by the presence of epichlorohydrin and to a lesser extent by glycidol, intermediates of dehalogenation pathway.

  19. Bioremoval of Basic Violet 3 and Acid Blue 93 by Pseudomonas putida and its adsorption isotherms and kinetics.

    Science.gov (United States)

    Arunarani, A; Chandran, Preethy; Ranganathan, B V; Vasanthi, N S; Sudheer Khan, S

    2013-02-01

    Basic Violet 3 and Acid Blue 93 are the most important group of synthetic colourants extensively used in textile industries for dyeing cotton, wool, silk and nylon. Release of these dye pollutants in to the environment adversely affects the human health and aquatic organisms. The present study we used Pseudomonas putida MTCC 4910 for the adsorptive removal of Basic Violet 3 and Acid Blue 93 from the aqueous solutions. The pH (4-9) and NaCl concentrations (1mM-1M) did not influence the adsorption process. The equilibrium adsorption process fitted well to Freundlich model than Langmuir model. The kinetics of adsorption fitted well by pseudo-second-order. Thus in the present study an attempt has been made to exploit the dye removal capability of P. putida MTCC 4910, and it was found to be an efficient microbe that could be used for bio removal of dyes from textile effluents. Copyright © 2012 Elsevier B.V. All rights reserved.

  20. Genetic and functional characterization of the gene cluster directing the biosynthesis of putisolvin I and II in Pseudomonas putida strain PCL1445

    NARCIS (Netherlands)

    Dubern, J.F.; Coppoolse, E.R.; Stiekema, W.J.; Bloemberg, G.V.

    2008-01-01

    Pseudomonas putida PCL1445 secretes two cyclic lipopeptides, putisolvin I and putisolvin II, which possess a surface-tension-reducing ability, and are able to inhibit biofilm formation and to break down biofilms of Pseudomonas species including Pseudomonas aeruginosa. The putisolvin synthetase gene

  1. Potential of the TCE-degrading endophyte Pseudomonas putida W619-TCE to improve plant growth and reduce TCE phytotoxicity and evapotranspiration in poplar cuttings

    International Nuclear Information System (INIS)

    Weyens, Nele; Truyens, Sascha; Dupae, Joke; Newman, Lee; Taghavi, Safiyh; Lelie, Daniel van der; Carleer, Robert; Vangronsveld, Jaco

    2010-01-01

    The TCE-degrading poplar endophyte Pseudomonas putida W619-TCE was inoculated in poplar cuttings, exposed to 0, 200 and 400 mg l -1 TCE, that were grown in two different experimental setups. During a short-term experiment, plants were grown hydroponically in half strength Hoagland nutrient solution and exposed to TCE for 3 days. Inoculation with P. putida W619-TCE promoted plant growth, reduced TCE phytotoxicity and reduced the amount of TCE present in the leaves. During a mid-term experiment, plants were grown in potting soil and exposed to TCE for 3 weeks. Here, inoculation with P. putida W619-TCE had a less pronounced positive effect on plant growth and TCE phytotoxicity, but resulted in strongly reduced amounts of TCE in leaves and roots of plants exposed to 400 mg l -1 TCE, accompanied by a lowered evapotranspiration of TCE. Dichloroacetic acid (DCAA) and trichloroacetic acid (TCAA), which are known intermediates of TCE degradation, were not detected. - The endophyte P. putida W619-TCE degrades TCE during its transport through the xylem, leading to reduced TCE concentrations in poplar, and decreased TCE evapotranspiration.

  2. Antimicrobial effect of Al2O3, Ag and Al2O3/Ag thin films on Escherichia coli and Pseudomonas putida

    International Nuclear Information System (INIS)

    Angelov, O; Stoyanova, D; Ivanova, I; Todorova, S

    2016-01-01

    The influence of Al 2 O 3 , Ag and Al 2 O 3 /Ag thin films on bacterial growth of Gramnegative bacteria Pseudomonas putida and Escherichia coli is studied. The nanostructured thin films are deposited on glass substrates without intentional heating through r.f. magnetron sputtering in Ar atmosphere of Al 2 O 3 and Ag targets or through sequential sputtering of Al 2 O 3 and Ag targets, respectively. The individual Ag thin films (thickness 8 nm) have a weak bacteriostatic effect on Escherichia coli expressed as an extended adaptive phase of the bacteria up to 5 hours from the beginning of the experiment, but the final effect is only 10 times lower bacterial density than in the control. The individual Al 2 O 3 film (20 nm) has no antibacterial effect against two strains E. coli - industrial and pathogenic. The Al 2 O 3 /Ag bilayer films (Al 2 O 3 20 nm/Ag 8 nm) have strong bactericidal effect on Pseudomonas putida and demonstrate an effective time of disinfection for 2 hours. The individual films Al2O3 and Ag have not pronounced antibacterial effect on Pseudomonas putida . A synergistic effect of Al2O3/Ag bilayer films in formation of oxidative species on the surface in contact with the bacterial suspension could be a reason for their antimicrobial effect on E. coli and P. putida . (paper)

  3. Expression of recombinant Pseudomonas stutzeri di-heme cytochrome c(4) by high-cell-density fed-batch cultivation of Pseudomonas putida

    DEFF Research Database (Denmark)

    Thuesen, Marianne Hallberg; Nørgaard, Allan; Hansen, Anne Merete

    2003-01-01

    The gene of the di-heme protein cytochrome c(4) from Pseudomonas stutzeri was expressed in Pseudomonas putida. High-yield expression of the protein was achieved by high-cell-density fed-batch cultivation using an exponential glucose feeding strategy. The recombinant cytochrome c(4) protein...

  4. Field release of genetically modified Pseudomonas putida WCS358r : molecular analysis of effects on microbial communities in the rhizosphere of wheat

    NARCIS (Netherlands)

    Viebahn, Mareike

    2005-01-01

    Genetically modified microorganisms (GMMs) with enhanced biocontrol activity are attractive to apply in agriculture. To investigate potential ecological risks of field introduction of GMMs, effects of P. putida strain WCS358r and two genetically modified derivatives of this strain on the indigenous

  5. Involvement of specialized DNA polymerases Pol II, Pol IV and DnaE2 in DNA replication in the absence of Pol I in Pseudomonas putida

    International Nuclear Information System (INIS)

    Sidorenko, Julia; Jatsenko, Tatjana; Saumaa, Signe; Teras, Riho; Tark-Dame, Mariliis; Horak, Rita; Kivisaar, Maia

    2011-01-01

    The majority of bacteria possess a different set of specialized DNA polymerases than those identified in the most common model organism Escherichia coli. Here, we have studied the ability of specialized DNA polymerases to substitute Pol I in DNA replication in Pseudomonas putida. Our results revealed that P. putida Pol I-deficient cells have severe growth defects in LB medium, which is accompanied by filamentous cell morphology. However, growth of Pol I-deficient bacteria on solid rich medium can be restored by reduction of reactive oxygen species in cells. Also, mutants with improved growth emerge rapidly. Similarly to the initial Pol I-deficient P. putida, its adapted derivatives express a moderate mutator phenotype, which indicates that DNA replication carried out in the absence of Pol I is erroneous both in the original Pol I-deficient bacteria and the adapted derivatives. Analysis of the spectra of spontaneous Rif r mutations in P. putida strains lacking different DNA polymerases revealed that the presence of specialized DNA polymerases Pol II and Pol IV influences the frequency of certain base substitutions in Pol I-proficient and Pol I-deficient backgrounds in opposite ways. Involvement of another specialized DNA polymerase DnaE2 in DNA replication in Pol I-deficient bacteria is stimulated by UV irradiation of bacteria, implying that DnaE2-provided translesion synthesis partially substitutes the absence of Pol I in cells containing heavily damaged DNA.

  6. Transposon mutations in the flagella biosynthetic pathway of the solvent-tolerant Pseudomonas putida S12 result in a decreased expression of solvent efflux genes

    NARCIS (Netherlands)

    Kieboom, J; Bruinenberg, R; Keizer-Gunnink, [No Value; de Bont, JAM

    2001-01-01

    Fourteen solvent-sensitive transposon mutants were generated from the solvent-tolerant Pseudomonas putida strain S12 by applying the TnMOD-KmO mutagenesis system. These mutants were unable to grow in the presence of octanol and toluene. By cloning the region flanking the transposon insertion point a

  7. Identification and characterization of an N-acylhomoserine lactone-dependent quorum-sensing system in Pseudomonas putida strain IsoF

    DEFF Research Database (Denmark)

    Steidle, A.; Allesen-Holm, M.; Riedel, K.

    2002-01-01

    Recent reports have shown that several strains of Pseudomonas putida produce N-acylhomoserine lactones (AHLs). These signal molecules enable bacteria to coordinately express certain phenotypic traits in a density-dependent manner in a process referred to as quorum sensing. In this study we have...

  8. Potential of the TCE-degrading endophyte Pseudomonas putida W619-TCE to improve plant growth and reduce TCE phytotoxicity and evapotranspiration in poplar cuttings

    Energy Technology Data Exchange (ETDEWEB)

    Weyens, N.; van der Lelie, D.; Truyens, S.; Dupae, J.; Newman, L.; Taghavi, S.; Carleer, R.; Vangronsveld, J.

    2010-09-01

    The TCE-degrading poplar endophyte Pseudomonas putida W619-TCE was inoculated in poplar cuttings, exposed to 0, 200 and 400 mg l{sup -1} TCE, that were grown in two different experimental setups. During a short-term experiment, plants were grown hydroponically in half strength Hoagland nutrient solution and exposed to TCE for 3 days. Inoculation with P. putida W619-TCE promoted plant growth, reduced TCE phytotoxicity and reduced the amount of TCE present in the leaves. During a mid-term experiment, plants were grown in potting soil and exposed to TCE for 3 weeks. Here, inoculation with P. putida W619-TCE had a less pronounced positive effect on plant growth and TCE phytotoxicity, but resulted in strongly reduced amounts of TCE in leaves and roots of plants exposed to 400 mg l{sup -1} TCE, accompanied by a lowered evapotranspiration of TCE. Dichloroacetic acid (DCAA) and trichloroacetic acid (TCAA), which are known intermediates of TCE degradation, were not detected. The endophyte P. putida W619-TCE degrades TCE during its transport through the xylem, leading to reduced TCE concentrations in poplar, and decreased TCE evapotranspiration.

  9. Biofilm as a production platform for heterologous production of rhamnolipids by the non‑pathogenic strain Pseudomonas putida KT2440

    DEFF Research Database (Denmark)

    Wigneswaran, Vinoth; Nielsen, Kristian Fog; Sternberg, Claus

    2016-01-01

    of the rhlAB operon resulting in different levels of rhamnolipid production. Biosynthesis of rhamnolipids in P. putida decreased bacterial growth rate but stimulated biofilm formation by enhancing cell motility. Continuous rhamnolipid production in a biofilm was achieved using flow cell technology...

  10. The impact of ColRS two-component system and TtgABC efflux pump on phenol tolerance of Pseudomonas putida becomes evident only in growing bacteria

    Directory of Open Access Journals (Sweden)

    Kivisaar Maia

    2010-04-01

    Full Text Available Abstract Background We have recently found that Pseudomonas putida deficient in ColRS two-component system is sensitive to phenol and displays a serious defect on solid glucose medium where subpopulation of bacteria lyses. The latter phenotype is significantly enhanced by the presence of phenol in growth medium. Here, we focused on identification of factors affecting phenol tolerance of the colR-deficient P. putida. Results By using transposon mutagenesis approach we identified a set of phenol-tolerant derivatives of colR-deficient strain. Surprisingly, half of independent phenol tolerant clones possessed miniTn5 insertion in the ttgABC operon. However, though inactivation of TtgABC efflux pump significantly enhanced phenol tolerance, it did not affect phenol-enhanced autolysis of the colR mutant on glucose medium indicating that phenol- and glucose-caused stresses experienced by the colR-deficient P. putida are not coupled. Inactivation of TtgABC pump significantly increased the phenol tolerance of the wild-type P. putida as well. Comparison of phenol tolerance of growing versus starving bacteria revealed that both ColRS and TtgABC systems affect phenol tolerance only under growth conditions and not under starvation. Flow cytometry analysis showed that phenol strongly inhibited cell division and to some extent also caused cell membrane permeabilization to propidium iodide. Single cell analysis of populations of the ttgC- and colRttgC-deficient strains revealed that their membrane permeabilization by phenol resembles that of the wild-type and the colR mutant, respectively. However, cell division of P. putida with inactivated TtgABC pump seemed to be less sensitive to phenol than that of the parental strain. At the same time, cell division appeared to be more inhibited in the colR-mutant strain than in the wild-type P. putida. Conclusions ColRS signal system and TtgABC efflux pump are involved in the phenol tolerance of P. putida. However, as

  11. Evaluation of NGS and RT-PCR Methods for ALK Rearrangement in European NSCLC Patients: Results from the European Thoracic Oncology Platform Lungscape Project.

    Science.gov (United States)

    Letovanec, Igor; Finn, Stephen; Zygoura, Panagiota; Smyth, Paul; Soltermann, Alex; Bubendorf, Lukas; Speel, Ernst-Jan; Marchetti, Antonio; Nonaka, Daisuke; Monkhorst, Kim; Hager, Henrik; Martorell, Miguel; Sejda, Aleksandra; Cheney, Richard; Hernandez-Losa, Javier; Verbeken, Eric; Weder, Walter; Savic, Spasenija; Di Lorito, Alessia; Navarro, Atilio; Felip, Enriqueta; Warth, Arne; Baas, Paul; Meldgaard, Peter; Blackhall, Fiona; Dingemans, Anne-Marie; Dienemann, Hendrik; Dziadziuszko, Rafal; Vansteenkiste, Johan; O'Brien, Cathal; Geiger, Thomas; Sherlock, Jon; Schageman, Jeoffrey; Dafni, Urania; Kammler, Roswitha; Kerr, Keith; Thunnissen, Erik; Stahel, Rolf; Peters, Solange

    2018-03-01

    The reported prevalence of ALK receptor tyrosine kinase gene (ALK) rearrangement in NSCLC ranges from 2% to 7%. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proved to be a reproducible and sensitive technique. Reverse-transcriptase polymerase chain reaction (RT-PCR) has also been advocated, and most recently, the advent of targeted next-generation sequencing (NGS) for ALK and other fusions has become possible. This study compares anaplastic lymphoma kinase (ALK) evaluation with all four techniques in resected NSCLC from the large European Thoracic Oncology Platform Lungscape cohort. A total of 96 cases from the European Thoracic Oncology Platform Lungscape iBiobank, with any ALK immunoreactivity were examined by FISH, central RT-PCR, and NGS. An H-score higher than 120 defines IHC positivity. RNA was extracted from the same formalin-fixed, paraffin-embedded tissues. For RT-PCR, primers covered the most frequent ALK translocations. For NGS, the Oncomine Solid Tumour Fusion Transcript Kit (Thermo Fisher Scientific, Waltham, MA) was used. The concordance was assessed using the Cohen κ coefficient (two-sided α ≤ 5%). NGS provided results for 77 of the 95 cases tested (81.1%), whereas RT-PCR provided results for 77 of 96 (80.2%). Concordance occurred in 55 cases of the 60 cases tested with all four methods (43 ALK negative and 12 ALK positive). Using ALK copositivity for IHC and FISH as the criterion standard, we derived a sensitivity for RT-PCR/NGS of 70.0%/85.0%, with a specificity of 87.1%/79.0%. When either RT-PCR or NGS was combined with IHC, the sensitivity remained the same, whereas the specificity increased to 88.7% and 83.9% respectively. NGS evaluation with the Oncomine Solid Tumour Fusion transcript kit and RT-PCR proved to have high sensitivity and specificity, advocating their use in routine practice. For maximal sensitivity and specificity, ALK status should be

  12. EGFR, ALK, RET, KRAS and BRAF alterations in never-smokers with non-small cell lung cancer.

    Science.gov (United States)

    Dong, Y U; Ren, Weihong; Qi, Jun; Jin, B O; Li, Ying; Tao, Huiqing; Xu, Ren; Li, Yanqing; Zhang, Qinxian; Han, Baohui

    2016-04-01

    Non-small cell lung cancer (NSCLC), caused by various mutations in a spectrum of cancer driver genes, may have distinct pathological characteristics and drug responses. Extensive genetic screening and pathological characterization is required for the design of customized therapies to improve patient outcomes. Notably, NSCLC in never-smokers exhibits distinctive clinicopathological features, which are frequently associated with tumorigenic mutations, and thus may be treated as a unique disease entity. However, to the best of our knowledge, these mutations have not been extensively and accurately characterized in an NSCLC study with a large sample size. Therefore, the present study enrolled a large cohort of NSCLC patients, which consisted of 358 never-smokers, for the screening of genetic alterations in the epidermal growth factor receptor (EGFR), ret proto-oncogene (RET), anaplastic lymphoma kinase (ALK), Kirsten rat sarcoma viral oncogene homolog (KRAS) and B-Raf proto-oncogene serine/threonine kinase (BRAF) tumorigenic genes. It was identified that the mutation rate was 47.8, 7.5, 3.6, 1.4 and 0.3% for EGFR, ALK, KRAS, RET and BRAF, respectively. In addition, clinicopathological features associated with these mutations were characterized. EGFR mutations were more frequently observed in female and older patients. By contrast, KRAS mutations were more frequently detected in male patients, and ALK and RET translocations in younger patients. The cancer cells were frequently well-differentiated in carcinoma cases exhibiting EGFR mutations, however, were less differentiated in those with ALK translocations. In conclusion, the present study determined the frequency of oncogenic alterations and associated clinicopathological features in NSCLC exhibited by never-smokers using a large sample size. The results of the present study may enrich our knowledge of NSCLC in never-smokers and provide useful insights for improvement of the outcome of molecularly targeted therapies

  13. Unusual poly(3-hydroxyalkanoate) (PHA) biosynthesis behavior of Pseudomonas putida Bet001 and Delftia tsuruhatensis Bet002 isolated from palm oil mill effluent.

    Science.gov (United States)

    Razaif-Mazinah, Mohd Rafais Mohd; Anis, Siti Nor Syairah; Harun, Hazwani Izzati; Rashid, Khairunnisa Abdul; Annuar, Mohamad Suffian Mohamad

    2017-03-01

    Pseudomonas putida Bet001 and Delftia tsuruhatensis Bet002, isolated from palm oil mill effluent, accumulated poly(3-hydroxyalkanoates) (PHAs) when grown on aliphatic fatty acids, sugars, and glycerol. The substrates were supplied at 20:1 C/N mole ratio. Among C-even n-alkanoic acids, myristic acid gave the highest PHA content 26 and 28 wt% in P. putida and D. tsuruhatensis, respectively. Among C-odd n-alkanoic acids, undecanoic gave the highest PHA content at 40 wt% in P. putida and 46 wt% in D. tsuruhatensis on pentadecanoic acid. Sugar and glycerol gave <10 wt% of PHA content for both bacteria. Interestingly, D. tsuruhatensis accumulated both short- and medium-chain length PHA when supplied with n-alkanoic acids ranging from octanoic to lauric, sucrose, and glycerol with 3-hydroxybutyrate as the major monomer unit. In P. putida, the major hydroxyalkanoates unit was 3-hydroxyoctanoate and 3-hydroxydecanoate when grown on C-even acids. Conversely, 3-hydroxyheptanoate, 3-hydrxoynonanoate, and 3-hydroxyundecanoate were accumulated with C-odd acids. Weight-averaged molecular weight (M w ) was in the range of 53-81 kDa and 107-415 kDa for P. putida and D. tsuruhatensis, respectively. Calorimetric analyses indicated that both bacteria synthesized semicrystalline polymer with good thermal stability with degradation temperature (T d ) ranging from 178 to 282 °C. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  14. Photoautotrophic production of polyhydroxyalkanoates in a synthetic mixed culture of Synechococcus elongatus cscB and Pseudomonas putida cscAB.

    Science.gov (United States)

    Löwe, Hannes; Hobmeier, Karina; Moos, Manuel; Kremling, Andreas; Pflüger-Grau, Katharina

    2017-01-01

    One of the major challenges for the present and future generations is to find suitable substitutes for the fossil resources we rely on today. Cyanobacterial carbohydrates have been discussed as an emerging renewable feedstock in industrial biotechnology for the production of fuels and chemicals, showing promising production rates when compared to crop-based feedstock. However, intrinsic capacities of cyanobacteria to produce biotechnological compounds are limited and yields are low. Here, we present an approach to circumvent these problems by employing a synthetic bacterial co-culture for the carbon-neutral production of polyhydroxyalkanoates (PHAs) from CO 2 . The co-culture consists of two bio - modules : Bio - module I , in which the cyanobacterial strain Synechococcus elongatus cscB fixes CO 2 , converts it to sucrose, and exports it into the culture supernatant; and bio - module II , where this sugar serves as C-source for Pseudomonas putida cscAB and is converted to PHAs that are accumulated in the cytoplasm. By applying a nitrogen-limited process, we achieved a maximal PHA production rate of 23.8 mg/(L day) and a maximal titer of 156 mg/L. We will discuss the present shortcomings of the process and show the potential for future improvement. These results demonstrate the feasibility of mixed cultures of S. elongatus cscB and P. putida cscAB for PHA production, making room for the cornucopia of possible products that are described for P. putida . The construction of more efficient sucrose-utilizing P. putida phenotypes and the optimization of process conditions will increase yields and productivities and eventually close the gap in the contemporary process. In the long term, the co-culture may serve as a platform process, in which P. putida is used as a chassis for the implementation of synthetic metabolic pathways for biotechnological production of value-added products.

  15. Study on the efficiency of the two phase partitioning stirred tank bioreactor on the toluene filtration from the airstream by Pseudomonas putida via

    Directory of Open Access Journals (Sweden)

    2013-02-01

    Full Text Available Introduction: There are different methods for controlling gaseous pollutants formed from air pollution sources that one of the most economical and efficient of them, is bio-filtration. The purpose of this study is Toluene removal from airstream by using the pure Pseudomonas putida bacteria as a fluidized bed in a two phase partitioning stirred tank bioreactor.Toluene ( Metyle benzene is one of the aromatic compounds which uses as a chemical solvent.low to moderate concentration of Toluene causes fatigue, dizziness, weakness,unbalance behaviour, memory loss, insomnia, loss of appetite, loss of vision and hearing. .Material and Method: In this experimental study at first, pure Pseudomonas putida in an aqueous phase containing nutrients and trace elements solution was duplicated and accustomed with Toluene. then solution contained microorganisms with 10% silicon oil was entered to bioreactor. The amount of CO2 and pollutant concentrations in the entrance and exhaust of bioreactor containing Pseudomonas putida was studied during 17 days for each variable. .Result: Experimental findings showed that in the 0.06 m3/h and 0.12 m3/h flow rate, the efficiency of bioreactor containing Pseudomonas putida in the concentration ranges of 283 Mg/m3 to 4710 Mg/m3 was at least 97% and 25% respectively. Statistical analysis (ANOVA showed that in two flow rates of 0.06 m3/h and 0.12 m3/h removal efficiency and mineralization percentage had significant differences .(Pvalue =0.01. .Conclusion: Achieving high efficiencies in pollutants removal was because of the prepared optimum conditions for Pseudomonas putida in the two phase partitioning stirred tank bioreactor with 10% organic phase.

  16. Environmental tobacco smoke exposure and EGFR and ALK alterations in never smokers' lung cancer. Results from the LCRINS study.

    Science.gov (United States)

    Torres-Durán, María; Ruano-Ravina, Alberto; Kelsey, Karl T; Parente-Lamelas, Isaura; Leiro-Fernández, Virginia; Abdulkader, Ihab; Provencio, Mariano; Abal-Arca, José; Castro-Añón, Olalla; Montero-Martínez, Carmen; Vidal-García, Iria; Amenedo, Margarita; Golpe-Gómez, Antonio; Martínez, Cristina; Guzmán-Taveras, Rosirys; Mejuto-Martí, María José; Fernández-Villar, Alberto; Barros-Dios, Juan Miguel

    2017-12-28

    Environmental tobacco smoke (ETS) exposure is a main risk factor of lung cancer in never smokers. Epidermal Growth Factor Receptor (EGFR) mutations and ALK translocations are more frequent in never smokers' lung cancer than in ever-smokers. We performed a multicenter case-control study to assess if ETS exposure is associated with the presence of EGFR mutations and its types and if ALK translocations were related with ETS exposure. All patients were never smokers and had confirmed lung cancer diagnosis. ETS exposure during childhood showed a negative association on the probability of EGRF mutation though not significant. Exposure during adulthood, at home or at workplace, did not show any association with EGFR mutation. The mutation type L858R seemed the most associated with a lower probability of EGFR alterations for ETS exposure at home in adult life. There is no apparent association between ETS exposure and ALK translocation. These results might suggest that ETS exposure during childhood or at home in adult life could influence the EGFR mutations profile in lung cancer in never smokers, reducing the probability of presenting EFGR mutation. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Large-scale production of poly(3-hydroxyoctanoic acid) by Pseudomonas putida GPo1 and a simplified downstream process.

    Science.gov (United States)

    Elbahloul, Yasser; Steinbüchel, Alexander

    2009-02-01

    The suitability of Pseudomonas putida GPo1 for large-scale cultivation and production of poly(3-hydroxyoctanoate) (PHO) was investigated in this study. Three fed-batch cultivations of P. putida GPo1 at the 350- or 400-liter scale in a bioreactor with a capacity of 650 liters were done in mineral salts medium containing initially 20 mM sodium octanoate as the carbon source. The feeding solution included ammonium octanoate, which was fed at a relatively low concentration to promote PHO accumulation under nitrogen-limited conditions. During cultivation, the pH was regulated by addition of NaOH, NH(4)OH, or octanoic acid, which was used as an additional carbon source. Partial O(2) pressure (pO(2)) was adjusted to 20 to 40% by controlling the airflow and stirrer speed. Under the optimized conditions, P. putida GPo1 was able to grow to cell densities as high as 18, 37, and 53 g cells (dry mass) (CDM) per liter containing 49, 55, and 60% (wt/wt) of PHO, respectively. The resulting 40 kg CDM from these three cultivations was used directly for extraction of PHO. Three different methods of extraction of PHO were applied. From these, only acetone extraction showed better performance and resulted in 94% recovery of the PHO contents of cells. A novel mixture of precipitation solvents composed of 70% (vol/vol) methanol and 70% (vol/vol) ethanol was identified in this study. The ratio of PHO concentrate to the mixture was 0.2:1 (vol/vol) and allowed complete precipitation of PHO as white flakes. However, at a ratio of 1:1 (vol/vol) of the solvent mixture to PHO concentrate, a highly purified PHO was obtained. Precipitation yielded a dough-like polymeric material which was cast into thin layers and then shredded into small strips to allow evaporation of the remaining solvents. Gas chromatographic analysis revealed a purity of about 99% +/- 0.2% (wt/wt) of the polymer, which consisted mainly of 3-hydroxyoctanoic acid (96 mol%).

  18. Cytology specimens offer an effective alternative to formalin-fixed tissue as demonstrated by novel automated detection for ALK break-apart FISH testing and immunohistochemistry in lung adenocarcinoma.

    Science.gov (United States)

    Rosenblum, Frida; Hutchinson, Lloyd M; Garver, Joann; Woda, Bruce; Cosar, Ediz; Kurian, Elizabeth M

    2014-11-01

    Minimally invasive sampling by cytology or core needle biopsy often provides an initial diagnosis for treatment in patients with lung nodules. From these limited specimens, multiple molecular studies are frequently requested. Current guidelines from the US Food and Drug Administration recommend using formalin-fixed paraffin-embedded tissue sections for the detection of anaplastic lymphoma kinase (ALK) gene rearrangement by fluorescence in situ hybridization (FISH). The authors compared alcohol-fixed and formalin-fixed cytology specimens using a novel automated detection for ALK rearrangements by FISH and immunohistochemistry (IHC). ALK FISH testing was performed on 129 lung adenocarcinomas from 71 cytology cases and 58 biopsy/resection specimens using Papanicolaou staining with integrated cytomorphology. IHC with the ALK D5F3 antibody was performed on cases with residual material (88 of 129 cases). The mean age of the patients was 66 years; there were 62 women and 67 men. ALK gene rearrangement was present in 4% of cytology specimens (3 of 71 specimens) and 7% of surgical specimens (4 of 58 specimens). FISH in 13 cases was technically unsuccessful. Of the 7 FISH-positive cases, only 2 cytology cases (4%) and 2 surgical cases (6%) were found to be positive with the ALK antibody, demonstrating 80% concordance. The one case found to be negative for ALK by IHC demonstrated a variant rearrangement of the ALK 2p23 gene locus by FISH. The results of the current study validate the usefulness of alcohol-fixed and/or formalin-fixed cytology specimens for ALK rearrangement by a novel automated FISH method. IHC using the D5F3 antibody for ALK is specific in this limited cohort. The authors also demonstrated that alcohol-fixed cytology specimens can be used for ALK rearrangement by automated FISH, alone or in conjunction with IHC. © 2014 American Cancer Society.

  19. The efficacy and safety of alectinib in the treatment of ALK+ NSCLC: a systematic review and meta-analysis

    Directory of Open Access Journals (Sweden)

    Fan JS

    2018-03-01

    Full Text Available Junsheng Fan,1,2,* Zengfei Xia,1,* Xiaoli Zhang,1,* Yuqing Chen,1 Ruolan Qian,1 Sihan Liu,1 Danming You,1 Jian Zhang,1 Peng Luo1 1Department of Oncology, Zhujiang Hospital of Southern Medical University, Guangzhou, China; 2Department of Respiratory Medicine, Shanghai Tenth People’s Hospital, Tongji University, Shanghai, China *These authors contributed equally to this work Background: Alectinib is a second-generation anaplastic lymphoma kinase (ALK inhibitor approved by the US Food and Drug Administration to treat crizotinib-refractory non-small cell lung cancer. We performed this meta-analysis to synthesize the results of different clinical trials to evaluate the efficacy and safety of alectinib. Methods: A search of 3 databases, including PubMed, Web of Science, and the Cochrane Library, was performed from the inception of each database through September 5, 2017. We have pooled the overall response rate (ORR, disease control rate, progression-free survival, and intracranial ORR to evaluate the efficacy of alectinib. Discontinuation rate, rate of dose reduction or interruption due to adverse events as well as the incidence of several adverse events were aggregated to evaluate its safety. Results: A total of 8 studies with 626 patients have been included in our study. The pooled efficacy parameters are as follows: ORR 70% (95% CI: 57% to 82%, disease control rate 88% (95% CI: 82% to 94%, progression-free survival 9.36 months (95% CI: 7.38% to 11.34%, and intracranial ORR 52% (95% CI: 45% to 59%. ALK inhibitor-naïve patients tend to have better responses than crizotinib-pretreated patients. The aggregate discontinuation rate is 7% (95% CI: 4% to 10%, and the pooled rate of dose reduction or interruption is 33% (95% CI: 24% to 42%. The incidences of most adverse events were relatively low, while the incidences of 2 frequently reported adverse events, myalgia (18% and anemia (25%, were even higher than with the first-generation ALK inhibitor

  20. Response to crizotinib in a non-small-cell lung cancer patient harboring an EML4-ALK fusion with an atypical LTBP1 insertion

    Directory of Open Access Journals (Sweden)

    Aguado C

    2018-03-01

    Full Text Available Cristina Aguado,1,* Maria-de-los-Llanos Gil,2,* Zaira Yeste,1 Ana Giménez-Capitán,1 Cristina Teixidó,1 Niki Karachaliou,2 Santiago Viteri,2 Rafael Rosell,1,2 Miguel A Molina-Vila1 1Laboratory of Oncology, Pangaea Oncology, 2Dr Rosell Oncology Institute, Quirón Dexeus University Hospital, Barcelona, Spain *These authors contributed equally to this work Abstract: Fusion of the anaplastic lymphoma receptor tyrosine kinase gene (ALK with the echinoderm microtubule-associated protein 4 gene (EML4 is the second most common actionable alteration in non-small-cell lung cancer, with a frequency of 5%. Here, we present a case of an EML4-ALK-positive patient with an atypical in-frame insertion from the LTBP1 gene in the canonical junction of variant 1. The patient was a 39-year-old never-smoker female diagnosed with Stage IV lung adenocarcinoma. A core biopsy was negative for EGFR and KRAS mutations but positive for ALK immunohistochemistry and fluorescence in situ hybridization. When submitted to nCounter, the sample showed a 3'/5' imbalance indicative of an ALK rearrangement, but failed to give a positive signal for any of the variants tested. Finally, a band with a molecular weight higher than expected appeared after reverse transcriptase-polymerase chain reaction analysis. When Sanger sequencing was performed, the band revealed an atypical EML4-ALK fusion gene with an in-frame 129 bp insertion. A 115 bp segment of the insertion corresponded to an intronic region of LTBP1, a gene located in the short arm of chromosome 2, between ALK and EML4. The patient received crizotinib and showed a good therapeutic response that is still ongoing after 12 months. Our result suggests that short in-frame insertions of other genes in the EML4-ALK junction do not affect the sensitivity of the EML4-ALK fusion protein to crizotinib. Keywords: lung cancer, NSCLC, EML4-ALK, LTBP1, crizotinib, targeted therapy

  1. Sustainable production of valuable compound 3-succinoyl-pyridine by genetically engineering Pseudomonas putida using the tobacco waste.

    Science.gov (United States)

    Wang, Weiwei; Xu, Ping; Tang, Hongzhi

    2015-11-17

    Treatment of solid and liquid tobacco wastes with high nicotine content remains a longstanding challenge. Here, we explored an environmentally friendly approach to replace tobacco waste disposal with resource recovery by genetically engineering Pseudomonas putida. The biosynthesis of 3-succinoyl-pyridine (SP), a precursor in the production of hypotensive agents, from the tobacco waste was developed using whole cells of the engineered Pseudomonas strain, S16dspm. Under optimal conditions in fed-batch biotransformation, the final concentrations of product SP reached 9.8 g/L and 8.9 g/L from aqueous nicotine solution and crude suspension of the tobacco waste, respectively. In addition, the crystal compound SP produced from aqueous nicotine of the tobacco waste in batch biotransformation was of high purity and its isolation yield on nicotine was 54.2%. This study shows a promising route for processing environmental wastes as raw materials in order to produce valuable compounds.

  2. Production of Medium Chain Length Polyhydroxyalkanoates From Oleic Acid Using Pseudomonas putida PGA1 by Fed Batch Culture

    Directory of Open Access Journals (Sweden)

    Sidik Marsudi

    2010-10-01

    Full Text Available Bacterial polyhydroxyalkanoates (PHAs are a class of p0lymers currently receiving much attention because of their potential as renewable and biodegradable plastics. A wide variety of bacteria has been reported to produce PHAs including Pseudomonas strains. These strains are known as versatile medium chain length PHAs (PHAs-mcl producers using fatty acids as carbon source. Oleic acid was used to produce PHAs-mcl using Pseudomonas putida PGA 1 by continuous feeding of both nitrogen and carbon source, in a fed batch culture. During cell growth, PHAs also accumulated, indicating that PHA production in this organism is growth associated. Residual cell increased until the nitrogen source was depleted. At the end of fermentation, final cell concentration, PHA content, and roductivity were 30.2 g/L, 44.8 % of cell dry weight, and 0.188 g/l/h, respectively.

  3. Expression, crystallization and preliminary diffraction studies of the Pseudomonas putida cytochrome P450cam operon repressor CamR

    International Nuclear Information System (INIS)

    Maenaka, Katsumi; Fukushi, Kouji; Aramaki, Hironori; Shirakihara, Yasuo

    2005-01-01

    The P. putida cytochrome P450cam operon repressor CamR has been expressed in E. coli and crystallized in space group P2 1 2 1 2. The Pseudomonas putida cam repressor (CamR) is a homodimeric protein that binds to the camO DNA operator to inhibit the transcription of the cytochrome P450cam operon camDCAB. CamR has two functional domains: a regulatory domain and a DNA-binding domain. The binding of the inducer d-camphor to the regulatory domain renders the DNA-binding domain unable to bind camO. Native CamR and its selenomethionyl derivative have been overproduced in Escherichia coli and purified. Native CamR was crystallized under the following conditions: (i) 12–14% PEG 4000, 50 mM Na PIPES, 0.1 M KCl, 1% glycerol pH 7.3 at 288 K with and without camphor and (ii) 1.6 M P i , 50 mM Na PIPES, 2 mM camphor pH 6.7 at 278 K. The selenomethionyl derivative CamR did not crystallize under either of these conditions, but did crystallize using 12.5% PEG MME 550, 25 mM Na PIPES, 2.5 mM MgCl 2 pH 7.3 at 298 K. Preliminary X-ray diffraction studies revealed the space group to be orthorhombic (P2 1 2 1 2), with unit-cell parameters a = 48.0, b = 73.3, c = 105.7 Å. Native and selenomethionyl derivative data sets were collected to 3 Å resolution at SPring-8 and the Photon Factory

  4. Fructose 1-phosphate is the one and only physiological effector of the Cra (FruR) regulator of Pseudomonas putida.

    Science.gov (United States)

    Chavarría, Max; Durante-Rodríguez, Gonzalo; Krell, Tino; Santiago, César; Brezovsky, Jan; Damborsky, Jiri; de Lorenzo, Víctor

    2014-01-01

    Fructose-1-phosphate (F1P) is the preferred effector of the catabolite repressor/activator (Cra) protein of the soil bacterium Pseudomonas putida but its ability to bind other metabolic intermediates in vivo is unclear. The Cra protein of this microorganism (Cra(PP)) was submitted to mobility shift assays with target DNA sequences (the PfruB promoter) and candidate effectors fructose-1,6-bisphosphate (FBP), glucose 6-phosphate (G6P), and fructose-6-phosphate (F6P). 1 mM F1P was sufficient to release most of the Cra protein from its operators but more than 10 mM of FBP or G6P was required to free the same complex. However, isothermal titration microcalorimetry failed to expose any specific interaction between Cra(PP) and FBP or G6P. To solve this paradox, transcriptional activity of a PfruB-lacZ fusion was measured in wild-type and ΔfruB cells growing on substrates that change the intracellular concentrations of F1P and FBP. The data indicated that PfruB activity was stimulated by fructose but not by glucose or succinate. This suggested that Cra(PP) represses expression in vivo of the cognate fruBKA operon in a fashion dependent just on F1P, ruling out any other physiological effector. Molecular docking and dynamic simulations of the Cra-agonist interaction indicated that both metabolites can bind the repressor, but the breach in the relative affinity of Cra(PP) for F1P vs FBP is three orders of magnitude larger than the equivalent distance in the Escherichia coli protein. This assigns the Cra protein of P. putida the sole role of transducing the presence of fructose in the medium into a variety of direct and indirect physiological responses.

  5. A reduction in growth rate of Pseudomonas putida KT2442 counteracts productivity advances in medium-chain-length polyhydroxyalkanoate production from gluconate

    Directory of Open Access Journals (Sweden)

    Zinn Manfred

    2011-04-01

    Full Text Available Abstract Background The substitution of plastics based on fossil raw material by biodegradable plastics produced from renewable resources is of crucial importance in a context of oil scarcity and overflowing plastic landfills. One of the most promising organisms for the manufacturing of medium-chain-length polyhydroxyalkanoates (mcl-PHA is Pseudomonas putida KT2440 which can accumulate large amounts of polymer from cheap substrates such as glucose. Current research focuses on enhancing the strain production capacity and synthesizing polymers with novel material properties. Many of the corresponding protocols for strain engineering rely on the rifampicin-resistant variant, P. putida KT2442. However, it remains unclear whether these two strains can be treated as equivalent in terms of mcl-PHA production, as the underlying antibiotic resistance mechanism involves a modification in the RNA polymerase and thus has ample potential for interfering with global transcription. Results To assess PHA production in P. putida KT2440 and KT2442, we characterized the growth and PHA accumulation on three categories of substrate: PHA-related (octanoate, PHA-unrelated (gluconate and poor PHA substrate (citrate. The strains showed clear differences of growth rate on gluconate and citrate (reduction for KT2442 > 3-fold and > 1.5-fold, respectively but not on octanoate. In addition, P. putida KT2442 PHA-free biomass significantly decreased after nitrogen depletion on gluconate. In an attempt to narrow down the range of possible reasons for this different behavior, the uptake of gluconate and extracellular release of the oxidized product 2-ketogluconate were measured. The results suggested that the reason has to be an inefficient transport or metabolization of 2-ketogluconate while an alteration of gluconate uptake and conversion to 2-ketogluconate could be excluded. Conclusions The study illustrates that the recruitment of a pleiotropic mutation, whose effects might

  6. mRNA processing in yeast

    International Nuclear Information System (INIS)

    Stevens, A.

    1982-01-01

    Investigations in this laboratory center on basic enzymatic reactions of RNA. Still undefined are reactions involved in the conversion of precursors of mRA (pre-mRNA) to mRNA in eukaryotes. The pre-mRNA is called heterogeneous nuclear RNA and is 2 to 6 times larger than mRNA. The conversion, called splicing, involves a removal of internal sequences called introns by endoribonuclease action followed by a rejoining of the 3'- and 5'-end fragments, called exons, by ligating activity. It has not been possible yet to study the enzymes involved in vitro. Also undefined are reactions involved in the turnover or discarding of certain of the pre-mRNA molecules. Yeast is a simple eukaryote and may be expected to have the same, but perhaps simpler, processing reactions as the higher eukaryotes. Two enzymes involved in the processing of pre-mRNA and mRNA in yeast are under investigation. Both enzymes have been partially purified from ribonucleoprotein particles of yeast. The first is a unique decapping enzyme which cleaves [ 3 H]m 7 Gppp [ 14 C]RNA-poly (A) of yeast, yielding [ 3 H]m 7 GDP and is suggested by the finding that the diphosphate product, m 7 GpppA(G), and UDP-glucose are not hydrolyzed. The second enzyme is an endoribonuclease which converts both the [ 3 H] and [ 14 C] labels of [ 3 H]m 7 Gppp[ 14 C]RNA-poly(A) from an oligo(dT)-cellulose bound form to an unbound, acid-insoluble form. Results show that the stimulation involves an interaction of the labeled RNA with the small nuclear RNA. The inhibition of the enzyme by ethidium bromide and its stimulation by small nuclear RNA suggest that it may be a processing ribonuclease, requiring specific double-stranded features in its substrate. The characterization of the unique decapping enzyme and endoribonuclease may help to understand reactions involved in the processing of pre-mRNA and mRNA in eukaryotes

  7. Fine needle aspiration cytology of ALK1(-), CD30+ anaplastic large cell lymphoma post renal transplantation: a case report and literature review.

    Science.gov (United States)

    Balachandran, Indra; Walker, Joe W; Broman, Jerry

    2010-03-01

    Post transplant lymphoproliferative disorders (PTLD) complicates the course of 0.3 to 3% of renal transplant patients receiving immunosuppression. Epstein-Barr virus (EBV) related non-Hodgkin's lymphomas of B-cell type is more common than those of T-cell origin. CD30 positive Anaplastic Large Cell Lymphoma (ALCL) is a Non-Hodgkin's lymphoma (B or T cell type) that accounts for a small percentage of PTLD's. ALCL of T-cell type are a spectrum of disease ranging from primary cutaneous to systemic nodal ALCL. The systemic nodal ALCL is further subdivided into anaplastic lymphoma kinase-1 (ALK-1) positive or negative. ALK-1 protein is a gene fusion product of translocation (2;5) and carries prognostic implications. We present an unusual manifestation of ALK-1 negative CD30 positive ALCL in a post renal transplant patient in FNA cytology with all supportive adjuvant studies and differential diagnoses and review the cytology literature on this topic.

  8. Inflammatory myofibroblastic tumors of the lung carrying a chimeric A2M-ALK gene: report of 2 infantile cases and review of the differential diagnosis of infantile pulmonary lesions.

    Science.gov (United States)

    Tanaka, Mio; Kohashi, Kenichi; Kushitani, Kei; Yoshida, Misa; Kurihara, Sho; Kawashima, Masumi; Ueda, Yuka; Souzaki, Ryota; Kinoshita, Yoshiaki; Oda, Yoshinao; Takeshima, Yukio; Hiyama, Eiso; Taguchi, Tomoaki; Tanaka, Yukichi

    2017-08-01

    We report 2 infantile cases of pulmonary tumor carrying a chimeric A2M-ALK gene. A2M-ALK is a newly identified anaplastic lymphoma kinase (ALK)-related chimeric gene from a tumor diagnosed as fetal lung interstitial tumor (FLIT). FLIT is a recently recognized infantile pulmonary lesion defined as a mass-like lesion that morphologically resembles the fetal lung. Grossly, FLIT characteristically appears as a well-circumscribed spongy mass, whereas the tumors in these patients were solid and firm. Histologically, the tumors showed intrapulmonary lesions composed of densely proliferating polygonal or spindle-shaped mesenchymal cells with diffuse and dense infiltrations of inflammatory cells forming microcystic or micropapillary structures lined by thyroid transcription factor 1-positive pneumocytes, favoring inflammatory myofibroblastic tumor rather than FLIT. The proliferating cells were immunoreactive for ALK, and A2M-ALK was identified in both tumors with reverse-transcription polymerase chain reaction. The dense infiltration of inflammatory cells, immunoreactivity for ALK, and identification of an ALK-related chimeric gene suggested a diagnosis of inflammatory myofibroblastic tumor. Histologically, most reported FLITs show sparse inflammatory infiltrates and a relatively low density of interstitial cells in the septa, although prominent infiltration of inflammatory cells and high cellularity of interstitial cells are seen in some FLITs. The present cases suggest that ALK rearrangements, including the chimeric A2M-ALK gene, may be present in these infantile pulmonary lesions, especially those with inflammatory cell infiltration. We propose that these infantile pulmonary lesions containing a chimeric A2M-ALK gene be categorized as a specific type of inflammatory myofibroblastic tumor that develops exclusively in neonates and infants. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Broad host range ProUSER vectors enable fast characterization of inducible promoters and optimization of p-coumaric acid production in Pseudomonas putida KT2440

    DEFF Research Database (Denmark)

    Calero Valdayo, Patricia; Ingemann Jensen, Sheila; Nielsen, Alex Toftgaard

    2016-01-01

    Pseudomonas putida KT2440 has gained increasing interest as a host for the production of biochemicals. Because of the lack of a systematic characterization of inducible promoters in this strain, we generated ProUSER broad-host-expression plasmids that facilitate fast uracil-based cloning. A set...... of ProUSER-reporter vectors was further created to characterize different inducible promoters. The PrhaB and Pm promoters were orthogonal and showed titratable, high, and homogeneous expression. To optimize the production of p-coumaric acid, P. putida was engineered to prevent degradation of tyrosine...... and p-coumaric acid. Pm and PrhaB were used to control the expression of a tyrosine ammonia lyase or AroG* and TyrA* involved in tyrosine production, respectively. Pathway expression was optimized by modulating inductions, resulting in small-scale p-coumaric acid production of 1.2 mM, the highest...

  10. Principles of mRNA transport in yeast.

    Science.gov (United States)

    Heym, Roland Gerhard; Niessing, Dierk

    2012-06-01

    mRNA localization and localized translation is a common mechanism by which cellular asymmetry is achieved. In higher eukaryotes the mRNA transport machinery is required for such diverse processes as stem cell division and neuronal plasticity. Because mRNA localization in metazoans is highly complex, studies at the molecular level have proven to be cumbersome. However, active mRNA transport has also been reported in fungi including Saccharomyces cerevisiae, Ustilago maydis and Candida albicans, in which these events are less difficult to study. Amongst them, budding yeast S. cerevisiae has yielded mechanistic insights that exceed our understanding of other mRNA localization events to date. In contrast to most reviews, we refrain here from summarizing mRNA localization events from different organisms. Instead we give an in-depth account of ASH1 mRNA localization in budding yeast. This approach is particularly suited to providing a more holistic view of the interconnection between the individual steps of mRNA localization, from transcriptional events to cytoplasmic mRNA transport and localized translation. Because of our advanced mechanistic understanding of mRNA localization in yeast, the present review may also be informative for scientists working, for example, on mRNA localization in embryogenesis or in neurons.

  11. PHOX2B-mediated regulation of ALK expression: in vitro identification of a functional relationship between two genes involved in neuroblastoma.

    Directory of Open Access Journals (Sweden)

    Tiziana Bachetti

    Full Text Available BACKGROUND: Neuroblastoma (NB is a severe pediatric tumor originating from neural crest derivatives and accounting for 15% of childhood cancer mortality. The heterogeneous and complex genetic etiology has been confirmed with the identification of mutations in two genes, encoding for the receptor tyrosine kinase Anaplastic Lymphoma Kinase (ALK and the transcription factor Paired-like Homeobox 2B (PHOX2B, in a limited proportion of NB patients. Interestingly, these two genes are overexpressed in the great majority of primary NB samples and cell lines. These observations led us to test the hypothesis of a regulatory or functional relationship between ALK and PHOX2B underlying NB pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Following this possibility, we first confirmed a striking correlation between the transcription levels of ALK, PHOX2B and its direct target PHOX2A in a panel of NB cell lines. Then, we manipulated their expression in NB cell lines by siRNA-mediated knock-down and forced over-expression of each gene under analysis. Surprisingly, PHOX2B- and PHOX2A-directed siRNAs efficiently downregulated each other as well as ALK gene and, consistently, the enhanced expression of PHOX2B in NB cells yielded an increment of ALK protein. We finally demonstrated that PHOX2B drives ALK gene transcription by directly binding its promoter, which therefore represents a novel PHOX2B target. CONCLUSIONS/SIGNIFICANCE: These findings provide a compelling explanation of the concurrent involvement of these two genes in NB pathogenesis and are going to foster a better understanding of molecular interactions at the base of the disease. Moreover, this work opens new perspectives for NBs refractory to conventional therapies that may benefit from the design of novel therapeutic RNAi-based approaches for multiple gene targets.

  12. DNA alkylation lesions and their repair in human cells: modification of the comet assay with 3-methyladenine DNA glycosylase (AlkD).

    Science.gov (United States)

    Hašplová, Katarína; Hudecová, Alexandra; Magdolénová, Zuzana; Bjøras, Magnar; Gálová, Eliška; Miadoková, Eva; Dušinská, Mária

    2012-01-05

    3-methyladenine DNA glycosylase (AlkD) belongs to a new family of DNA glycosylases; it initiates repair of cytotoxic and promutagenic alkylated bases (its main substrates being 3-methyladenine and 7-methylguanine). The modification of the comet assay (single cell gel electrophoresis) using AlkD enzyme thus allows assessment of specific DNA alkylation lesions. The resulting baseless sugars are alkali-labile, and under the conditions of the alkaline comet assay they appear as DNA strand breaks. The alkylating agent methyl methanesulfonate (MMS) was used to induce alkylation lesions and to optimize conditions for the modified comet assay method with AlkD on human lymphoblastoid (TK6) cells. We also studied cellular and in vitro DNA repair of alkylated bases in DNA in TK6 cells after treatment with MMS. Results from cellular repair indicate that 50% of DNA alkylation is repaired in the first 60 min. The in vitro repair assay shows that while AlkD recognises most alkylation lesions after 60 min, a cell extract from TK6 cells recognises most of the MMS-induced DNA adducts already in the first 15 min of incubation, with maximum detection of lesions after 60 min' incubation. Additionally, we tested the in vitro repair capacity of human lymphocyte extracts from 5 individuals and found them to be able to incise DNA alkylations in the same range as AlkD. The modification of the comet assay with AlkD can be useful for in vitro and in vivo genotoxicity studies to detect alkylation damage and repair and also for human biomonitoring and molecular epidemiology studies. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  13. Isolation and characterization of bacteria from mercury contaminated sites in Rio Grande do Sul, Brazil, and assessment of methylmercury removal capability of a Pseudomonas putida V1 strain.

    Science.gov (United States)

    Cabral, Lucélia; Giovanella, Patrícia; Gianello, Clésio; Bento, Fátima Menezes; Andreazza, Robson; Camargo, Flávio Anastácio Oliveira

    2013-06-01

    Methylmercury (MeHg) is one of the most dangerous heavy metal for living organisms that may be found in environment. Given the crescent industrialization of Brazil and considering that mercury is a residue of several industrial processes, there is an increasing need to encounter and develop remediation approaches of mercury contaminated sites. The aim of this study was to isolate and characterize methylmercury resistant bacteria from soils and sludge sewage from Rio Grande do Sul, Brazil. Sixteen bacteria were isolated from these contaminated sites and some isolates were highly resistant to methylmercury (>8.7 μM). All the isolates were identified by 16S rDNA. Pseudomonas putida V1 was able to volatilize approximately 90 % of methylmercury added to growth media and to resist to copper, lead, nickel, chromate, zinc, cobalt, manganese and barium. In the presence of high concentrations of methylmercury (12 μM), cell growth was limited, but P. putida V1 was still able to remove up to 29 % of this compound from culture medium. This bacterium removed an average of 77 % of methylmercury from culture medium with pH in the range 4.0-6.0. In addition, methylmercury was efficiently removed (>80 %) in temperature of 21-25 °C. Polymerase chain reactions indicated the presence of merA but not merB in P. putida V1. The growth and ability of P. putida V1 to remove methylmercury in a wide range of pH (4.0 and 8.0) and temperature (10-35 °C), its tolerance to other heavy metals and ability to grow in the presence of up to 11.5 μM of methylmercury, suggest this strain as a new potential resource for degrading methylmercury contaminated sites.

  14. Draft Genome Sequence Analysis of a Pseudomonas putida W15Oct28 Strain with Antagonistic Activity to Gram-Positive and Pseudomonas sp. Pathogens

    Science.gov (United States)

    Ye, Lumeng; Hildebrand, Falk; Dingemans, Jozef; Ballet, Steven; Laus, George; Matthijs, Sandra; Berendsen, Roeland; Cornelis, Pierre

    2014-01-01

    Pseudomonas putida is a member of the fluorescent pseudomonads known to produce the yellow-green fluorescent pyoverdine siderophore. P. putida W15Oct28, isolated from a stream in Brussels, was found to produce compound(s) with antimicrobial activity against the opportunistic pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and the plant pathogen Pseudomonas syringae, an unusual characteristic for P. putida. The active compound production only occurred in media with low iron content and without organic nitrogen sources. Transposon mutants which lost their antimicrobial activity had the majority of insertions in genes involved in the biosynthesis of pyoverdine, although purified pyoverdine was not responsible for the antagonism. Separation of compounds present in culture supernatants revealed the presence of two fractions containing highly hydrophobic molecules active against P. aeruginosa. Analysis of the draft genome confirmed the presence of putisolvin biosynthesis genes and the corresponding lipopeptides were found to contribute to the antimicrobial activity. One cluster of ten genes was detected, comprising a NAD-dependent epimerase, an acetylornithine aminotransferase, an acyl CoA dehydrogenase, a short chain dehydrogenase, a fatty acid desaturase and three genes for a RND efflux pump. P. putida W15Oct28 genome also contains 56 genes encoding TonB-dependent receptors, conferring a high capacity to utilize pyoverdines from other pseudomonads. One unique feature of W15Oct28 is also the presence of different secretion systems including a full set of genes for type IV secretion, and several genes for type VI secretion and their VgrG effectors. PMID:25369289

  15. Pseudomonas wadenswilerensis sp. nov. and Pseudomonas reidholzensis sp. nov., two novel species within the Pseudomonas putida group isolated from forest soil.

    Science.gov (United States)

    Frasson, David; Opoku, Michael; Picozzi, Tara; Torossi, Tanja; Balada, Stefanie; Smits, Theo H M; Hilber, Urs

    2017-08-01

    Within the frame of a biotechnological screening, we isolated two Pseudomonas strains from forest soil. 16S rRNA gene sequence analysis indicated that strain CCOS 864T shared 99.8 % similarity with Pseudomonas donghuensis HYST, while strain CCOS 865T shared 99.0 % similarity with Pseudomonas putida DSM 291T and lower similarity with other P. putida group type strains. Based on multilocus sequence analysis, the two strains were genotypically distinct from each other, each forming a separate clade. Strains CCOS 864T and CCOS 865T were Gram-stain-negative, motile and rod-shaped, growing at a temperature range of 4-37 °C. Strain CCOS 864T could be phenotypically distinguished from P. putida group species by the combination of gelatinase-positive reaction and positive growth on N-acetyl-d-glucosamine, p-hydroxyphenylacetic acid and inosine but lack of fluorescein production on King's B medium, while strain CCOS 865T could be distinguished from P. putida group species by the combination of positive growth with saccharic acid and negative growth with p-hydroxyphenylacetic acid and l-pyroglutamic acid. The major polar lipid for both strains was phosphatidylethanolamine; the major quinone was ubiquinone Q-9. DNA-DNA hybridization and average nucleotide identities confirmed the novel species status for the two strains. The DNA G+C contents of CCOS 864T and CCOS 865T were 62.1 and 63.8 mol%, respectively. The phenotypic, phylogenetic and DNA-DNA relatedness data support the suggestion that CCOS 864T and CCOS 865T represent two novel Pseudomonas species. The names Pseudomonas wadenswilerensis sp. nov. (type strain CCOS 864T=LMG 29327T) and Pseudomonas reidholzensis sp. nov. (type strain CCOS 865T=LMG 29328T) are proposed.

  16. A newly isolated Pseudomonas putida S-1 strain for batch-mode-propanethiol degradation and continuous treatment of propanethiol-containing waste gas

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Dong-Zhi, E-mail: cdz@zjut.edu.cn [College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032 (China); Sun, Yi-Ming; Han, Li-Mei [College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032 (China); Chen, Jing [College of Food and Pharmacy, Zhejiang Ocean University, Zhoushan 316004 (China); Ye, Jie-Xu; Chen, Jian-Meng [College of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou 310032 (China)

    2016-01-25

    Highlights: • A novel strain capable of effectively degrading 1-propanethiol (PT) was isolated. • Cells could be feasibly cultured in nutrition-rich media for PT degradation. • A possible pathway for PT degradation was proposed. • Pseudomonas putida S-1 could degrade mixed pollutants with diauxic growth. • Continuous removal of gaseous PT with or without isopropanol was demonstrated. - Abstract: Pseudomonas putida S-1 was isolated from activated sludge. This novel strain was capable of degrading malodorous 1-propanethiol (PT). PT degradation commenced with no lag phase by cells pre-grown in nutrition-rich media, such as Luria–Bertani (LB), and PT-contained mineral medium at specific growth rates of 0.10–0.19 h{sup −1}; this phenomenon indicated the operability of a large-scale cell culture. A possible PT degradation pathway was proposed on the basis of the detected metabolites, including dipropyl disulfide, 3-hexanone, 2-hexanone, 3-hexanol, 2-hexanol, S{sup 0}, SO{sub 4}{sup 2−}, and CO{sub 2}. P. putida S-1 could degrade mixed pollutants containing PT, diethyl disulfide, isopropyl alcohol, and acetaldehyde, and LB-pre-cultured cells underwent diauxic growth. Waste gas contaminated with 200–400 mg/m{sup 3} PT was continuously treated by P. putida S-1 pre-cultured in LB medium in a completely stirred tank reactor. The removal efficiencies exceeded 88% when PT stream was mixed with 200 mg/m{sup 3} isopropanol; by contrast, the removal efficiencies decreased to 60% as the empty bed residence time was shortened from 40 s to 20 s.

  17. Co-Culture with Listeria monocytogenes within a Dual-Species Biofilm Community Strongly Increases Resistance of Pseudomonas putida to Benzalkonium Chloride

    Science.gov (United States)

    Giaouris, Efstathios; Chorianopoulos, Nikos; Doulgeraki, Agapi; Nychas, George-John

    2013-01-01

    Biofilm formation is a phenomenon occurring almost wherever microorganisms and surfaces exist in close proximity. This study aimed to evaluate the possible influence of bacterial interactions on the ability of Listeria monocytogenes and Pseudomonas putida to develop a dual-species biofilm community on stainless steel (SS), as well as on the subsequent resistance of their sessile cells to benzalkonium chloride (BC) used in inadequate (sub-lethal) concentration (50 ppm). The possible progressive adaptability of mixed-culture biofilms to BC was also investigated. To accomplish these, 3 strains per species were left to develop mixed-culture biofilms on SS coupons, incubated in daily renewable growth medium for a total period of 10 days, under either mono- or dual-species conditions. Each day, biofilm cells were exposed to disinfection treatment. Results revealed that the simultaneous presence of L. monocytogenes strongly increased the resistance of P. putida biofilm cells to BC, while culture conditions (mono-/dual-species) did not seem to significantly influence the resistance of L. monocytogenes biofilm cells. BC mainly killed L. monocytogenes cells when this was applied against the dual-species sessile community during the whole incubation period, despite the fact that from the 2nd day this community was mainly composed (>90%) of P. putida cells. No obvious adaptation to BC was observed in either L. monocytogenes or P. putida biofilm cells. Pulsed field gel electrophoresis (PFGE) analysis showed that the different strains behaved differently with regard to biofilm formation and antimicrobial resistance. Such knowledge on the physiological behavior of mixed-culture biofilms could provide the information necessary to control their formation. PMID:24130873

  18. Co-culture with Listeria monocytogenes within a dual-species biofilm community strongly increases resistance of Pseudomonas putida to benzalkonium chloride.

    Directory of Open Access Journals (Sweden)

    Efstathios Giaouris

    Full Text Available Biofilm formation is a phenomenon occurring almost wherever microorganisms and surfaces exist in close proximity. This study aimed to evaluate the possible influence of bacterial interactions on the ability of Listeria monocytogenes and Pseudomonas putida to develop a dual-species biofilm community on stainless steel (SS, as well as on the subsequent resistance of their sessile cells to benzalkonium chloride (BC used in inadequate (sub-lethal concentration (50 ppm. The possible progressive adaptability of mixed-culture biofilms to BC was also investigated. To accomplish these, 3 strains per species were left to develop mixed-culture biofilms on SS coupons, incubated in daily renewable growth medium for a total period of 10 days, under either mono- or dual-species conditions. Each day, biofilm cells were exposed to disinfection treatment. Results revealed that the simultaneous presence of L. monocytogenes strongly increased the resistance of P. putida biofilm cells to BC, while culture conditions (mono-/dual-species did not seem to significantly influence the resistance of L. monocytogenes biofilm cells. BC mainly killed L. monocytogenes cells when this was applied against the dual-species sessile community during the whole incubation period, despite the fact that from the 2nd day this community was mainly composed (>90% of P. putida cells. No obvious adaptation to BC was observed in either L. monocytogenes or P. putida biofilm cells. Pulsed field gel electrophoresis (PFGE analysis showed that the different strains behaved differently with regard to biofilm formation and antimicrobial resistance. Such knowledge on the physiological behavior of mixed-culture biofilms could provide the information necessary to control their formation.

  19. A newly isolated Pseudomonas putida S-1 strain for batch-mode-propanethiol degradation and continuous treatment of propanethiol-containing waste gas

    International Nuclear Information System (INIS)

    Chen, Dong-Zhi; Sun, Yi-Ming; Han, Li-Mei; Chen, Jing; Ye, Jie-Xu; Chen, Jian-Meng

    2016-01-01

    Highlights: • A novel strain capable of effectively degrading 1-propanethiol (PT) was isolated. • Cells could be feasibly cultured in nutrition-rich media for PT degradation. • A possible pathway for PT degradation was proposed. • Pseudomonas putida S-1 could degrade mixed pollutants with diauxic growth. • Continuous removal of gaseous PT with or without isopropanol was demonstrated. - Abstract: Pseudomonas putida S-1 was isolated from activated sludge. This novel strain was capable of degrading malodorous 1-propanethiol (PT). PT degradation commenced with no lag phase by cells pre-grown in nutrition-rich media, such as Luria–Bertani (LB), and PT-contained mineral medium at specific growth rates of 0.10–0.19 h"−"1; this phenomenon indicated the operability of a large-scale cell culture. A possible PT degradation pathway was proposed on the basis of the detected metabolites, including dipropyl disulfide, 3-hexanone, 2-hexanone, 3-hexanol, 2-hexanol, S"0, SO_4"2"−, and CO_2. P. putida S-1 could degrade mixed pollutants containing PT, diethyl disulfide, isopropyl alcohol, and acetaldehyde, and LB-pre-cultured cells underwent diauxic growth. Waste gas contaminated with 200–400 mg/m"3 PT was continuously treated by P. putida S-1 pre-cultured in LB medium in a completely stirred tank reactor. The removal efficiencies exceeded 88% when PT stream was mixed with 200 mg/m"3 isopropanol; by contrast, the removal efficiencies decreased to 60% as the empty bed residence time was shortened from 40 s to 20 s.

  20. Spotlight on crizotinib in the first-line treatment of ALK-positive advanced non-small-cell lung cancer: patients selection and perspectives

    Directory of Open Access Journals (Sweden)

    Giroux Leprieur E

    2016-06-01

    Full Text Available Etienne Giroux Leprieur,1,2 Vincent Fallet,3,4 Jacques Cadranel,3,4 Marie Wislez3,4 1Respiratory Diseases and Thoracic Oncology Department, APHP-Ambroise Paré Hospital, Boulogne-Billancourt, France; 2EA4340 Laboratory, UVSQ, Paris-Saclay University, France; 3Respiratory Diseases Department, APHP – Tenon Hospital, Paris, France; 4Sorbonne University, GRC 04, UPMC Univ Paris 06, France Abstract: Around 4% of advanced non-small-cell lung cancers (NSCLCs have an ALK rearrangement at the time of diagnosis. This molecular feature is more frequent in young patients, with no/light smoking habit and with adenocarcinoma pathological subtype. Crizotinib is a tyrosine kinase inhibitor, targeting ALK, ROS1, RON, and MET. The preclinical efficacy results led to a fast-track clinical development. The US Food and Drug Administration (FDA approval was achieved after the Phase I clinical trial in 2011 in ALK-rearranged advanced NSCLC progressing after a first-line treatment. In 2013, the randomized Phase III trial PROFILE-1007 confirmed the efficacy of crizotinib in ALK-rearranged NSCLC, compared to cytotoxic chemotherapy, in second-line setting or more. In 2014, the PROFILE-1014 trial showed the superiority of crizotinib in the first-line setting compared to the pemetrexed platinum doublet chemotherapy. The response rate was 74%, and the progression-free survival was 10.9 months with crizotinib. Based on these results, crizotinib received approval from the FDA and European Medicines Agency for first-line treatment of ALK-rearranged NSCLC. The various molecular mechanisms at the time of the progression (ALK mutations or amplification, ALK-independent mechanisms encourage performing re-biopsy at the time of progression under crizotinib. The best treatment strategy at the progression (crizotinib continuation beyond progression, switch to second-generation tyrosine kinase inhibitors, or cytotoxic chemotherapy depends on the phenotype of the progression, the

  1. Contribution of transcription-coupled DNA repair to MMS-induced mutagenesis in E. coli strains deficient in functional AlkB protein.

    Science.gov (United States)

    Wrzesiński, Michał; Nieminuszczy, Jadwiga; Sikora, Anna; Mielecki, Damian; Chojnacka, Aleksandra; Kozłowski, Marek; Krwawicz, Joanna; Grzesiuk, Elzbieta

    2010-06-01

    In Escherichia coli the alkylating agent methyl methanesulfonate (MMS) induces defense systems (adaptive and SOS responses), DNA repair pathways, and mutagenesis. We have previously found that AlkB protein induced as part of the adaptive (Ada) response protects cells from the genotoxic and mutagenic activity of MMS. AlkB is a non-heme iron (II), alpha-ketoglutarate-dependent dioxygenase that oxidatively demethylates 1meA and 3meC lesions in DNA, with recovery of A and C. Here, we studied the impact of transcription-coupled DNA repair (TCR) on MMS-induced mutagenesis in E. coli strain deficient in functional AlkB protein. Measuring the decline in the frequency of MMS-induced argE3-->Arg(+) revertants under transient amino acid starvation (conditions for TCR induction), we have found a less effective TCR in the BS87 (alkB(-)) strain in comparison with the AB1157 (alkB(+)) counterpart. Mutation in the mfd gene encoding the transcription-repair coupling factor Mfd, resulted in weaker TCR in MMS-treated and starved AB1157 mfd-1 cells in comparison to AB1157 mfd(+), and no repair in BS87 mfd(-) cells. Determination of specificity of Arg(+) revertants allowed to conclude that MMS-induced 1meA and 3meC lesions, unrepaired in bacteria deficient in AlkB, are the source of mutations. These include AT-->TA transversions by supL suppressor formation (1meA) and GC-->AT transitions by supB or supE(oc) formation (3meC). The repair of these lesions is partly Mfd-dependent in the AB1157 mfd-1 and totally Mfd-dependent in the BS87 mfd-1 strain. The nucleotide sequence of the mfd-1 allele shows that the mutated Mfd-1 protein, deprived of the C-terminal translocase domain, is unable to initiate TCR. It strongly enhances the SOS response in the alkB(-)mfd(-) bacteria but not in the alkB(+)mfd(-) counterpart. Copyright 2010 Elsevier B.V. All rights reserved.

  2. Potential of the TCE-degrading endophyte Pseudomonas putida W619-TCE to improve plant growth and reduce TCE phytotoxicity and evapotranspiration in poplar cuttings.

    Science.gov (United States)

    Weyens, Nele; Truyens, Sascha; Dupae, Joke; Newman, Lee; Taghavi, Safiyh; van der Lelie, Daniel; Carleer, Robert; Vangronsveld, Jaco

    2010-09-01

    The TCE-degrading poplar endophyte Pseudomonas putida W619-TCE was inoculated in poplar cuttings, exposed to 0, 200 and 400 mg l(-1) TCE, that were grown in two different experimental setups. During a short-term experiment, plants were grown hydroponically in half strength Hoagland nutrient solution and exposed to TCE for 3 days. Inoculation with P. putida W619-TCE promoted plant growth, reduced TCE phytotoxicity and reduced the amount of TCE present in the leaves. During a mid-term experiment, plants were grown in potting soil and exposed to TCE for 3 weeks. Here, inoculation with P. putida W619-TCE had a less pronounced positive effect on plant growth and TCE phytotoxicity, but resulted in strongly reduced amounts of TCE in leaves and roots of plants exposed to 400 mg l(-1) TCE, accompanied by a lowered evapotranspiration of TCE. Dichloroacetic acid (DCAA) and trichloroacetic acid (TCAA), which are known intermediates of TCE degradation, were not detected. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  3. Pseudomonas putida growing at low temperature shows increased levels of CrcZ and CrcY sRNAs, leading to reduced Crc-dependent catabolite repression.

    Science.gov (United States)

    Fonseca, Pilar; Moreno, Renata; Rojo, Fernando

    2013-01-01

    The Crc protein of Pseudomonas inhibits the expression of genes involved in the transport and assimilation of a number of non-preferred carbon sources when preferred substrates are available, thus coordinating carbon metabolism. Crc acts by binding to target mRNAs, inhibiting their translation. In Pseudomonas putida, the amount of free Crc available is controlled by two sRNAs, CrcY and CrcZ, which bind to and sequester Crc. The levels of these sRNAs vary according to metabolic conditions. Pseudomonas putida grows optimally at 30°C, but can also thrive at 10°C. The present work shows that when cells grow exponentially at 10°C, the repressive effect of Crc on many genes is significantly reduced compared with that seen at 30°C. Total Crc levels were similar at both temperatures, but those of CrcZ and CrcY were significantly higher at 10°C. Therefore, Crc-mediated repression may, at least in part, be reduced at 10°C because the fraction of Crc protein sequestered by CrcZ and CrcY is larger, reducing the amount of free Crc available to bind its targets. This may help P. putida to face cold stress. The results reported might help understanding the behaviour of this bacterium in bioremediation or rhizoremediation strategies at low temperatures. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  4. Proof of concept for the simplified breakdown of cellulose by combining Pseudomonas putida strains with surface displayed thermophilic endocellulase, exocellulase and β-glucosidase.

    Science.gov (United States)

    Tozakidis, Iasson E P; Brossette, Tatjana; Lenz, Florian; Maas, Ruth M; Jose, Joachim

    2016-06-10

    The production and employment of cellulases still represents an economic bottleneck in the conversion of lignocellulosic biomass to biofuels and other biocommodities. This process could be simplified by displaying the necessary enzymes on a microbial cell surface. Such an approach, however, requires an appropriate host organism which on the one hand can withstand the rough environment coming along with lignocellulose hydrolysis, and on the other hand does not consume the generated glucose so that it remains available for subsequent fermentation steps. The robust soil bacterium Pseudomonas putida showed a strongly reduced uptake of glucose above a temperature of 50 °C, while remaining structurally intact hence recyclable, which makes it suitable for cellulose hydrolysis at elevated temperatures. Consequently, three complementary, thermophilic cellulases from Ruminiclostridium thermocellum were displayed on the surface of the bacterium. All three enzymes retained their activity on the cell surface. A mixture of three strains displaying each one of these enzymes was able to synergistically hydrolyze filter paper at 55 °C, producing 20 μg glucose per mL cell suspension in 24 h. We could establish Pseudomonas putida as host for the surface display of cellulases, and provided proof-of-concept for a fast and simple cellulose breakdown process at elevated temperatures. This study opens up new perspectives for the application of P. putida in the production of biofuels and other biotechnological products.

  5. Effects of impurities in biodiesel-derived glycerol on growth and expression of heavy metal ion homeostasis genes and gene products in Pseudomonas putida LS46.

    Science.gov (United States)

    Fu, Jilagamazhi; Sharma, Parveen; Spicer, Vic; Krokhin, Oleg V; Zhang, Xiangli; Fristensky, Brian; Wilkins, John A; Cicek, Nazim; Sparling, Richard; Levin, David B

    2015-07-01

    Biodiesel production-derived waste glycerol (WG) was previously investigated as potential carbon source for medium chain length polyhydroxyalkanoate (mcl-PHA) production by Pseudomonas putida LS46. In this study, we evaluated the effect of impurities in the WG on P. putida LS46 physiology during exponential growth and corresponding changes in transcription and protein expression profiles compared with cells grown on pure, reagent grade glycerol. High concentration of metal ions, such as Na(+), and numbers of heavy metals ion, such as copper, ion, zinc, were detected in biodiesel-derived WG. Omics analysis from the corresponding cultures suggested altered expression of genes involved in transport and metabolism of ammonia and heavy metal ions. Expression of three groups of heavy metal homeostasis genes was significantly changed (mostly upregulated) in WG cultures and included the following: copper-responded cluster 1 and 2 genes, primarily containing cusABC; two copies of copAB and heavy metal translocating P-type ATPase; Fur-regulated, TonB-dependent siderophore receptor; and several cobalt/zinc/cadmium transporters. Expression of these genes suggests regulation of intracellular concentrations of heavy metals during growth on biodiesel-derived glycerol. Finally, a number of genes involved in adapting to, or metabolizing free fatty acids and other nonheavy metal contaminants, such as Na(+), were also upregulated in P. putida LS46 grown on biodiesel-derived glycerol.

  6. The prevalence of ALK rearrangement in pulmonary adenocarcinomas in an unselected Caucasian population from a defined catchment area

    DEFF Research Database (Denmark)

    Skov, Birgit G; Clementsen, Paul; Larsen, Klaus R

    2017-01-01

    . METHODS AND RESULTS: All patients diagnosed in the population of the greater Copenhagen area were included, irrespective of gender, age, smoking habits, stage or type of available diagnostic material. Tumours were stained with immunohistochemistry (clone 5A4). Immunohistochemistry-positive tumours were......AIMS: To assess the prevalence of EML4-ALK rearrangement gene measured by immunohistochemistry in an unselected population-based consecutive cohort of patients with adenocarcinoma of the lung (ACL), and the correlation with smoking history, thyroid transcription factor 1 (TTF1), gender and age...

  7. n-Alkane and clofibrate, a peroxisome proliferator, activate transcription of ALK2 gene encoding cytochrome P450alk2 through distinct cis-acting promoter elements in Candida maltosa

    International Nuclear Information System (INIS)

    Kogure, Takahisa; Takagi, Masamichi; Ohta, Akinori

    2005-01-01

    The ALK2 gene, encoding one of the n-alkane-hydroxylating cytochromes P450 in Candida maltosa, is induced by n-alkanes and a peroxisome proliferator, clofibrate. Deletion analysis of this gene's promoter revealed two cis-acting elements-an n-alkane-responsive element (ARE2) and a clofibrate-responsive element (CRE2)-that partly overlap in sequence but have distinct functions. ARE2-mediated activation responded to n-alkanes but not to clofibrate and was repressed by glucose. CRE2-mediated activation responded to polyunsaturated fatty acids and steroid hormones as well as to peroxisome proliferators but not to n-alkanes, and it was not repressed by glucose. Both elements mediated activation by oleic acid. Mutational analysis demonstrated that three CCG sequences in CRE2 were critical to the activation by clofibrate as well as to the in vitro binding of a specific protein to this element. These findings suggest that ALK2 is induced by peroxisome proliferators and steroid hormones through a specific CRE2-mediated regulatory mechanism

  8. In situ, real-time catabolic gene expression: Extraction and characterization of naphthalene dioxygenase mRNA transcripts from groundwater

    International Nuclear Information System (INIS)

    Wilson, M.S.; Bakermans, C.; Madsen, E.L.

    1999-01-01

    The authors developed procedures for isolating and characterizing in situ-transcribed mRNA from groundwater microorganisms catabolizing naphthalene at a coal tar waste-contaminated site. Groundwater was pumped through 0.22-microm-pore-size filters, which were then frozen to dry ice-ethanol. RNA was extracted from the frozen filters by boiling sodium dodecyl sulfate lysis and acidic phenol-chloroform extraction. Transcript characterization was performed with a series of PCR primers designed to amplify nahAc homologs. Several primer pairs were found to amplify nahAc homologs representing the entire diversity of the naphthalene-degrading genes. The environmental RNA extract was reverse transcribed, and the resultant mixture of cDNAs was amplified by PCR. A digoxigenin-labeled probe mixture was produced by PCR amplification of groundwater cDNA. This probe mixture hybridized under stringent conditions with the corresponding PCR products from naphthalene-degrading bacteria carrying a variety of nahAc homologs, indicating that diverse dioxygenase transcripts had been retrieved from groundwater. Diluted and undiluted cDNA preparations were independently amplified, and 28 of the resulting PCR products were cloned and sequenced. Sequence comparisons revealed two major groups related to the dioxygenase genes ndoB and dntAc, previously cloned from Pseudomonas putida NCIB 9816-4 and Burkholderia sp. strain DNT, respectively. A distinctive subgroup of sequences was found only in experiments performed with the undiluted cDNA preparation. To the authors' knowledge, these results are the first to directly document in situ transcription of genes encoding naphthalene catabolism at a contaminated site by indigenous microorganisms. The retrieved sequences represent greater diversity than has been detected at the study site by culture-based approaches

  9. mRNA Cancer Vaccines-Messages that Prevail.

    Science.gov (United States)

    Grunwitz, Christian; Kranz, Lena M

    2017-01-01

    During the last decade, mRNA became increasingly recognized as a versatile tool for the development of new innovative therapeutics. Especially for vaccine development, mRNA is of outstanding interest and numerous clinical trials have been initiated. Strikingly, all of these studies have proven that large-scale GMP production of mRNA is feasible and concordantly report a favorable safety profile of mRNA vaccines. Induction of T-cell immunity is a multi-faceted process comprising antigen acquisition, antigen processing and presentation, as well as immune stimulation. The effectiveness of mRNA vaccines is critically dependent on making the antigen(s) of interest available to professional antigen-presenting cells, especially DCs. Efficient delivery of mRNA into DCs in vivo remains a major challenge in the mRNA vaccine field. This review summarizes the principles of mRNA vaccines and highlights the importance of in vivo mRNA delivery and recent advances in harnessing their therapeutic potential.

  10. CYP3A5 mRNA degradation by nonsense-mediated mRNA decay.

    Science.gov (United States)

    Busi, Florent; Cresteil, Thierry

    2005-09-01

    The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g.31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudoexon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.

  11. Growth on octane alters the membrane lipid fatty acids of Pseudomonas oleovorans due to the induction of alkB and synthesis of octanol.

    Science.gov (United States)

    Chen, Q; Janssen, D B; Witholt, B

    1995-01-01

    Growth of Pseudomonas oleovorans GPo1, which contains the OCT plasmid, on octane results in changes in the membrane phospholipid fatty acid composition. These changes were not found for GPo12, an OCT-plasmid-cured variant of GPo1, during growth in the presence or absence of octane, implying the involvement of OCT-plasmid-encoded functions. When recombinant strain GPo12(pGEc47) carrying the alk genes from the OCT plasmid was grown on octane, the cells showed the same changes in fatty acid composition as those found for GPo1, indicating that such changes result from induction and expression of the alk genes. This finding was corroborated by inducing GPo12(pGEc47) with dicyclopropylketone (DCPK), a gratuitous inducer of the alk genes. Further experiments showed that the increase of the mean acyl chain length of fatty acids is related to the expression of alkB, which encodes a major integral membrane protein, while the formation of trans unsaturated fatty acids mainly results from the effects of 1-octanol, an octane oxidation product. PMID:7592483

  12. Diversity of alkane degrading bacteria associated with plants in a petroleum oil-contaminated environment and expression of alkane monooxygenase (alkB) genes

    Science.gov (United States)

    Andria, V.; Yousaf, S.; Reichenauer, T. G.; Smalla, K.; Sessitsch, A.

    2009-04-01

    Among twenty-six different plant species, Italian ryegrass (Lolium multiflorum var. Taurus), Birdsfoot trefoil (Lotus corniculatus var. Leo), and the combination of both plants performed well in a petroleum oil contaminated soil. Hydrocarbon degrading bacteria were isolated from the rhizosphere, root interior and shoot interior and subjected to the analysis of 16S rRNA, the 16S and 23S rRNA intergenic spacer region and alkane hydroxylase genes. Higher numbers of culturable, degrading bacteria were associated with Italian ryegrass, which were also characterized by a higher diversity, particularly in the plant interior. Only half of the isolated bacteria hosted known alkane hydroxylase genes (alkB and cytochrome P153-like). Our results indicated that alkB genes have spread through horizontal gene transfer, particularly in the Italian ryegrass rhizosphere, and suggested mobility of catabolic genes between Gram-negative and Gram-positive bacteria. We furthermore studied the colonization behaviour of selected hydrocarbon-degrading strains (comprising an endopyhte and a rhizosphere strain) as well as the expression of their alkane monooxygenase genes in association with Italian ryegrass. Results showed that the endophyte strain better colonized the plant, particularly the plant interior, and also showed higher expression of alkB genes suggesting a more efficient degradation of the pollutant. Furthermore, plants inoculated with the endophyte were better able to grow in the presence of diesel. The rhizosphere strain colonized primarily the rhizosphere and showed low alkB gene expression in the plant interior.

  13. MPN- and Real-Time-Based PCR Methods for the Quantification of Alkane Monooxygenase Homologous Genes (alkB) in Environmental Samples

    Science.gov (United States)

    Pérez-de-Mora, Alfredo; Schulz, Stephan; Schloter, Michael

    Hydrocarbons are major contaminants of soil ecosystems as a result of uncontrolled oil spills and wastes disposal into the environment. Ecological risk assessment and remediation of affected sites is often constrained due to lack of suitable prognostic and diagnostic tools that provide information of abiotic-biotic interactions occurring between contaminants and biological targets. Therefore, the identification and quantification of genes involved in the degradation of hydrocarbons may play a crucial role for evaluating the natural attenuation potential of contaminated sites and the development of successful bioremediation strategies. Besides other gene clusters, the alk operon has been identified as a major player for alkane degradation in different soils. An oxygenase gene (alkB) codes for the initial step of the degradation of aliphatic alkanes under aerobic conditions. In this work, we present an MPN- and a real-time PCR method for the quantification of the bacterial gene alkB (coding for rubredoxin-dependent alkane monooxygenase) in environmental samples. Both approaches enable a rapid culture-independent screening of the alkB gene in the environment, which can be used to assess the intrinsic natural attenuation potential of a site or to follow up the on-going progress of bioremediation assays.

  14. Expression of Pseudomonas aeruginosa transposable phages in Pseudomonas putida cells. I. Establishment of lysogeny and lytic growth efficiency

    Energy Technology Data Exchange (ETDEWEB)

    Gorbunova, S.A.; Yanenko, A.S.; Akhverdyan, V.Z.; Reulets, M.A.; Krylov, V.N.

    1986-03-01

    Expression of the genomes of Pseudomonas aeruginosa transposable phages (TP) in the cells of a heterologous host, P. putida PpGl, was studied. A high efficiency of TP lytic growth in PpGl cells was obtained both after zygotic induction following RP4::TP plasmid transfer and after thermoinduction of PpGl cells lysogenic for thermoinducible prophage D3112cts15. Characteristic for PpGl cells was a high TP yield (20-25 phage D3112cts15 particles per cell), which was evidence of a high level of TP transposition in cells of this species. The frequency of RP4::TP transfer into PpGl and PA01 cells was equal, but the lysogeny detection rat was somewhat lower in PpGl. Pseudomonas aeruginosa TP can integrate into the PpGl chromosome, producing inducible lysogens. The presence of RP4 is not necessary for the expression of the TP genome in PpGl cells. The D3112cts15 TP may be used for interspecific transduction of plasmids and chromosomal markers.

  15. Growth stimulation of Bacillus cereus and Pseudomonas putida using nanostructured ZnO thin film as transducer element

    Energy Technology Data Exchange (ETDEWEB)

    Loukanov, Alexandre, E-mail: loukanov@mail.saitama-u.ac.jp [Saitama University, Department of Chemistry, Faculty of Science (Japan); Filipov, Chavdar [University of Forestry, Department of Infectious pathology, hygiene, technology and control of food stuffs of animal origin, Faculty of Veterinary Medicine (Bulgaria); Valcheva, Violeta [Bulgarian Academy of Science, Department of Infectious Diseases, Institute of microbiology (Bulgaria); Lecheva, Marta [University of Mining and Geology “St. Ivan Rilski”, Laboratory of Engineering NanoBiotechnology, Department of Engineering Geoecology (Bulgaria); Emin, Saim [University of Nova Gorica, Materials Research Laboratory (Slovenia)

    2015-04-15

    The semiconductor zinc oxide nanomaterial (ZnO or ZnO:H) is widely used in advanced biosensor technology for the design of highly-sensitive detector elements for various applications. In the attempt to evaluate its effect on common microorganisms, two types of nanostructured transducer films have been used (average diameter 600–1000 nm). They have been prepared by using both wet sol–gel method and magnetron sputtering. Their polycrystalline structure and specific surface features have been analyzed by X-ray diffraction (XRD), scanning electron microscope, and atomic force microscope. The assessment of growth stimulation of bacteria was determined using epifluorescent microscope by cell staining with Live/Dead BacLight kit. In our experiments, the growth stimulation of Gram-positive and Gram-negative bacteria on nanostructured ZnO film is demonstrated by Bacillus cereus and Pseudomonas putida. These two bacterial species have been selected, because they are well known and studied in biosensor technologies, with structural difference of their cell walls. These pathogens are easy for with common source in the liquid food or some commercial products. Our data has revealed that the method of transducer film preparation influences strongly bacterial inhibition and division. These results present the transforming signal precisely, when ZnO is used in biosensor applications.

  16. Expression of Pseudomonas aeruginosa transposable phages in Pseudomonas putida cells. I. Establishment of lysogeny and lytic growth efficiency

    International Nuclear Information System (INIS)

    Gorbunova, S.A.; Yanenko, A.S.; Akhverdyan, V.Z.; Reulets, M.A.; Krylov, V.N.

    1986-01-01

    Expression of the genomes of Pseudomonas aeruginosa transposable phages (TP) in the cells of a heterologous host, P. putida PpGl, was studied. A high efficiency of TP lytic growth in PpGl cells was obtained both after zygotic induction following RP4::TP plasmid transfer and after thermoinduction of PpGl cells lysogenic for thermoinducible prophage D3112cts15. Characteristic for PpGl cells was a high TP yield (20-25 phage D3112cts15 particles per cell), which was evidence of a high level of TP transposition in cells of this species. The frequency of RP4::TP transfer into PpGl and PA01 cells was equal, but the lysogeny detection rat was somewhat lower in PpGl. Pseudomonas aeruginosa TP can integrate into the PpGl chromosome, producing inducible lysogens. The presence of RP4 is not necessary for the expression of the TP genome in PpGl cells. The D3112cts15 TP may be used for interspecific transduction of plasmids and chromosomal markers

  17. Growth stimulation of Bacillus cereus and Pseudomonas putida using nanostructured ZnO thin film as transducer element

    International Nuclear Information System (INIS)

    Loukanov, Alexandre; Filipov, Chavdar; Valcheva, Violeta; Lecheva, Marta; Emin, Saim

    2015-01-01

    The semiconductor zinc oxide nanomaterial (ZnO or ZnO:H) is widely used in advanced biosensor technology for the design of highly-sensitive detector elements for various applications. In the attempt to evaluate its effect on common microorganisms, two types of nanostructured transducer films have been used (average diameter 600–1000 nm). They have been prepared by using both wet sol–gel method and magnetron sputtering. Their polycrystalline structure and specific surface features have been analyzed by X-ray diffraction (XRD), scanning electron microscope, and atomic force microscope. The assessment of growth stimulation of bacteria was determined using epifluorescent microscope by cell staining with Live/Dead BacLight kit. In our experiments, the growth stimulation of Gram-positive and Gram-negative bacteria on nanostructured ZnO film is demonstrated by Bacillus cereus and Pseudomonas putida. These two bacterial species have been selected, because they are well known and studied in biosensor technologies, with structural difference of their cell walls. These pathogens are easy for with common source in the liquid food or some commercial products. Our data has revealed that the method of transducer film preparation influences strongly bacterial inhibition and division. These results present the transforming signal precisely, when ZnO is used in biosensor applications

  18. Growth stimulation of Bacillus cereus and Pseudomonas putida using nanostructured ZnO thin film as transducer element

    Science.gov (United States)

    Loukanov, Alexandre; Filipov, Chavdar; Valcheva, Violeta; Lecheva, Marta; Emin, Saim

    2015-04-01

    The semiconductor zinc oxide nanomaterial (ZnO or ZnO:H) is widely used in advanced biosensor technology for the design of highly-sensitive detector elements for various applications. In the attempt to evaluate its effect on common microorganisms, two types of nanostructured transducer films have been used (average diameter 600-1000 nm). They have been prepared by using both wet sol-gel method and magnetron sputtering. Their polycrystalline structure and specific surface features have been analyzed by X-ray diffraction (XRD), scanning electron microscope, and atomic force microscope. The assessment of growth stimulation of bacteria was determined using epifluorescent microscope by cell staining with Live/Dead BacLight kit. In our experiments, the growth stimulation of Gram-positive and Gram-negative bacteria on nanostructured ZnO film is demonstrated by Bacillus cereus and Pseudomonas putida. These two bacterial species have been selected, because they are well known and studied in biosensor technologies, with structural difference of their cell walls. These pathogens are easy for with common source in the liquid food or some commercial products. Our data has revealed that the method of transducer film preparation influences strongly bacterial inhibition and division. These results present the transforming signal precisely, when ZnO is used in biosensor applications.

  19. Influence of the Hfq and Crc global regulators on the control of iron homeostasis in Pseudomonas putida.

    Science.gov (United States)

    Sánchez-Hevia, Dione L; Yuste, Luis; Moreno, Renata; Rojo, Fernando

    2018-04-30

    Metabolically versatile bacteria use catabolite repression control to select their preferred carbon sources, thus optimizing carbon metabolism. In pseudomonads, this occurs through the combined action of the proteins Hfq and Crc, which form stable tripartite complexes at target mRNAs, inhibiting their translation. The activity of Hfq/Crc is antagonised by small RNAs of the CrcZ family, the amounts of which vary according to carbon availability. The present work examines the role of Pseudomonas putida Hfq protein under conditions of low-level catabolite repression, in which Crc protein would have a minor role since it is sequestered by CrcZ/CrcY. The results suggest that, under these conditions, Hfq remains operative and plays an important role in iron homeostasis. In this scenario, Crc appears to participate indirectly by helping CrcZ/CrcY to control the amount of free Hfq in the cell. Iron homeostasis in pseudomonads relies on regulatory elements such as the Fur protein, the PrrF1-F2 sRNAs, and several extracytoplasmic sigma factors. Our results show that the absence of Hfq is paralleled by a reduction in PrrF1-F2 small RNAs. Hfq thus provides a regulatory link between iron and carbon metabolism, coordinating the iron supply to meet the needs of the enzymes operational under particular nutritional regimes. This article is protected by copyright. All rights reserved. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

  20. Growth kinetics, effect of carbon substrate in biosynthesis of mcl-PHA by Pseudomonas putida Bet001.

    Science.gov (United States)

    Gumel, A M; Annuar, M S M; Heidelberg, T

    2014-01-01

    Growth associated biosynthesis of medium chain length poly-3-hydroxyalkanoates (mcl-PHA) in Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. Models with substrate inhibition terms described well the kinetics of its growth. Selected fatty acids (C8:0 to C18:1) and ammonium were used as carbon and nitrogen sources during growth and PHA biosynthesis, resulting in PHA accumulation of about 50 to 69% (w/w) and PHA yields ranging from 10.12 g L(-1) to 15.45 g L(-1), respectively. The monomer composition of the PHA ranges from C4 to C14, and was strongly influenced by the type of carbon substrate fed. Interestingly, an odd carbon chain length (C7) monomer was also detected when C18:1 was fed. Polymer showed melting temperature (T m) of 42.0 (± 0.2) °C, glass transition temperature (T g) of -1.0 (± 0.2) °C and endothermic melting enthalpy of fusion (ΔHf) of 110.3 (± 0.1) J g(-1). The molecular weight (M w) range of the polymer was relatively narrow between 55 to 77 kDa.

  1. Growth kinetics, effect of carbon substrate in biosynthesis of mcl-PHA by Pseudomonas putida Bet001

    Directory of Open Access Journals (Sweden)

    A.M. Gumel

    2014-06-01

    Full Text Available Growth associated biosynthesis of medium chain length poly-3-hydroxyalkanoates (mcl-PHA in Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. Models with substrate inhibition terms described well the kinetics of its growth. Selected fatty acids (C8:0 to C18:1 and ammonium were used as carbon and nitrogen sources during growth and PHA biosynthesis, resulting in PHA accumulation of about 50 to 69% (w/w and PHA yields ranging from 10.12 g L-1 to 15.45 g L-1, respectively. The monomer composition of the PHA ranges from C4 to C14, and was strongly influenced by the type of carbon substrate fed. Interestingly, an odd carbon chain length (C7 monomer was also detected when C18:1 was fed. Polymer showed melting temperature (Tm of 42.0 (± 0.2 °C, glass transition temperature (Tg of -1.0 (± 0.2 °C and endothermic melting enthalpy of fusion (ΔHf of 110.3 (± 0.1 J g-1. The molecular weight (Mw range of the polymer was relatively narrow between 55 to 77 kDa.

  2. Directed evolution of toluene dioxygenase from Pseudomonas putida for improved selectivity toward cis-indandiol during indene bioconversion.

    Science.gov (United States)

    Zhang, N; Stewart, B G; Moore, J C; Greasham, R L; Robinson, D K; Buckland, B C; Lee, C

    2000-10-01

    Toluene dioxygenase (TDO) from Pseudomonas putida F1 converts indene to a mixture of cis-indandiol (racemic), 1-indenol, and 1-indanone. The desired product, cis-(1S,2R)-indandiol, is a potential key intermediate in the chemical synthesis of indinavir sulfate (Crixivan), Merck's HIV-1 protease inhibitor for the treatment of AIDS. To reduce the undesirable byproducts 1-indenol and 1-indanone formed during indene bioconversion, the recombinant TDO expressed in Escherichia coli was evolved by directed evolution using the error-prone polymerase chain reaction (epPCR) method. High-throughput fluorometric and spectrophotometric assays were developed for rapid screening of the mutant libraries in a 96-well format. Mutants with reduced 1-indenol by-product formation were identified, and the individual indene bioconversion product profiles of the selected mutants were confirmed by HPLC. Changes in the amino acid sequence of the mutant enzymes were identified by analyzing the nucleotide sequence of the genes. A mutant with the most desirable product profile from each library, defined as the most reduced 1-indenol concentration and with the highest cis-(1S,2R)-indandiol enantiomeric excess, was used to perform each subsequent round of mutagenesis. After three rounds of mutagenesis and screening, mutant 1C4-3G was identified to have a threefold reduction in 1-indenol formation over the wild type (20% vs 60% of total products) and a 40% increase of product (cis-indandiol) yield.

  3. Indole-based assay to assess the effect of ethanol on Pseudomonas putida F1 dioxygenase activity.

    Science.gov (United States)

    da Silva, Márcio Luis Busi; Alvarez, Pedro J J

    2010-06-01

    Toluene dioxygenase (TDO) is ubiquitous in nature and has a broad substrate range, including benzene, toluene, ethylbenzene and xylenes (BTEX). Pseudomonas putida F1 (PpF1) induced on toluene is known to produce indigo from indole through the activity of TDO. In this work, a spectrophotometric assay previously developed to measure indole to indigo production rates was modified to characterize the effects of various ethanol concentrations on toluene aerobic biodegradation activity and assess catabolite repression of TDO. Indigo production rate by cells induced on toluene alone was 0.0012 +/- 0.0006 OD(610) min(-1). The presence of ethanol did not fully repress TDO activity when toluene was also available as a carbon source. However, indigo production rates by PpF1 grown on ethanol:toluene mixtures (3:1 w/w) decreased by approximately 50%. Overall, the proposed spectrophotometric assay is a simple approach to quantify TDO activity, and demonstrates how the presence of ethanol in groundwater contaminated with reformulated gasoline is likely to interfere with naturally occurring microorganisms from fully expressing their aerobic catabolic potential towards hydrocarbons bioremediation.

  4. Frequent conjugative transfer accelerates adaptation of a broad-host-range plasmid to an unfavorable Pseudomonas putida host.

    Science.gov (United States)

    Heuer, Holger; Fox, Randal E; Top, Eva M

    2007-03-01

    IncP-1 plasmids are known to be promiscuous, but it is not understood if they are equally well adapted to various species within their host range. Moreover, little is known about their fate in bacterial communities. We determined if the IncP-1beta plasmid pB10 was unstable in some Proteobacteria, and whether plasmid stability was enhanced after long-term carriage in a single host and when regularly switched between isogenic hosts. Plasmid pB10 was found to be very unstable in Pseudomonas putida H2, and conferred a high cost (c. 20% decrease in fitness relative to the plasmid-free host). H2(pB10) was then evolved under conditions that selected for plasmid maintenance, with or without regular plasmid transfer (host-switching). When tested in the ancestral host, the evolved plasmids were more stable and their cost was significantly reduced (9% and 16% for plasmids from host-switched and nonswitched lineages, respectively). Our findings suggest that IncP-1 plasmids can rapidly adapt to an unfavorable host by improving their overall stability, and that regular conjugative transfer accelerates this process.

  5. Characterization and Genome Analysis of a Nicotine and Nicotinic Acid-Degrading Strain Pseudomonas putida JQ581 Isolated from Marine.

    Science.gov (United States)

    Li, Aiwen; Qiu, Jiguo; Chen, Dongzhi; Ye, Jiexu; Wang, Yuhong; Tong, Lu; Jiang, Jiandong; Chen, Jianmeng

    2017-05-31

    The presence of nicotine and nicotinic acid (NA) in the marine environment has caused great harm to human health and the natural environment. Therefore, there is an urgent need to use efficient and economical methods to remove such pollutants from the environment. In this study, a nicotine and NA-degrading bacterium-strain JQ581-was isolated from sediment from the East China Sea and identified as a member of Pseudomonas putida based on morphology, physio-biochemical characteristics, and 16S rDNA gene analysis. The relationship between growth and nicotine/NA degradation suggested that strain JQ581 was a good candidate for applications in the bioaugmentation treatment of nicotine/NA contamination. The degradation intermediates of nicotine are pseudooxynicotine (PN) and 3-succinoyl-pyridine (SP) based on UV, high performance liquid chromatography, and liquid chromatography-mass spectrometry analyses. However, 6-hydroxy-3-succinoyl-pyridine (HSP) was not detected. NA degradation intermediates were identified as 6-hydroxynicotinic acid (6HNA). The whole genome of strain JQ581 was sequenced and analyzed. Genome sequence analysis revealed that strain JQ581 contained the gene clusters for nicotine and NA degradation. This is the first report where a marine-derived Pseudomonas strain had the ability to degrade nicotine and NA simultaneously.

  6. Preparation and characterization of monodisperse microcapsules with alginate and bentonite via external gelation technique encapsulating Pseudomonas putida Rs-198.

    Science.gov (United States)

    Li, Xuan; Wu, Zhansheng; He, Yanhui; Ye, Bang-Ce; Wang, Jun

    2017-10-01

    This paper evaluated the external gelation technique for preparing microcapsules. The microcapsules were consisted of Pseudomonas putida Rs-198 (Rs-198) core and sodium alginate (NaAlg)-bentonite (Bent) shell. Different emulsification rotation speeds and core/shell ratios were used to prepare the microcapsules of each formulation. The near-spherical microcapsules were monodisperse with a mean diameter of 25-100 μm and wrinkled surfaces. Fourier transform infrared spectrophotometry (FTIR) and thermogravimetric analysis (TGA) revealed the physical mixture of the wall material and the superior thermal stability of the microcapsules. Percentage yield, water content, and encapsulation efficiency were evaluated and correlated with the changes in emulsification rotation speed and core/shell ratio. In vitro release experiments demonstrated that 60% of the bacteria were released from the NaAlg-Bent microcapsules within three days. Considerably better survival was observed for encapsulated cells compared to free cells, especially in pH 4.0 and 10.0. In summary, the desired properties of microcapsules can be obtained by external gelation technique and the microcapsules on the bacteria had a good protective effect.

  7. Effect of exopolymers on oxidative dissolution of natural rhodochrosite by Pseudomonas putida strain MnB1: An electrochemical study

    International Nuclear Information System (INIS)

    Wang, Huawei; Zhang, Daoyong; Song, Wenjuan; Pan, Xiangliang; Al-Misned, Fahad A.; Golam Mortuza, M.

    2015-01-01

    Highlights: • The biogeochemical behavior of natural rhodochrosite was investigated by electrochemical methods. • Bacterial exopolymers contributed to the increasing dissolution of natural rhodochrosite. • Oxidative dissolution of natural rhodochrosite was well explained by Tafel and EIS analysis. - Abstract: Oxidative dissolution of natural rhodochrosite by the Mn(II) oxidizing bacterium Pseudomonas putida strain MnB1 was investigated based on batch and electrochemical experiments using natural rhodochrosite as the working electrode. Tafel curves and batch experiments revealed that bacterial exopolymers (EPS) significantly increased dissolution of natural rhodochrosite. The corrosion current significantly increased with reaction time for EPS treatment. However, the corrosion process was blocked in the presence of cells plus extra EPS due to formation of the passivation layer. Moreover, the scanning electron microscopy and the energy dispersive spectroscopy (SEM–EDS) results showed that the surface of the natural rhodochrosite was notably changed in the presence of EPS alone or/and bacterial cells. This study is helpful for understanding the role of EPS in bacterially oxidation of Mn(II). It also indicates that the Mn(II) oxidizing bacteria may exert their effects on Mn(II) cycle and other biological and biogeochemical processes much beyond their local ambient environment because of the catalytically dissolution of solid Mn(II) by EPS and the possible long distance transport of the detached EPS

  8. Presence of albumin mRNA precursors in nuclei of analbuminemic rat liver lacking cytoplasmic albumin mRNA.

    OpenAIRE

    Esumi, H; Takahashi, Y; Sekiya, T; Sato, S; Nagase, S; Sugimura, T

    1982-01-01

    Analbuminemic rats, which lack serum albumin, were previously found to have no albumin mRNA in the cytoplasm of the liver. In the present study, the existence of nuclear albumin mRNA precursors in the liver of analbuminemic rats was examined by RNA X cDNA hybridization kinetics. Albumin mRNA precursors were present in the nuclei of analbuminemic rat liver at almost normal levels, despite the absence of albumin mRNA from the cytoplasm. Nuclear RNA of analbuminemic rat liver was subjected to el...

  9. The HSP90 inhibitor 17-AAG synergizes with doxorubicin and U0126 in anaplastic large cell lymphoma irrespective of ALK expression.

    Science.gov (United States)

    Georgakis, Georgios V; Li, Yang; Rassidakis, Georgios Z; Medeiros, L Jeffrey; Younes, Anas

    2006-12-01

    Heat shock protein 90 (HSP90) chaperones and maintains the molecular integrity of a variety of signal transduction proteins, including the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) oncogenic protein, a genetic abnormality that is frequently observed in anaplastic large cell lymphoma (ALCL) cells. Here we demonstrate that HSP90 is overexpressed in primary and cultured ALK-positive and ALK-negative ALCL cells, and we evaluate the potential role of the small molecule inhibitor of HSP90, 17-allylamino-17-demethoxygeldanamycin (17-AAG) in treating ALCL. The antiproliferative effect of 17-AAG-cultured cells was determined by MTS assay. Apoptosis and cell-cycle arrest were determined by Annexin-V/propidium iodide and propidium iodide staining, respectively, and fluorescein-activated cell sorting analysis. Expression of HSP90 was evaluated by immunohistochemistry, and molecular changes were determined by Western blot. Treatment of cultured ALCL cells with 17-AAG induced cell-cycle arrest and apoptosis, irrespective of ALK expression. At the molecular level, 17-AAG induced degradation of ALK and Akt proteins, dephosphorylated extracellular signal-regulated kinase, and degraded the cell-cycle regulatory protein cyclin D1 and its cyclin-dependent kinases, CDK4 and CDK6, but had a differential effect on p27 and p53 proteins. Inhibition of extracellular signal-regulated kinase phosphorylation by the mitogen activated protein kinase inhibitor U0126 induced cell death in all ALCL cell lines, and sublethal concentration 17-AAG showed synergistic antiproliferative effects when combined with U0126 or doxorubicin. Our data demonstrate that targeting HSP90 function by 17-AAG may offer a novel therapeutic strategy for ALCL, either as single-agent activity or by combining 17-AAG with conventional or targeted therapeutic schemes.

  10. Amphiregulin triggered epidermal growth factor receptor activation confers in vivo crizotinib-resistance of EML4-ALK lung cancer and circumvention by epidermal growth factor receptor inhibitors.

    Science.gov (United States)

    Taniguchi, Hirokazu; Takeuchi, Shinji; Fukuda, Koji; Nakagawa, Takayuki; Arai, Sachiko; Nanjo, Shigeki; Yamada, Tadaaki; Yamaguchi, Hiroyuki; Mukae, Hiroshi; Yano, Seiji

    2017-01-01

    Crizotinib, a first-generation anaplastic lymphoma kinase (ALK) tyrosine-kinase inhibitor, is known to be effective against echinoderm microtubule-associated protein-like 4 (EML4)-ALK-positive non-small cell lung cancers. Nonetheless, the tumors subsequently become resistant to crizotinib and recur in almost every case. The mechanism of the acquired resistance needs to be deciphered. In this study, we established crizotinib-resistant cells (A925LPE3-CR) via long-term administration of crizotinib to a mouse model of pleural carcinomatous effusions; this model involved implantation of the A925LPE3 cell line, which harbors the EML4-ALK gene rearrangement. The resistant cells did not have the secondary ALK mutations frequently occurring in crizotinib-resistant cells, and these cells were cross-resistant to alectinib and ceritinib as well. In cell clone #2, which is one of the clones of A925LPE3-CR, crizotinib sensitivity was restored via the inhibition of epidermal growth factor receptor (EGFR) by means of an EGFR tyrosine-kinase inhibitor (erlotinib) or an anti-EGFR antibody (cetuximab) in vitro and in the murine xenograft model. Cell clone #2 did not have an EGFR mutation, but the expression of amphiregulin (AREG), one of EGFR ligands, was significantly increased. A knockdown of AREG with small interfering RNAs restored the sensitivity to crizotinib. These data suggest that overexpression of EGFR ligands such as AREG can cause resistance to crizotinib, and that inhibition of EGFR signaling may be a promising strategy to overcome crizotinib resistance in EML4-ALK lung cancer. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  11. Crizotinib-Resistant ROS1 Mutations Reveal a Predictive Kinase Inhibitor Sensitivity Model for ROS1- and ALK-Rearranged Lung Cancers.

    Science.gov (United States)

    Facchinetti, Francesco; Loriot, Yohann; Kuo, Mei-Shiue; Mahjoubi, Linda; Lacroix, Ludovic; Planchard, David; Besse, Benjamin; Farace, Françoise; Auger, Nathalie; Remon, Jordi; Scoazec, Jean-Yves; André, Fabrice; Soria, Jean-Charles; Friboulet, Luc

    2016-12-15

    The identification of molecular mechanisms conferring resistance to tyrosine kinase inhibitor (TKI) is a key step to improve therapeutic results for patients with oncogene addiction. Several alterations leading to EGFR and anaplastic lymphoma kinase (ALK) resistance to TKI therapy have been described in non-small cell lung cancer (NSCLC). Only two mutations in the ROS1 kinase domain responsible for crizotinib resistance have been described in patients thus far. A patient suffering from a metastatic NSCLC harboring an ezrin (EZR)-ROS1 fusion gene developed acquired resistance to the ALK/ROS1 inhibitor crizotinib. Molecular analysis (whole-exome sequencing, CGH) and functional studies were undertaken to elucidate the mechanism of resistance. Based on this case, we took advantage of the structural homology of ROS1 and ALK to build a predictive model for drug sensitivity regarding future ROS1 mutations. Sequencing revealed a dual mutation, S1986Y and S1986F, in the ROS1 kinase domain. Functional in vitro studies demonstrated that ROS1 harboring either the S1986Y or the S1986F mutation, while conferring resistance to crizotinib and ceritinib, was inhibited by lorlatinib (PF-06463922). The patient's clinical response confirmed the potency of lorlatinib against S1986Y/F mutations. The ROS1 S1986Y/F and ALK C1156Y mutations are homologous and displayed similar sensitivity patterns to ALK/ROS1 TKIs. We extended this analogy to build a model predicting TKI efficacy against potential ROS1 mutations. Clinical evidence, in vitro validation, and homology-based prediction provide guidance for treatment decision making for patients with ROS1-rearranged NSCLC who progressed on crizotinib. Clin Cancer Res; 22(24); 5983-91. ©2016 AACR. ©2016 American Association for Cancer Research.

  12. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis

    DEFF Research Database (Denmark)

    Krakauer, Martin; Sorensen, P; Khademi, M

    2008-01-01

    BACKGROUND: Interferon (IFN)-beta therapy in multiple sclerosis (MS) has been suggested to promote a deviation from T lymphocyte production of pathogenic Th1 cytokines to less detrimental Th2 cytokines, but this is still controversial. We studied patterns of in vivo blood mononuclear cell (MNC...... of any Th1 or Th2 cytokines. The largest changes in cytokine mRNA levels occurred early (~9-12 h) after an IFN-beta injection. CONCLUSION: We found no evidence of a Th1- or Th2-mRNA-promoting effect of IFN-beta therapy. The therapeutic effect of IFN-beta is more likely attributable to the induction...

  13. Production of medium-chain-length polyhydroxyalkanoates by sequential feeding of xylose and octanoic acid in engineered Pseudomonas putida KT2440

    Directory of Open Access Journals (Sweden)

    Le Meur Sylvaine

    2012-08-01

    Full Text Available Abstract Background Pseudomonas putida KT2440 is able to synthesize large amounts of medium-chain-length polyhydroxyalkanoates (mcl-PHAs. To reduce the substrate cost, which represents nearly 50% of the total PHA production cost, xylose, a hemicellulose derivate, was tested as the growth carbon source in an engineered P. putida KT2440 strain. Results The genes encoding xylose isomerase (XylA and xylulokinase (XylB from Escherichia coli W3110 were introduced into P. putida KT2440. The recombinant KT2440 exhibited a XylA activity of 1.47 U and a XylB activity of 0.97 U when grown on a defined medium supplemented with xylose. The cells reached a maximum specific growth rate of 0.24 h-1 and a final cell dry weight (CDW of 2.5 g L-1 with a maximal yield of 0.5 g CDW g-1 xylose. Since no mcl-PHA was accumulated from xylose, mcl-PHA production can be controlled by the addition of fatty acids leading to tailor-made PHA compositions. Sequential feeding strategy was applied using xylose as the growth substrate and octanoic acid as the precursor for mcl-PHA production. In this way, up to 20% w w-1 of mcl-PHA was obtained. A yield of 0.37 g mcl-PHA per g octanoic acid was achieved under the employed conditions. Conclusions Sequential feeding of relatively cheap carbohydrates and expensive fatty acids is a practical way to achieve more cost-effective mcl-PHA production. This study is the first reported attempt to produce mcl-PHA by using xylose as the growth substrate. Further process optimizations to achieve higher cell density and higher productivity of mcl-PHA should be investigated. These scientific exercises will undoubtedly contribute to the economic feasibility of mcl-PHA production from renewable feedstock.

  14. The homogentisate pathway: a central catabolic pathway involved in the degradation of L-phenylalanine, L-tyrosine, and 3-hydroxyphenylacetate in Pseudomonas putida.

    Science.gov (United States)

    Arias-Barrau, Elsa; Olivera, Elías R; Luengo, José M; Fernández, Cristina; Galán, Beatriz; García, José L; Díaz, Eduardo; Miñambres, Baltasar

    2004-08-01

    Pseudomonas putida metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate. Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate. Whereas the phh, tyr, and hpd genes are not linked in the P. putida genome, the hmgABC genes appear to form a single transcriptional unit. Gel retardation assays and lacZ translational fusion experiments have shown that hmgR encodes a specific repressor that controls the inducible expression of the divergently transcribed hmgABC catabolic genes, and homogentisate is the inducer molecule. Footprinting analysis revealed that HmgR protects a region in the Phmg promoter that spans a 17-bp palindromic motif and an external direct repetition from position -16 to position 29 with respect to the transcription start site. The HmgR protein is thus the first IclR-type regulator that acts as a repressor of an aromatic catabolic pathway. We engineered a broad-host-range mobilizable catabolic cassette harboring the hmgABC, hpd, and tyrB genes that allows heterologous bacteria to use Tyr as a unique carbon and energy source. Remarkably, we show here that the catabolism of 3-hydroxyphenylacetate in P. putida U funnels also into the homogentisate central pathway, revealing that the hmg cluster is a key catabolic trait for biodegradation of a small number of aromatic compounds.

  15. Identification and characterization of genes encoding polycyclic aromatic hydrocarbon dioxygenase and polycyclic aromatic hydrocarbon dihydrodiol dehydrogenase in Pseudomonas putida OUS82.

    OpenAIRE

    Takizawa, N; Kaida, N; Torigoe, S; Moritani, T; Sawada, T; Satoh, S; Kiyohara, H

    1994-01-01

    Naphthalene and phenanthrene are transformed by enzymes encoded by the pah gene cluster of Pseudomonas putida OUS82. The pahA and pahB genes, which encode the first and second enzymes, dioxygenase and cis-dihydrodiol dehydrogenase, respectively, were identified and sequenced. The DNA sequences showed that pahA and pahB were clustered and that pahA consisted of four cistrons, pahAa, pahAb, pahAc, and pahAd, which encode ferredoxin reductase, ferredoxin, and two subunits of the iron-sulfur prot...

  16. BMP4 and BMP7 Suppress StAR and Progesterone Production via ALK3 and SMAD1/5/8-SMAD4 in Human Granulosa-Lutein Cells.

    Science.gov (United States)

    Zhang, Han; Klausen, Christian; Zhu, Hua; Chang, Hsun-Ming; Leung, Peter C K

    2015-11-01

    Adequate production of progesterone by the corpus luteum is critical to the successful establishment of pregnancy. In animal models, bone morphogenetic protein (BMP) 4 and BMP7 have been shown to suppress either basal or gonadotropin-induced progesterone production, depending on the species examined. However, the effects of BMP4 and BMP7 on progesterone production in human granulosa cells are unknown. In the present study, we used immortalized (SVOG) and primary human granulosa-lutein cells to investigate the effects of BMP4 and BMP7 on steroidogenic acute regulatory protein (StAR) expression and progesterone production and to examine the underlying molecular mechanism. Treatment of primary and immortalized human granulosa cells with recombinant BMP4 or BMP7 decreased StAR expression and progesterone accumulation. In SVOG cells, the suppressive effects of BMP4 and BMP7 on StAR expression were blocked by pretreatment with inhibitors of activin receptor-like kinase (ALK)2/3/6 (dorsomorphin) or ALK2/3 (DMH1) but not ALK4/5/7 (SB-431542). Moreover, small interfering RNA-mediated depletion of ALK3, but not ALK2 or ALK6, reversed the effects of BMP4 and BMP7 on StAR expression. Likewise, BMP4- and BMP7-induced phosphorylation of SMAD 1/5/8 was reversed by treatment with DMH1 or small interfering RNA targeting ALK3. Knockdown of SMAD4, the essential common SMAD for BMP/TGF-β signaling, abolished the effects of BMP4 and BMP7 on StAR expression. Our results suggest that BMP4 and BMP7 down-regulate StAR and progesterone production via ALK3 and SMAD1/5/8-SMAD4 signaling in human granulosa-lutein cells.

  17. Lung cancer mutation profile of EGFR, ALK, and KRAS: Meta-analysis and comparison of never and ever smokers.

    Science.gov (United States)

    Chapman, Aaron M; Sun, Kathie Y; Ruestow, Peter; Cowan, Dallas M; Madl, Amy K

    2016-12-01

    Lung cancer is the leading cause of cancer-related mortality. While the majority of lung cancers are associated with tobacco smoke, approximately 10-15% of U.S. lung cancers occur in never smokers. Evidence suggests that lung cancer in never smokers appears to be a distinct disease caused by driver mutations which are different than the genetic pathways observed with lung cancer in smokers. A meta-analysis of human epidemiologic data was conducted to evaluate the profile of common or therapy-targetable mutations in lung cancers of never and ever smokers. Epidemiologic studies (N=167) representing over 63,000 lung cancer cases were identified and used to calculate summary odds ratios for lung cancer in never and ever smokers containing gene mutations: EGFR, chromosomal rearrangements and fusion of EML4 and ALK, and KRAS. This analysis also considered the effect of histopathology, smoking status, sex, and ethnicity. There were significantly increased odds of presenting the EGFR and ALK-EML4 mutations in 1) adenocarcinomas compared to non-small cell lung cancer and 2) never smokers compared to ever smokers. The prevalence of EGFR mutations was higher in Asian women as compared to women of Caucasian/Mixed ethnicity. As the smoking history increased, there was a decreased odds for exhibiting the EGFR mutation, particularly for cases >30 pack-years. Compared to ever smokers, never smokers had a decreased odds of KRAS mutations among those of Caucasian/Mixed ethnicity (OR=0.22, 95% CI: 0.17-0.29) and those of Asian ethnicity (OR=0.39, 95% CI: 0.30-0.50). Our findings show that key driver mutations and several patient features are highly prevalent in lung cancers of never smokers. These associations may be helpful as patient demographic models are developed to predict successful outcomes of targeted therapeutic interventions NSCLC. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  18. Early pneumothorax as a feature of response to crizotinib therapy in a patient with ALK rearranged lung adenocarcinoma

    International Nuclear Information System (INIS)

    Gennatas, Spyridon; Stanway, Susana J; Thomas, Robert; Min, Toon; Shah, Riyaz; O’Brien, Mary ER; Popat, Sanjay

    2013-01-01

    Single arm phase 1 and 2 studies on Crizotinib in ALK-positive patients so far have shown rapid and durable responses. Spontaneous pneumothoraces as a result of response to anti-cancer therapy are rare in oncology but have been documented in a number of tumour types including lung cancer. This includes cytotoxic chemotherapy as well as molecular targeted agents such as gefitinib and Bevacizumab. These often require chest drain insertion or surgical intervention with associated morbidity and mortality. They have also been associated with response to treatment. This is the first report we are aware of documenting pneumothorax as response to crizotinib therapy. A 48-year-old Caucasian male presented with a Stage IV, TTF1 positive, EGFR wild-type adenocarcinoma of the lung. He received first line chemotherapy with three cycles of cisplatin-pemetrexed chemotherapy with a differential response, and then second-line erlotinib for two months before further radiological evidence of disease progression. Further analysis of his diagnostic specimen identified an ALK rearrangement by fluorescence in situ hybridization (FISH). He was commenced on crizotinib therapy 250 mg orally twice daily. At his 4-week assessment he had a chest radiograph that identified a large left-sided pneumothorax with disease response evident on the right. Chest CT confirmed a 50% left-sided pneumothorax on a background of overall disease response. A chest tube was inserted with complete resolution of the pneumothorax that did not recur following its removal. Our case demonstrates this potential complication of crizotinib therapy and we therefore recommend that pneumothorax be considered in patients on crizotinib presenting with high lung metastatic burden and with worsening dyspnoea

  19. Spatial Pattern of Copper Phosphate Precipitation Involves in Copper Accumulation and Resistance of Unsaturated Pseudomonas putida CZ1 Biofilm.

    Science.gov (United States)

    Chen, Guangcun; Lin, Huirong; Chen, Xincai

    2016-12-28

    Bacterial biofilms are spatially structured communities that contain bacterial cells with a wide range of physiological states. The spatial distribution and speciation of copper in unsaturated Pseudomonas putida CZ1 biofilms that accumulated 147.0 mg copper per g dry weight were determined by transmission electron microscopy coupled with energy dispersive X-ray analysis, and micro-X-ray fluorescence microscopy coupled with micro-X-ray absorption near edge structure (micro-XANES) analysis. It was found that copper was mainly precipitated in a 75 μm thick layer as copper phosphate in the middle of the biofilm, while there were two living cell layers in the air-biofilm and biofilm-medium interfaces, respectively, distinguished from the copper precipitation layer by two interfaces. The X-ray absorption fine structure analysis of biofilm revealed that species resembling Cu₃(PO₄)₂ predominated in biofilm, followed by Cu-Citrate- and Cu-Glutathione-like species. Further analysis by micro-XANES revealed that 94.4% of copper were Cu₃(PO₄)₂-like species in the layer next to the air interface, whereas the copper species of the layer next to the medium interface were composed by 75.4% Cu₃(PO₄)₂, 10.9% Cu-Citrate-like species, and 11.2% Cu-Glutathione-like species. Thereby, it was suggested that copper was initially acquired by cells in the biofilm-air interface as a citrate complex, and then transported out and bound by out membranes of cells, released from the copper-bound membranes, and finally precipitated with phosphate in the extracellular matrix of the biofilm. These results revealed a clear spatial pattern of copper precipitation in unsaturated biofilm, which was responsible for the high copper tolerance and accumulation of the biofilm.

  20. Biosynthesis and characterization of polyhydroxyalkanoates copolymers produced by Pseudomonas putida Bet001 isolated from palm oil mill effluent.

    Directory of Open Access Journals (Sweden)

    Ahmad Mohammed Gumel

    Full Text Available The biosynthesis and characterization of medium chain length poly-3-hydroxyalkanoates (mcl-PHA produced by Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. The biosynthesis of mcl-PHA in this newly isolated microorganism follows a growth-associated trend. Mcl-PHA accumulation ranging from 49.7 to 68.9% on cell dry weight (CDW basis were observed when fatty acids ranging from octanoic acid (C(8:0 to oleic acid (C(18:1 were used as sole carbon and energy source. Molecular weight of the polymer was found to be ranging from 55.7 to 77.7 kDa. Depending on the type of fatty acid used, the (1H NMR and GCMSMS analyses of the chiral polymer showed a composition of even and odd carbon atom chain with monomer length of C4 to C14 with C8 and C10 as the principal monomers. No unsaturated monomer was detected. Thermo-chemical analyses showed the accumulated PHA to be semi-crystalline polymer with good thermal stability, having a thermal degradation temperature (T(d of 264.6 to 318.8 (± 0.2 (oC, melting temperature (T(m of 43. (± 0.2 (oC, glass transition temperature (T(g of -1.0 (± 0.2 (oC and apparent melting enthalpy of fusion (ΔH(f of 100.9 (± 0.1 J g(-1.

  1. Biosynthesis and characterization of polyhydroxyalkanoates copolymers produced by Pseudomonas putida Bet001 isolated from palm oil mill effluent.

    Science.gov (United States)

    Gumel, Ahmad Mohammed; Annuar, Mohamad Suffian Mohamad; Heidelberg, Thorsten

    2012-01-01

    The biosynthesis and characterization of medium chain length poly-3-hydroxyalkanoates (mcl-PHA) produced by Pseudomonas putida Bet001 isolated from palm oil mill effluent was studied. The biosynthesis of mcl-PHA in this newly isolated microorganism follows a growth-associated trend. Mcl-PHA accumulation ranging from 49.7 to 68.9% on cell dry weight (CDW) basis were observed when fatty acids ranging from octanoic acid (C(8:0)) to oleic acid (C(18:1)) were used as sole carbon and energy source. Molecular weight of the polymer was found to be ranging from 55.7 to 77.7 kDa. Depending on the type of fatty acid used, the (1)H NMR and GCMSMS analyses of the chiral polymer showed a composition of even and odd carbon atom chain with monomer length of C4 to C14 with C8 and C10 as the principal monomers. No unsaturated monomer was detected. Thermo-chemical analyses showed the accumulated PHA to be semi-crystalline polymer with good thermal stability, having a thermal degradation temperature (T(d)) of 264.6 to 318.8 (± 0.2) (o)C, melting temperature (T(m)) of 43. (± 0.2) (o)C, glass transition temperature (T(g)) of -1.0 (± 0.2) (o)C and apparent melting enthalpy of fusion (ΔH(f)) of 100.9 (± 0.1) J g(-1).

  2. Genetic analysis of plant endophytic Pseudomonas putida BP25 and chemo-profiling of its antimicrobial volatile organic compounds.

    Science.gov (United States)

    Sheoran, Neelam; Valiya Nadakkakath, Agisha; Munjal, Vibhuti; Kundu, Aditi; Subaharan, Kesavan; Venugopal, Vibina; Rajamma, Suseelabhai; Eapen, Santhosh J; Kumar, Aundy

    2015-04-01

    Black pepper associated bacterium BP25 was isolated from root endosphere of apparently healthy cultivar Panniyur-5 that protected black pepper against Phytophthora capsici and Radopholus similis - the major production constraints. The bacterium was characterized and mechanisms of its antagonistic action against major pathogens are elucidated. The polyphasic phenotypic analysis revealed its identity as Pseudomonas putida. Multi locus sequence typing revealed that the bacterium shared gene sequences with several other isolates representing diverse habitats. Tissue localization assays exploiting green fluorescence protein expression clearly indicated that PpBP25 endophytically colonized not only its host plant - black pepper, but also other distantly related plants such as ginger and arabidopsis. PpBP25 colonies could be enumerated from internal tissues of plants four weeks post inoculation indicated its stable establishment and persistence in the plant system. The bacterium inhibited broad range of pathogens such as Phytophthora capsici, Pythium myriotylum, Giberella moniliformis, Rhizoctonia solani, Athelia rolfsii, Colletotrichum gloeosporioides and plant parasitic nematode, Radopholus similis by its volatile substances. GC/MS based chemical profiling revealed presence of Heneicosane; Tetratetracontane; Pyrrolo [1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl); Tetracosyl heptafluorobutyrate; 1-3-Eicosene, (E)-; 1-Heneicosanol; Octadecyl trifluoroacetate and 1-Pentadecene in PpBP25 metabolite. Dynamic head space GC/MS analysis of airborne volatiles indicated the presence of aromatic compounds such as 1-Undecene;Disulfide dimethyl; Pyrazine, methyl-Pyrazine, 2,5-dimethyl-; Isoamyl alcohol; Pyrazine, methyl-; Dimethyl trisulfide, etc. The work paved way for profiling of broad spectrum antimicrobial VOCs in endophytic PpBP25 for crop protection. Copyright © 2015 Elsevier GmbH. All rights reserved.

  3. Genetic Dissection of the Regulatory Network Associated with High C-di-GMP Levels in Pseudomonas putida KT2440

    Directory of Open Access Journals (Sweden)

    María Isabel Ramos-González

    2016-07-01

    Full Text Available Most bacteria grow in nature forming multicellular structures named biofilms. The bacterial second messenger cyclic diguanosine monophosphate (c-di-GMP is a key player in the regulation of the transition from planktonic to sessile lifestyles and this regulation is crucial in the development of biofilms. In Pseudomonas putida KT2440, Rup4959, a multidomain response regulator with diguanylate cyclase activity, when overexpressed causes an increment in the intracellular levels of c-di-GMP that gives rise to a pleiotropic phenotype consisting of increased biofilm formation and crinkly colony morphology. In a broad genomic screen we have isolated mutant derivatives that lose the crinkly morphology, designed as cfc (crinkle free colony. A total of nineteen different genes have been identified as being related with the emergence of the cfc phenotype either because the expression or functionality of Rup4959 is compromised, or due to a lack of transduction of the c-di-GMP signal to downstream elements involved in the acquisition of the phenotype. Discernment between these possibilities was investigated by using a c-di-GMP biosensor and by HPLC-MS quantification of the second messenger. Interestingly five of the identified genes encode proteins with AAA+ ATPase domain. Among the bacterial determinants found in this screen are the global transcriptional regulators GacA, AlgU and FleQ and two enzymes involved in the arginine biosynthesis pathway. We present evidences that this pathway seems to be an important element to both the availability of the free pool of the second messenger c-di-GMP and to its further transduction as a signal for biosynthesis of biopolimers. In addition we have identified an uncharacterized hybrid sensor histidine kinase whose phosphoaceptor conserved histidine residue has been shown in this work to be required for in vivo activation of the orphan response regulator Rup4959, which suggests these two elements constitute a two

  4. Cra regulates the cross-talk between the two branches of the phosphoenolpyruvate : phosphotransferase system of Pseudomonas putida.

    Science.gov (United States)

    Chavarría, Max; Fuhrer, Tobias; Sauer, Uwe; Pflüger-Grau, Katharina; de Lorenzo, Víctor

    2013-01-01

    The gene that encodes the catabolite repressor/activator, Cra (FruR), of Pseudomonas putida is divergent from the fruBKA operon for the uptake of fructose via the phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS(Fru)). The expression of the fru cluster has been studied in cells growing on substrates that change the intracellular concentrations of fructose-1-P (F1P), the principal metabolic intermediate that counteracts the DNA-binding ability of Cra on an upstream operator. While the levels of the regulator were not affected by any of the growth conditions tested, the transcription of fruB was stimulated by fructose but not by the gluconeogenic substrate, succinate. The analysis of the P(fruB) promoter activity in a strain lacking the Cra protein and the determination of key metabolites revealed that this regulator represses the expression of PTS(Fru) in a fashion that is dependent on the endogenous concentrations of F1P. Because FruB (i.e. the EI-HPr-EIIA(Fru) polyprotein) can deliver a high-energy phosphate to the EIIA(Ntr) (PtsN) enzyme of the PTS(Ntr) branch, the cross-talk between the two phosphotransferase systems was examined under metabolic regimes that allowed for the high or low transcription of the fruBKA operon. While fructose caused cross-talk, succinate prevented it almost completely. Furthermore, PtsN phosphorylation by FruB occurred in a Δcra mutant regardless of growth conditions. These results traced the occurrence of the cross-talk to intracellular pools of Cra effectors, in particular F1P. The Cra/F1P duo seems to not only control the expression of the PTS(Fru) but also checks the activity of the PTS(Ntr) in vivo. © 2012 Society for Applied Microbiology and Blackwell Publishing Ltd.

  5. Two Distinct Pathways for Metabolism of Theophylline and Caffeine Are Coexpressed in Pseudomonas putida CBB5▿ †

    Science.gov (United States)

    Yu, Chi Li; Louie, Tai Man; Summers, Ryan; Kale, Yogesh; Gopishetty, Sridhar; Subramanian, Mani

    2009-01-01

    Pseudomonas putida CBB5 was isolated from soil by enrichment on caffeine. This strain used not only caffeine, theobromine, paraxanthine, and 7-methylxanthine as sole carbon and nitrogen sources but also theophylline and 3-methylxanthine. Analyses of metabolites in spent media and resting cell suspensions confirmed that CBB5 initially N demethylated theophylline via a hitherto unreported pathway to 1- and 3-methylxanthines. NAD(P)H-dependent conversion of theophylline to 1- and 3-methylxanthines was also detected in the crude cell extracts of theophylline-grown CBB5. 1-Methylxanthine and 3-methylxanthine were subsequently N demethylated to xanthine. CBB5 also oxidized theophylline and 1- and 3-methylxanthines to 1,3-dimethyluric acid and 1- and 3-methyluric acids, respectively. However, these methyluric acids were not metabolized further. A broad-substrate-range xanthine-oxidizing enzyme was responsible for the formation of these methyluric acids. In contrast, CBB5 metabolized caffeine to theobromine (major metabolite) and paraxanthine (minor metabolite). These dimethylxanthines were further N demethylated to xanthine via 7-methylxanthine. Theobromine-, paraxanthine-, and 7-methylxanthine-grown cells also metabolized all of the methylxanthines mentioned above via the same pathway. Thus, the theophylline and caffeine N-demethylation pathways converged at xanthine via different methylxanthine intermediates. Xanthine was eventually oxidized to uric acid. Enzymes involved in theophylline and caffeine degradation were coexpressed when CBB5 was grown on theophylline or on caffeine or its metabolites. However, 3-methylxanthine-grown CBB5 cells did not metabolize caffeine, whereas theophylline was metabolized at much reduced levels to only methyluric acids. To our knowledge, this is the first report of theophylline N demethylation and coexpression of distinct pathways for caffeine and theophylline degradation in bacteria. PMID:19447909

  6. Root inoculation with Pseudomonas putida KT2440 induces transcriptional and metabolic changes and systemic resistance in maize plants

    Directory of Open Access Journals (Sweden)

    Chantal ePlanchamp

    2015-01-01

    Full Text Available Pseudomonas putida KT2440 (KT2440 rhizobacteria colonize a wide range of plants. They have been extensively studied for their capacity to adhere to maize seeds, to tolerate toxic secondary metabolites produced by maize roots and to be attracted by maize roots. However, the response of maize plants to KT2440 colonization has not been investigated yet. Maize roots were inoculated with KT2440 and the local (roots and systemic (leaves early plant responses were investigated. The colonization behavior of KT2440 following application to maize seedlings was investigated and transcriptional analysis of stress- and defense-related genes as well as metabolite profiling of local and systemic maize tissues of KT2440-inoculated were performed. The local and systemic responses differed and more pronounced changes were observed in roots compared to leaves. Early in the interaction roots responded via jasmonic acid- and abscisic acid-dependent signaling. Interestingly, during later steps, the salicylic acid pathway was suppressed. Metabolite profiling revealed the importance of plant phospholipids in KT2440-maize interactions. An additional important maize secondary metabolite, a form of benzoxazinone, was also found to be differently abundant in roots three days after KT2440 inoculation. However, the transcriptional and metabolic changes observed in bacterized plants early during the interaction were minor and became even less pronounced with time, indicating an accommodation state of the plant to the presence of KT2440. Since the maize plants reacted to the presence of KT2440 in the rhizosphere, we also investigated the ability of these bacteria to trigger induced systemic resistance (ISR against the maize anthracnose fungus Colletotrichum graminicola. The observed resistance was expressed as strongly reduced leaf necrosis and fungal development in infected bacterized plants compared to non-bacterized controls, showing the potential of KT2440 to act as

  7. Full-length mRNA sequencing uncovers a widespread coupling between transcription initiation and mRNA processing.

    Science.gov (United States)

    Anvar, Seyed Yahya; Allard, Guy; Tseng, Elizabeth; Sheynkman, Gloria M; de Klerk, Eleonora; Vermaat, Martijn; Yin, Raymund H; Johansson, Hans E; Ariyurek, Yavuz; den Dunnen, Johan T; Turner, Stephen W; 't Hoen, Peter A C

    2018-03-29

    The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription initiation, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing. In MCF-7 breast cancer cells, we find 2700 genes with interdependent alternative transcription initiation, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules, including examples of coupling between transcription start sites and polyadenylation sites. The analysis of three human primary tissues (brain, heart and liver) reveals similar patterns of interdependency between transcription initiation and mRNA processing events. We predict thousands of novel open reading frames from full-length mRNA sequences and obtained evidence for their translation by shotgun proteomics. The mapping database rescues 358 previously unassigned peptides and improves the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improves proteogenomics analysis of MCF-7 cells. Our findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription initiation and mRNA processing.

  8. Expression of calmodulin mRNA in rat olfactory neuroepithelium.

    Science.gov (United States)

    Biffo, S; Goren, T; Khew-Goodall, Y S; Miara, J; Margolis, F L

    1991-04-01

    A calmodulin (CaM) cDNA was isolated by differential hybridization screening of a lambda gt10 library prepared from rat olfactory mucosa. This cDNA fragment, containing most of the open reading frame of the rat CaMI gene, was subcloned and used to characterize steady-state expression of CaM mRNA in rat olfactory neuroepithelium and bulb. Within the bulb mitral cells are the primary neuronal population expressing CaM mRNA. The major CaM mRNA expressed in the olfactory mucosa is 1.7 kb with smaller contributions from mRNAs of 4.0 and 1.4 kb. CaM mRNA was primarily associated with the olfactory neurons and, despite the cellular complexity of the tissue and the known involvement of CaM in diverse cellular processes, was only minimally evident in sustentacular cells, gland cells or respiratory epithelium. Following bulbectomy CaM mRNA declines in the olfactory neuroepithelium as does olfactory marker protein (OMP) mRNA. In contrast to the latter, CaM mRNA makes a partial recovery by one month after surgery. These results, coupled with those from in situ hybridization, indicate that CaM mRNA is expressed in both mature and immature olfactory neurons. The program regulating CaM gene expression in olfactory neurons is distinct from those controlling expression of B50/GAP43 in immature, or OMP in mature, neurons respectively.

  9. Toxicity of fungal-generated silver nanoparticles to soil-inhabiting Pseudomonas putida KT2440, a rhizospheric bacterium responsible for plant protection and bioremediation

    International Nuclear Information System (INIS)

    Gupta, Indarchand R.; Anderson, Anne J.; Rai, Mahendra

    2015-01-01

    Highlights: • This study incorporates the mycosynthesis of AgNPs and their characterisation by various methods. • A first attempt demonstrating the toxicity assessment of AgNPs on beneficial soil microbe. • Use of biosensor in Pseudomonas putida KT2440, gave accurate antimicrobial results. - Abstract: Silver nanoparticles have attracted considerable attention due to their beneficial properties. But toxicity issues associated with them are also rising. The reports in the past suggested health hazards of silver nanoparticles at the cellular, molecular, or whole organismal level in eukaryotes. Whereas, there is also need to examine the exposure effects of silver nanoparticle to the microbes, which are beneficial to humans as well as environment. The available literature suggests the harmful effects of physically and chemically synthesised silver nanoparticles. The toxicity of biogenically synthesized nanoparticles has been less studied than physically and chemically synthesised nanoparticles. Hence, there is a greater need to study the toxic effects of biologically synthesised silver nanoparticles in general and mycosynthesized nanoparticles in particular. In the present study, attempts have been made to assess the risk associated with the exposure of mycosynthesized silver nanoparticles on a beneficial soil microbe Pseudomonas putida. KT2440. The study demonstrates mycosynthesis of silver nanoparticles and their characterisation by UV–vis spectrophotometry, FTIR, X-ray diffraction, nanosight LM20 – a particle size distribution analyzer and TEM. Silver nanoparticles obtained herein were found to exert the hazardous effect at the concentration of 0.4 μg/ml, which warrants further detailed investigations concerning toxicity

  10. The complete genomic sequence of lytic bacteriophage gh-1 infecting Pseudomonas putida--evidence for close relationship to the T7 group

    International Nuclear Information System (INIS)

    Kovalyova, Irina V.; Kropinski, Andrew M.

    2003-01-01

    The genome of the lytic Pseudomonas putida bacteriophage gh-1 is linear double-stranded DNA containing 37,359 bp with 216-bp direct terminal repeats. Like other members of the T7 group, the gh-1 genome contains regions of high homology to T7 interspersed with nonhomologous regions that contain small open reading frames of unknown function. The genome shares 31 genes in common with other members of the T7 group, including RNA polymerase, and an additional 12 unique putative genes. A major difference between gh-1 and other members of this group is the absence of any open reading frames between the left direct terminal repeat and gene 1. Sequence analysis of the gh-1 genome also revealed the presence of 10 putative phage promoters with a consensus sequence similar to the promoters of T3 and phiYeO3-12 (consensus: TAAAAACCCTCACTRTGGCHSCM). P. putida mutants resistant to gh-1 were demonstrated to have an altered lipopolysaccharide structure, indicating that members of this group use lipopolysaccharide as their cellular receptor

  11. Microcalorimetric and potentiometric titration studies on the adsorption of copper by P. putida and B. thuringiensis and their composites with minerals

    International Nuclear Information System (INIS)

    Fang Linchuan; Cai Peng; Li Pengxiang; Wu Huayong; Liang Wei; Rong Xingmin; Chen Wenli; Huang Qiaoyun

    2010-01-01

    In order to have a better understanding of the interactions of heavy metals with bacteria and minerals in soil and associated environments, isothermal titration calorimetry (ITC), potentiometric titration and equilibrium sorption experiments were conducted to investigate the adsorption behavior of Cu(II) by Bacillus thuringiensis, Pseudomonas putida and their composites with minerals. The interaction of montmorillonite with bacteria increased the reactive sites and resulted in greater adsorption for Cu(II) on their composites, while decreased adsorption sites and capacities for Cu(II) were observed on goethite-bacteria composites. A gram-positive bacterium B. thuringiensis played a more important role than a gram-negative bacterium P. putida in determining the properties of the bacteria-minerals interfaces. The enthalpy changes (ΔH ads ) from endothermic (6.14 kJ mol -1 ) to slightly exothermic (-0.78 kJ mol -1 ) suggested that Cu(II) is complexed with the anionic oxygen ligands on the surface of bacteria-mineral composites. Large entropies (32.96-58.89 J mol -1 K -1 ) of Cu(II) adsorption onto bacteria-mineral composites demonstrated the formation of inner-sphere complexes in the presence of bacteria. The thermodynamic data implied that Cu(II) mainly bound to the carboxyl and phosphoryl groups as inner-sphere complexes on bacteria and mineral-bacteria composites.

  12. Toxicity of fungal-generated silver nanoparticles to soil-inhabiting Pseudomonas putida KT2440, a rhizospheric bacterium responsible for plant protection and bioremediation

    Energy Technology Data Exchange (ETDEWEB)

    Gupta, Indarchand R. [Nanobiotechnology Laboratory, Department of Biotechnology, S.G.B. Amravati University, Amravati 444602, Maharashtra (India); Department of Biotechnology, Institute of Science, Nipat Niranjan Nagar, Caves Road, Aurangabad 431004, Maharashtra (India); Anderson, Anne J. [Department of Biology, Utah State University, Logan, Utah 84321 (United States); Rai, Mahendra, E-mail: mahendrarai@sgbau.ac.in [Nanobiotechnology Laboratory, Department of Biotechnology, S.G.B. Amravati University, Amravati 444602, Maharashtra (India); Laboratório de Química Biológica, Instituto de Química, UNICAMP, Cidade Universitária “Zefferino Vaz” Barão Geraldo, CEP 13083-970, Caixa Postal 6150, Campinas, SP (Brazil)

    2015-04-09

    Highlights: • This study incorporates the mycosynthesis of AgNPs and their characterisation by various methods. • A first attempt demonstrating the toxicity assessment of AgNPs on beneficial soil microbe. • Use of biosensor in Pseudomonas putida KT2440, gave accurate antimicrobial results. - Abstract: Silver nanoparticles have attracted considerable attention due to their beneficial properties. But toxicity issues associated with them are also rising. The reports in the past suggested health hazards of silver nanoparticles at the cellular, molecular, or whole organismal level in eukaryotes. Whereas, there is also need to examine the exposure effects of silver nanoparticle to the microbes, which are beneficial to humans as well as environment. The available literature suggests the harmful effects of physically and chemically synthesised silver nanoparticles. The toxicity of biogenically synthesized nanoparticles has been less studied than physically and chemically synthesised nanoparticles. Hence, there is a greater need to study the toxic effects of biologically synthesised silver nanoparticles in general and mycosynthesized nanoparticles in particular. In the present study, attempts have been made to assess the risk associated with the exposure of mycosynthesized silver nanoparticles on a beneficial soil microbe Pseudomonas putida. KT2440. The study demonstrates mycosynthesis of silver nanoparticles and their characterisation by UV–vis spectrophotometry, FTIR, X-ray diffraction, nanosight LM20 – a particle size distribution analyzer and TEM. Silver nanoparticles obtained herein were found to exert the hazardous effect at the concentration of 0.4 μg/ml, which warrants further detailed investigations concerning toxicity.

  13. Genetically engineered Pseudomonas putida X3 strain and its potential ability to bioremediate soil microcosms contaminated with methyl parathion and cadmium.

    Science.gov (United States)

    Zhang, Rong; Xu, Xingjian; Chen, Wenli; Huang, Qiaoyun

    2016-02-01

    A multifunctional Pseudomonas putida X3 strain was successfully engineered by introducing methyl parathion (MP)-degrading gene and enhanced green fluorescent protein (EGFP) gene in P. putida X4 (CCTCC: 209319). In liquid cultures, the engineered X3 strain utilized MP as sole carbon source for growth and degraded 100 mg L(-1) of MP within 24 h; however, this strain did not further metabolize p-nitrophenol (PNP), an intermediate metabolite of MP. No discrepancy in minimum inhibitory concentrations (MICs) to cadmium (Cd), copper (Cu), zinc (Zn), and cobalt (Co) was observed between the engineered X3 strain and its host strain. The inoculated X3 strain accelerated MP degradation in different polluted soil microcosms with 100 mg MP kg(-1) dry soil and/or 5 mg Cd kg(-1) dry soil; MP was completely eliminated within 40 h. However, the presence of Cd in the early stage of remediation slightly delayed MP degradation. The application of X3 strain in Cd-contaminated soil strongly affected the distribution of Cd fractions and immobilized Cd by reducing bioavailable Cd concentrations with lower soluble/exchangeable Cd and organic-bound Cd. The inoculated X3 strain also colonized and proliferated in various contaminated microcosms. Our results suggested that the engineered X3 strain is a potential bioremediation agent showing competitive advantage in complex contaminated environments.

  14. The Crc/CrcZ-CrcY global regulatory system helps the integration of gluconeogenic and glycolytic metabolism in Pseudomonas putida.

    Science.gov (United States)

    La Rosa, Ruggero; Nogales, Juan; Rojo, Fernando

    2015-09-01

    In metabolically versatile bacteria, carbon catabolite repression (CCR) facilitates the preferential assimilation of the most efficient carbon sources, improving growth rates and fitness. In Pseudomonas putida, the Crc and Hfq proteins and the CrcZ and CrcY small RNAs, which are believed to antagonize Crc/Hfq, are key players in CCR. Unlike that seen in other bacterial species, succinate and glucose elicit weak CCR in this bacterium. In the present work, metabolic, transcriptomic and constraint-based metabolic flux analyses were combined to clarify whether P. putida prefers succinate or glucose, and to identify the role of the Crc protein in the metabolism of these compounds. When provided simultaneously, succinate was consumed faster than glucose, although both compounds were metabolized. CrcZ and CrcY levels were lower when both substrates were present than when only one was provided, suggesting a role for Crc in coordinating metabolism of these compounds. Flux distribution analysis suggested that, when both substrates are present, Crc works to organize a metabolism in which carbon compounds flow in opposite directions: from glucose to pyruvate, and from succinate to pyruvate. Thus, our results support that Crc not only favours the assimilation of preferred compounds, but balances carbon fluxes, optimizing metabolism and growth. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

  15. Multiple Hfq-Crc target sites are required to impose catabolite repression on (methyl)phenol metabolism in Pseudomonas putida CF600.

    Science.gov (United States)

    Wirebrand, Lisa; Madhushani, Anjana W K; Irie, Yasuhiko; Shingler, Victoria

    2018-01-01

    The dmp-system encoded on the IncP-2 pVI150 plasmid of Pseudomonas putida CF600 confers the ability to assimilate (methyl)phenols. Regulation of the dmp-genes is subject to sophisticated control, which includes global regulatory input to subvert expression of the pathway in the presence of preferred carbon sources. Previously we have shown that in P. putida, translational inhibition exerted by the carbon repression control protein Crc operates hand-in-hand with the RNA chaperon protein Hfq to reduce translation of the DmpR regulator of the Dmp-pathway. Here, we show that Crc and Hfq co-target four additional sites to form riboprotein complexes within the proximity of the translational initiation sites of genes encoding the first two steps of the Dmp-pathway to mediate two-layered control in the face of selection of preferred substrates. Furthermore, we present evidence that Crc plays a hitherto unsuspected role in maintaining the pVI150 plasmid within a bacterial population, which has implications for (methyl)phenol degradation and a wide variety of other physiological processes encoded by the IncP-2 group of Pseudomonas-specific mega-plasmids. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

  16. Two small RNAs, CrcY and CrcZ, act in concert to sequester the Crc global regulator in Pseudomonas putida, modulating catabolite repression.

    Science.gov (United States)

    Moreno, Renata; Fonseca, Pilar; Rojo, Fernando

    2012-01-01

    The Crc protein is a translational repressor that recognizes a specific target at some mRNAs, controlling catabolite repression and co-ordinating carbon metabolism in pseudomonads. In Pseudomonas aeruginosa, the levels of free Crc protein are controlled by CrcZ, a sRNA that sequesters Crc, acting as an antagonist. We show that, in Pseudomonas putida, the levels of free Crc are controlled by CrcZ and by a novel 368 nt sRNA named CrcY. CrcZ and CrcY, which contain six potential targets for Crc, were able to bind Crc specifically in vitro. The levels of CrcZ and CrcY were low under conditions generating a strong catabolite repression, and increased strongly when catabolite repression was absent. Deletion of either crcZ or crcY had no effect on catabolite repression, but the simultaneous absence of both sRNAs led to constitutive catabolite repression that compromised growth on some carbon sources. Overproduction of CrcZ or CrcY significantly reduced repression. We propose that CrcZ and CrcY act in concert, sequestering and modulating the levels of free Crc according to metabolic conditions. The CbrA/CbrB two-component system activated crcZ transcription, but had little effect on crcY. CrcY was detected in P. putida, Pseudomonas fluorescens and Pseudomonas syringae, but not in P. aeruginosa. © 2011 Blackwell Publishing Ltd.

  17. In situ X-ray data collection from highly sensitive crystals of Pseudomonas putida PtxS in complex with DNA

    International Nuclear Information System (INIS)

    Pineda-Molina, E.; Daddaoua, A.; Krell, T.; Ramos, J. L.; García-Ruiz, J. M.; Gavira, J. A.

    2012-01-01

    The crystallization of both native P. putida transcriptional regulator PtxS and its complex with its DNA recognition sequence using the counter-diffusion method are reported. Pseudomonas putida PtxS is a member of the LacI protein family of transcriptional regulators involved in glucose metabolism. All genes involved in this pathway are clustered into two operons, kgu and gad. PtxS controls the expression of the kgu and gad operons as well as its own transcription. The PtxS operator is a perfect palindrome, 5′-TGAAACCGGTTTCA-3′, which is present in all three promoters. Crystallization of native PtxS failed, and PtxS–DNA crystals were finally produced by the counter-diffusion technique. A portion of the capillary used for crystal growth was attached to the end of a SPINE standard cap and directly flash-cooled in liquid nitrogen for diffraction tests. A full data set was collected with a beam size of 10 × 10 µm. The crystal belonged to the trigonal space group P3, with unit-cell parameters a = b = 213.71, c = 71.57 Å. Only unhandled crystals grown in capillaries of 0.1 mm inner diameter diffracted X-rays to 1.92 Å resolution

  18. ABILITY OF BACTERIAL CONSORTIUM: Bacillus coagulans, Bacilus licheniformis, Bacillus pumilus, Bacillus subtilis, Nitrosomonas sp. and Pseudomonas putida IN BIOREMEDIATION OF WASTE WATER IN CISIRUNG WASTE WATER TREATMENT PLANT

    Directory of Open Access Journals (Sweden)

    Ratu SAFITRI

    2015-10-01

    Full Text Available This study was conducted in order to determine the ability of bacterial consortium: Bacillus coagulans, Bacilus licheniformis, Bacillus pumilus, Bacillus subtilis, Nitrosomonas sp., and Pseudomonas putida in bioremediation of wastewater origin Cisirung WWTP. This study uses an experimental method completely randomized design (CRD, which consists of two treatment factors (8x8 factorial design. The first factor is a consortium of bacteria (K, consisting of 8 level factors (k1, k2, k3, k4, k5, k6, k7, and k8. The second factor is the time (T, consisting of a 7 level factors (t0, t1, t2, t3, t4, t5, t6, and t7. Test parameters consist of BOD (Biochemical Oxygen Demand, COD (Chemical Oxygen Demand, TSS (Total Suspended Solid, Ammonia and Population of Microbes during bioremediation. Data were analyzed by ANOVA, followed by Duncan test. The results of this study showed that the consortium of Bacillus pumilus, Bacillus subtilis, Bacillus coagulans, Nitrosomonas sp., and Pseudomonas putida with inoculum concentration of 5% (k6 is a consortium of the most effective in reducing BOD 71.93%, 64.30% COD, TSS 94.85%, and 88.58% of ammonia.

  19. Microcalorimetric and potentiometric titration studies on the adsorption of copper by P. putida and B. thuringiensis and their composites with minerals

    Energy Technology Data Exchange (ETDEWEB)

    Fang Linchuan [State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070 (China); Key Laboratory of Subtropical Agriculture and Environment, Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070 (China); Cai Peng; Li Pengxiang; Wu Huayong; Liang Wei; Rong Xingmin [Key Laboratory of Subtropical Agriculture and Environment, Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070 (China); Chen Wenli [State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070 (China); Huang Qiaoyun, E-mail: qyhuang@mail.hzau.edu.cn [State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070 (China); Key Laboratory of Subtropical Agriculture and Environment, Ministry of Agriculture, College of Resources and Environment, Huazhong Agricultural University, Wuhan 430070 (China)

    2010-09-15

    In order to have a better understanding of the interactions of heavy metals with bacteria and minerals in soil and associated environments, isothermal titration calorimetry (ITC), potentiometric titration and equilibrium sorption experiments were conducted to investigate the adsorption behavior of Cu(II) by Bacillus thuringiensis, Pseudomonas putida and their composites with minerals. The interaction of montmorillonite with bacteria increased the reactive sites and resulted in greater adsorption for Cu(II) on their composites, while decreased adsorption sites and capacities for Cu(II) were observed on goethite-bacteria composites. A gram-positive bacterium B. thuringiensis played a more important role than a gram-negative bacterium P. putida in determining the properties of the bacteria-minerals interfaces. The enthalpy changes ({Delta}H{sub ads}) from endothermic (6.14 kJ mol{sup -1}) to slightly exothermic (-0.78 kJ mol{sup -1}) suggested that Cu(II) is complexed with the anionic oxygen ligands on the surface of bacteria-mineral composites. Large entropies (32.96-58.89 J mol{sup -1} K{sup -1}) of Cu(II) adsorption onto bacteria-mineral composites demonstrated the formation of inner-sphere complexes in the presence of bacteria. The thermodynamic data implied that Cu(II) mainly bound to the carboxyl and phosphoryl groups as inner-sphere complexes on bacteria and mineral-bacteria composites.

  20. Microcalorimetric and potentiometric titration studies on the adsorption of copper by P. putida and B. thuringiensis and their composites with minerals.

    Science.gov (United States)

    Fang, Linchuan; Cai, Peng; Li, Pengxiang; Wu, Huayong; Liang, Wei; Rong, Xingmin; Chen, Wenli; Huang, Qiaoyun

    2010-09-15

    In order to have a better understanding of the interactions of heavy metals with bacteria and minerals in soil and associated environments, isothermal titration calorimetry (ITC), potentiometric titration and equilibrium sorption experiments were conducted to investigate the adsorption behavior of Cu(II) by Bacillus thuringiensis, Pseudomonas putida and their composites with minerals. The interaction of montmorillonite with bacteria increased the reactive sites and resulted in greater adsorption for Cu(II) on their composites, while decreased adsorption sites and capacities for Cu(II) were observed on goethite-bacteria composites. A gram-positive bacterium B. thuringiensis played a more important role than a gram-negative bacterium P. putida in determining the properties of the bacteria-minerals interfaces. The enthalpy changes (DeltaH(ads)) from endothermic (6.14 kJ mol(-1)) to slightly exothermic (-0.78 kJ mol(-1)) suggested that Cu(II) is complexed with the anionic oxygen ligands on the surface of bacteria-mineral composites. Large entropies (32.96-58.89 J mol(-1) K(-1)) of Cu(II) adsorption onto bacteria-mineral composites demonstrated the formation of inner-sphere complexes in the presence of bacteria. The thermodynamic data implied that Cu(II) mainly bound to the carboxyl and phosphoryl groups as inner-sphere complexes on bacteria and mineral-bacteria composites. Copyright 2010 Elsevier B.V. All rights reserved.

  1. Detoxification of corn stover and corn starch pyrolysis liquors by Pseudomonas putida and Streptomyces setonii suspended cells and plastic compost support biofilms.

    Science.gov (United States)

    Khiyami, Mohammad A; Pometto Iii, Anthony L; Brown, Robert C

    2005-04-20

    Plant biomass can be liquefied into fermentable sugars (levoglucosan then to glucose) for the production of ethanol, lactic acid, enzymes, and more by a process called pyrolysis. During the process microbial inhibitors are also generated. Pseudomonas putida (ATCC 17484) and Streptomyces setonii75Vi2 (ATCC 39116) were employed to degrade microbial inhibitors in diluted corn stover (Dcs) and diluted corn starch (Dst) pyrolysis liquors. The detoxification process evaluation included measuring total phenols and changes in UV spectra, a GC-MS analysis, and a bioassay, which employed Lactobacillus casei subsp. rhamosus (ATCC 11443) growth as an indicator of detoxification. Suspended-cell cultures illustrated limited detoxification ability of Dcs and Dst. P. putida and S. setoniiplastic compost support (PCS) biofilm continuous-stirred-tank-reactor pure cultures detoxified 10 and 25% (v/v) Dcs and Dst, whereas PCS biofilm mixed culture also partially detoxified 50% (v/v) Dcs and Dst in repeated batch culture. Therefore, PCS biofilm mixed culture is the process of choice to detoxify diluted pyrolysis liquors.

  2. Effects of Pseudomonas putida WCS358r and its genetically modified phenazine producing derivative on the Fusarium population in a field experiment, as determined by 18S rDNA analysis

    NARCIS (Netherlands)

    Leeflang, P.; Smit, E.; Glandorf, D.C.M.; Van Hannen, E.J.; Wernars, K.

    2002-01-01

    We measured effects of Pseudomonas putida WCS358r and its genetically modified phenazine producing derivative on the Fusarium population in the soil of a wheat field in the Netherlands. We used 18S rDNA analysis to study the Fusarium population through a strategy based on screening clone libraries

  3. A non-sense mutation in the putative anti-mutator gene ada/alkA of Mycobacterium tuberculosis and M. bovis isolates suggests convergent evolution

    Directory of Open Access Journals (Sweden)

    Gicquel Brigitte

    2007-05-01

    Full Text Available Abstract Background Previous studies have suggested that variations in DNA repair genes of W-Beijing strains may have led to transient mutator phenotypes which in turn may have contributed to host adaptation of this strain family. Single nucleotide polymorphism (SNP in the DNA repair gene mutT1 was identified in MDR-prone strains from the Central African Republic. A Mycobacteriumtuberculosis H37Rv mutant inactivated in two DNA repair genes, namely ada/alkA and ogt, was shown to display a hypermutator phenotype. We then looked for polymorphisms in these genes in Central African Republic strains (CAR. Results In this study, 55 MDR and 194 non-MDR strains were analyzed. Variations in DNA repair genes ada/alkA and ogt were identified. Among them, by comparison to M. tuberculosis published sequences, we found a non-sense variation in ada/alkA gene which was also observed in M. bovis AF2122 strain. SNPs that are present in the adjacent regions to the amber variation are different in M. bovis and in M. tuberculosis strain. Conclusion An Amber codon was found in the ada/alkA locus of clustered M. tuberculosis isolates and in M. bovis strain AF2122. This is likely due to convergent evolution because SNP differences between strains are incompatible with horizontal transfer of an entire gene. This suggests that such a variation may confer a selective advantage and be implicated in hypermutator phenotype expression, which in turn contributes to adaptation to environmental changes.

  4. Rearranged anaplastic lymphoma kinase (ALK) gene found for the first time in adult-onset papillary thyroid cancer cases among atomic bomb survivors

    International Nuclear Information System (INIS)

    Hamatani, K.; Mukai, M.; Takahashi, K.; Nakachi, K.; Kusunoki, Y.; Hayashi, Y.

    2012-01-01

    Full text of the publication follows: Thyroid cancer is one of the malignancies most strongly associated with ionizing radiation in humans. Epidemiology studies of atomic bomb (A-bomb) survivors have indicated that excess relative risk of papillary thyroid cancer per Gy was remarkably high in the survivors. We therefore aim to clarify mechanisms linking A-bomb radiation exposure and development of papillary thyroid cancer. Toward this end, we intend to clarify characteristics of gene alterations occurring in radiation-associated adult-onset papillary thyroid cancer from the Life Span Study cohort of A-bomb survivors. We have thus far found that with increased radiation dose, papillary thyroid cancer cases with chromosomal rearrangements (mainly RET/PTC rearrangements) significantly increased and papillary thyroid cancer cases with point mutations (mainly BRAF-V600E) significantly decreased. Papillary thyroid cancer cases with non-detected gene alterations that carried no mutations in RET, NTRK1, BRAF or RAS genes tended to increase with increased radiation dose. In addition, we found that relative frequency of these papillary thyroid cancer cases significantly decreased with time elapsed since exposure. Through analysis of papillary thyroid cancer cases with non-detected gene alterations, we recently discovered a new type of rearrangement for the first time in papillary thyroid cancer, i.e., rearranged anaplastic lymphoma kinase (ALK) gene, although identification of any partner gene(s) is needed. Specifically, rearrangement of ALK was found in 10 of 19 exposed papillary thyroid cancer cases with non-detected gene alterations but not in any of the six non-exposed papillary thyroid cancer cases. Furthermore, papillary thyroid cancer with ALK rearrangement was frequently found in the cases with high radiation dose or with short time elapsed since A-bomb exposure. These results suggest that chromosomal rearrangement, typically of RET and ALK, may play an important

  5. Quantitative determination of allergenic 5-alk(en)ylresorcinols in mango (Mangifera indica L.) peel, pulp, and fruit products by high-performance liquid chromatography.

    Science.gov (United States)

    Knödler, Matthias; Reisenhauer, Katharina; Schieber, Andreas; Carle, Reinhold

    2009-05-13

    Despite a number of serious case reports of mango dermatitis, no attempts at the identification and quantification of allergenic 5-alk(en)ylresorcinols in mango fruits have so far been made. Therefore, total alk(en)ylresorcinol content and relative homologue composition in 13 mango peel samples and 7 samples of mango pulp were determined by HPLC and LC-MS/MS analyses. Furthermore, mango puree and nectar prepared on pilot plant scale were also analyzed and compared with commercially available thermally preserved products. Depending on cultivar, alk(en)ylresorcinol contents ranged from 79.3 to 1850.5 mg/kg of dry matter (DM) in mango peels and from 4.9 to 187.3 mg/kg of DM in samples of mango pulp. The profile of alk(en)ylresorcinols was found to be highly characteristic, with an average homologue composition of C15:0 (6.1%), C15:1 (1.7%), C17:0 (1.1%), C17:1 (52.5%), C17:2 (33.4%), C17:3 (2.4%), C19:1 (2.1%), and C19:2 (0.8%). Mango puree samples prepared from peeled and unpeeled fruits revealed contents of 3.8 and 12.3 mg/kg of fresh weight, respectively. Content and homologue composition were not significantly affected during puree processing and thermal preservation. In nectar samples prepared from peeled and unpeeled fruits, contents of 1.4 and 4.6 mg/L, respectively, were found.

  6. Lobatin B inhibits NPM/ALK and NF-κB attenuating anaplastic-large-cell-lymphomagenesis and lymphendothelial tumour intravasation.

    Science.gov (United States)

    Kiss, Izabella; Unger, Christine; Huu, Chi Nguyen; Atanasov, Atanas Georgiev; Kramer, Nina; Chatruphonprasert, Waranya; Brenner, Stefan; McKinnon, Ruxandra; Peschel, Andrea; Vasas, Andrea; Lajter, Ildiko; Kain, Renate; Saiko, Philipp; Szekeres, Thomas; Kenner, Lukas; Hassler, Melanie R; Diaz, Rene; Frisch, Richard; Dirsch, Verena M; Jäger, Walter; de Martin, Rainer; Bochkov, Valery N; Passreiter, Claus M; Peter-Vörösmarty, Barbara; Mader, Robert M; Grusch, Michael; Dolznig, Helmut; Kopp, Brigitte; Zupko, Istvan; Hohmann, Judit; Krupitza, Georg

    2015-01-28

    An apolar extract of the traditional medicinal plant Neurolaena lobata inhibited the expression of the NPM/ALK chimera, which is causal for the majority of anaplastic large cell lymphomas (ALCLs). Therefore, an active principle of the extract, the furanoheliangolide sesquiterpene lactone lobatin B, was isolated and tested regarding the inhibition of ALCL expansion and tumour cell intravasation through the lymphendothelium. ALCL cell lines, HL-60 cells and PBMCs were treated with plant compounds and the ALK inhibitor TAE-684 to measure mitochondrial activity, proliferation and cell cycle progression and to correlate the results with protein- and mRNA-expression of selected gene products. Several endpoints indicative for cell death were analysed after lobatin B treatment. Tumour cell intravasation through lymphendothelial monolayers was measured and potential causal mechanisms were investigated analysing NF-κB- and cytochrome P450 activity, and 12(S)-HETE production. Lobatin B inhibited the expression of NPM/ALK, JunB and PDGF-Rβ, and attenuated proliferation of ALCL cells by arresting them in late M phase. Mitochondrial activity remained largely unaffected upon lobatin B treatment. Nevertheless, caspase 3 became activated in ALCL cells. Also HL-60 cell proliferation was attenuated whereas PBMCs of healthy donors were not affected by lobatin B. Additionally, tumour cell intravasation, which partly depends on NF-κB, was significantly suppressed by lobatin B most likely due to its NF-κB-inhibitory property. Lobatin B, which was isolated from a plant used in ethnomedicine, targets malignant cells by at least two properties: I) inhibition of NPM/ALK, thereby providing high specificity in combating this most prevalent fusion protein occurring in ALCL; II) inhibition of NF-κB, thereby not affecting normal cells with low constitutive NF-κB activity. This property also inhibits tumour cell intravasation into the lymphatic system and may provide an option to manage this

  7. Adverse renal effects of anaplastic lymphoma kinase inhibitors and the response to alectinib of an ALK+ lung cancer patient with renal dysfunction

    Directory of Open Access Journals (Sweden)

    Shimada M

    2017-06-01

    Full Text Available Midori Shimada,1,2 Minoru Fukuda,2,3 Masaaki Fukuda,2 Takeshi Kitazaki,2 Kohji Hashiguchi,2 Takaya Ikeda,1 Hiroyuki Yamaguchi,1 Katsumi Nakatomi,1 Kazuto Ashizawa,3 Hiroshi Mukae1 1Department of Respiratory Medicine, Unit of Translational Medicine, Nagasaki University Graduate School of Biomedical Sciences, 2Department of Respiratory Medicine, Japanese Red Cross Nagasaki Genbaku Hospital, 3Clinical Oncology Center, Nagasaki University Hospital, Nagasaki, Japan Abstract: A 62-year-old female patient with renal dysfunction and pulmonary adenocarcinoma developed postoperative recurrence and received carboplatin/pemetrexed and maintenance pemetrexed. As an anaplastic lymphoma kinase (ALK gene translocation was identified, the therapy was changed to crizotinib. However, the patient’s blood creatinine level increased, and her physical status worsened. Alectinib also induced exacerbation of renal dysfunction but was controlled by dose reduction of 140 mg twice daily for 2 weeks treatment and 2 weeks break were repeated, and exhibited a partial response for 16 months. Here, we describe the case in which alectinib treatment had beneficial clinical effects on ALK-positive lung adenocarcinoma, which controlled the adverse renal effects by dose reduction and drug breaks. Keywords: lung cancer, ALK, renal dysfunction, alectinib

  8. Effects of exogenous pyoverdines on Fe availability and their impacts on Mn(II) oxidation by Pseudomonas putida GB-1

    Science.gov (United States)

    Lee, Sung-Woo; Parker, Dorothy L.; Geszvain, Kati; Tebo, Bradley M.

    2014-01-01

    Pseudomonas putida GB-1 is a Mn(II)-oxidizing bacterium that produces pyoverdine-type siderophores (PVDs), which facilitate the uptake of Fe(III) but also influence MnO2 formation. Recently, a non-ribosomal peptide synthetase mutant that does not synthesize PVD was described. Here we identified a gene encoding the PVDGB-1 (PVD produced by strain GB-1) uptake receptor (PputGB1_4082) of strain GB-1 and confirmed its function by in-frame mutagenesis. Growth and other physiological responses of these two mutants and of wild type were compared during cultivation in the presence of three chemically distinct sets of PVDs (siderotypes n°1, n°2, and n°4) derived from various pseudomonads. Under iron-limiting conditions, Fe(III) complexes of various siderotype n°1 PVDs (including PVDGB-1) allowed growth of wild type and the synthetase mutant, but not the receptor mutant, confirming that iron uptake with any tested siderotype n°1 PVD depended on PputGB1_4082. Fe(III) complexes of a siderotype n°2 PVD were not utilized by any strain and strongly induced PVD synthesis. In contrast, Fe(III) complexes of siderotype n°4 PVDs promoted the growth of all three strains and did not induce PVD synthesis by the wild type, implying these complexes were utilized for iron uptake independent of PputGB1_4082. These differing properties of the three PVD types provided a way to differentiate between effects on MnO2 formation that resulted from iron limitation and others that required participation of the PVDGB-1 receptor. Specifically, MnO2 production was inhibited by siderotype n°1 but not n°4 PVDs indicating PVD synthesis or PputGB1_4082 involvement rather than iron-limitation caused the inhibition. In contrast, iron limitation was sufficient to explain the inhibition of Mn(II) oxidation by siderotype n°2 PVDs. Collectively, our results provide insight into how competition for iron via siderophores influences growth, iron nutrition and MnO2 formation in more complex environmental

  9. mRNA localization mechanisms in Trypanosoma cruzi.

    Directory of Open Access Journals (Sweden)

    Lysangela R Alves

    Full Text Available Asymmetric mRNA localization is a sophisticated tool for regulating and optimizing protein synthesis and maintaining cell polarity. Molecular mechanisms involved in the regulated localization of transcripts are widespread in higher eukaryotes and fungi, but not in protozoa. Trypanosomes are ancient eukaryotes that branched off early in eukaryote evolution. We hypothesized that these organisms would have basic mechanisms of mRNA localization. FISH assays with probes against transcripts coding for proteins with restricted distributions showed a discrete localization of the mRNAs in the cytoplasm. Moreover, cruzipain mRNA was found inside reservosomes suggesting new unexpected functions for this vacuolar organelle. Individual mRNAs were also mobilized to RNA granules in response to nutritional stress. The cytoplasmic distribution of these transcripts changed with cell differentiation, suggesting that localization mechanisms might be involved in the regulation of stage-specific protein expression. Transfection assays with reporter genes showed that, as in higher eukaryotes, 3'UTRs were responsible for guiding mRNAs to their final location. Our results strongly suggest that Trypanosoma cruzi have a core, basic mechanism of mRNA localization. This kind of controlled mRNA transport is ancient, dating back to early eukaryote evolution.

  10. Assessment of Qualitative and Quantitative Characters of Two Persian Clover Ecotypes Inoculated by Rhizobium leguminosarum biovartrifoli and Pesudomonas putida Bacteria

    Directory of Open Access Journals (Sweden)

    R Azamei

    2016-02-01

    Full Text Available Introduction Over the past decades, world attitude has changed towards the reduction of environmental pollutants. Harmful effects of synthetic fertilizers on environment have been identified. Bio-fertilizers are not harmful to the environment, but also they have favorable effects on plant growth processes. Soil biotechnology can be defined as the study of soil organisms and their metabolic processes which may have positive effects on plant yields. The main goal of this study is to asess the biotechnology fertilizers beneficial effects on soil organisms and their subsequently to maximize the yield. It is also our desire conside the soil quality, hygiene and environmental protection along this process. Among the strain of nitrogen-fixing bacteria, symbiotic bacteria such as rhizobium bacteria are important and essential in planning the sustainable farming systems. Several studies have shown that crop varieties which inoculated with rhizobium and pseudomonas were superior in yield production and performance. Material and Methods An experiment was designed as factorial performed in randomized complete block design (RCBD with three replications in Agricultural Research Center of Golpayegan (Isfahan during 2010 – 2011. the purpose of this study was to evaluate the effects of inoculation of two ecotypes of Persian clover by various strains of Rhizobium leguminosarum. Biovar trifoli bacteria accompanied with plant growth promoting rhizobacteria (PGPR Pseudomonas putida was employed to find certain qualitative and quantitative characteristics of clover yield, The main plots included two local ecotypes of Persian clover; Arak Haft Chin (V1 and Isfahan Haft Chin (V2, the subplots included inoculation by two strain of Rhizobium; Rb-3, Rb-13 and one strain of Pseudomonas; PS -168.4 cuts were performed during the experiment and 60 kg/ha seed was used for cultivation based on local knowledge. According to recommendations of the Institute of Soil and Water

  11. Protein Structure and the Sequential Structure of mRNA

    DEFF Research Database (Denmark)

    Brunak, Søren; Engelbrecht, Jacob

    1996-01-01

    entries in the Brookhaven Protein Data Bank produced 719 protein chains with matching mRNA sequence, amino acid sequence, and secondary structure assignment, By neural network analysis, we found strong signals in mRNA sequence regions surrounding helices and sheets, These signals do not originate from......A direct comparison of experimentally determined protein structures and their corresponding protein coding mRNA sequences has been performed, We examine whether real world data support the hypothesis that clusters of rare codons correlate with the location of structural units in the resulting...... protein, The degeneracy of the genetic code allows for a biased selection of codons which may control the translational rate of the ribosome, and may thus in vivo have a catalyzing effect on the folding of the polypeptide chain, A complete search for GenBank nucleotide sequences coding for structural...

  12. Increased IL-10 mRNA and IL-23 mRNA expression in multiple sclerosis: interferon-beta treatment increases IL-10 mRNA expression while reducing IL-23 mRNA expression

    DEFF Research Database (Denmark)

    Krakauer, M.; Sorensen, P.; Khademi, M.

    2008-01-01

    volunteers served to confirm initial findings. mRNA was analyzed by real-time reverse transcriptase polymerase chain reaction (PCR). RESULTS: We found elevated expression of interleukin (IL)-23 and IL-10 in untreated MS patients. IFN-beta therapy increased IL-10 and decreased IL-23 expression independently...... of the regulatory cytokine IL-10. The elevated IL-23 mRNA levels in MS patients are noteworthy in view of the newly discovered IL-23-driven Th17 T-cell subset, which is crucial in animal models of MS. Since IFN-beta therapy resulted in decreased IL-23 mRNA levels, the Th17 axis could be another target of IFN...

  13. Complexity on Acute Myeloid Leukemia mRNA Transcript Variant

    Directory of Open Access Journals (Sweden)

    Carlo Cattani

    2011-01-01

    Full Text Available This paper deals with the sequence analysis of acute myeloid leukemia mRNA. Six transcript variants of mlf1 mRNA, with more than 2000 bps, are analyzed by focusing on the autocorrelation of each distribution. Through the correlation matrix, some patches and similarities are singled out and commented, with respect to similar distributions. The comparison of Kolmogorov fractal dimension will be also given in order to classify the six variants. The existence of a fractal shape, patterns, and symmetries are discussed as well.

  14. Detection of melatonin receptor mRNA in human muscle

    International Nuclear Information System (INIS)

    Li Lei

    2004-01-01

    To verify the expression of melatonin receptor mRNA in human, muscle, muscle beside vertebrae was collected to obtain total RNA and the mRNA of melatonin receptor was detected by RT-PCR method. The electrophoretic results of RT-PCR products by mt 1 and MT 2 primer were all positive and the sequence is corresponding with human melatonin receptor cDNA. It suggests that melatonin may act on the muscle beside vertebrae directly and regulate its growth and development. (authors)

  15. Effective treatment with icotinib in lung adenocarcinoma with EGFR and ALK co-alterations and brain metastasis

    Directory of Open Access Journals (Sweden)

    Ye CY

    2016-10-01

    Full Text Available Chenyang Ye,1,* Ji Wang,2,* Shu Zheng,1 Ying Chai3 1Cancer Institute, Key Laboratory of Cancer Prevention and Intervention, National Ministry of Education, Provincial Key Laboratory of Molecular Biology in Medical Sciences, The Second Affiliated Hospital, Zhejiang University School of Medicine, 2Department of Surgical Oncology, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, 3Department of Thoracic Surgery, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, People’s Republic of China *These authors contributed equally to this work Abstract: We report a rare case of advanced lung cancer with epidermal growth factor receptor and anaplastic lymphoma kinase co-alterations and brain metastasis, in which icotinib treatment was effective for both the primary lung tumor and the brain metastasis. The patient achieved important clinical remission with a progression-free survival for two years. Our treatment strategy appears to be a promising therapeutic approach for this subgroup of patients. Keywords: lung cancer, brain metastasis, EGFR, ALK, icotinib

  16. Bone morphogenetic protein-9 suppresses growth of myeloma cells by signaling through ALK2 but is inhibited by endoglin

    International Nuclear Information System (INIS)

    Olsen, O E; Wader, K F; Misund, K; Våtsveen, T K; Rø, T B; Mylin, A K; Turesson, I; Størdal, B F; Moen, S H; Standal, T; Waage, A; Sundan, A; Holien, T

    2014-01-01

    Multiple myeloma is a malignancy of plasma cells predominantly located in the bone marrow. A number of bone morphogenetic proteins (BMPs) induce apoptosis in myeloma cells in vitro, and with this study we add BMP-9 to the list. BMP-9 has been found in human serum at concentrations that inhibit cancer cell growth in vitro. We here show that the level of BMP-9 in serum was elevated in myeloma patients (median 176 pg/ml, range 8–809) compared with healthy controls (median 110 pg/ml, range 8–359). BMP-9 was also present in the bone marrow and was able to induce apoptosis in 4 out of 11 primary myeloma cell samples by signaling through ALK2. BMP-9-induced apoptosis in myeloma cells was associated with c-MYC downregulation. The effects of BMP-9 were counteracted by membrane-bound (CD105) or soluble endoglin present in the bone marrow microenvironment, suggesting a mechanism for how myeloma cells can evade the tumor suppressing activity of BMP-9 in multiple myeloma

  17. Production of natural fragrance aromatic acids by coexpression of trans-anethole oxygenase and p-anisaldehyde dehydrogenase genes of Pseudomonas putida JYR-1 in Escherichia coli.

    Science.gov (United States)

    Han, Dongfei; Kurusarttra, Somwang; Ryu, Ji-Young; Kanaly, Robert A; Hur, Hor-Gil

    2012-12-05

    A gene encoding p-anisaldehyde dehydrogenase (PAADH), which catalyzes the oxidation of p-anisaldehyde to p-anisic acid, was identified to be clustered with the trans-anethole oxygenase (tao) gene in Pseudomonas putida JYR-1. Heterologously expressed PAADH in Escherichia coli catalyzed the oxidation of vanillin, veratraldehyde, and piperonal to the corresponding aromatic acids vanillic acid, veratric acid, and piperonylic acid, respectively. Coexpression of trans-anethole oxygenase (TAO) and PAADH in E. coli also resulted in the successful transformation of trans-anethole, isoeugenol, O-methyl isoeugenol, and isosafrole to p-anisic acid, vanillic acid, veratric acid, and piperonylic acid, respectively, which are compounds found in plants as secondary metabolites. Because of the relaxed substrate specificity and high transformation rates by coexpressed TAO and PAADH in E. coli , the engineered strain has potential to be applied in the fragrance industry.

  18. Application of Biosurfactants Produced by Pseudomonas putida using Crude Palm Oil (CPO) as Substrate for Crude Oil Recovery using Batch Method

    Science.gov (United States)

    Suryanti, V.; Handayani, D. S.; Masykur, A.; Septyaningsih, I.

    2018-03-01

    The application of biosurfactants which have been produced by Pseudomonas putida in nutrient broth medium supplemented with NaCl and crude palm oil (CPO) for oil recovery has been evaluated. The crude and purified biosurfactants have been examined for oil recovery from a laboratory oil-contaminated sand in agitated flask (batch method). Two synthetic surfactants and water as control was also performed for oil recovery as comparisons. Using batch method, the results showed that removing ability of crude oil from the oil-contaminated sand by purified and crude biosurfactants were 79.40±3.10 and 46.84±2.23 %, respectively. On other hand, the recoveries obtained with the SDS, Triton X-100 and water were 94.33±0.47, 74.84±7.39 and 34.42±1.21%respectively.

  19. Nitrogen regulation of the xyl genes of Pseudomonas putida mt-2 propagates into a significant effect of nitrate on m-xylene mineralization in soil

    DEFF Research Database (Denmark)

    Svenningsen, Nanna Bygvraa; Nicolaisen, Mette Haubjerg; Hansen, Hans Chr. Bruun

    2016-01-01

    nitrogen sensing status in both experimental systems. Hence, for nitrogen sources, regulatory patterns that emerge in soil reflect those observed in liquid cultures. The current study shows how distinct regulatory traits can lead to discrete environmental consequences; and it underpins that attempts......The nitrogen species available in the growth medium are key factors determining expression of xyl genes for biodegradation of aromatic compounds by Pseudomonas putida. Nitrogen compounds are frequently amended to promote degradation at polluted sites, but it remains unknown how regulation observed...... that NO3(-) compared with NH4(+) had a stimulating effect on xyl gene expression in pure culture as well as in soil, and that this stimulation was translated into increased m-xylene mineralization in soil. Furthermore, expression analysis of the nitrogen-regulated genes amtB and gdhA allowed us to monitor...

  20. Purification, crystallization and preliminary crystallographic analysis of DehI, a group I α-haloacid dehalogenase from Pseudomonas putida strain PP3

    Energy Technology Data Exchange (ETDEWEB)

    Schmidberger, Jason W. [School of Pharmacology and Medicine, University of Western Australia, Crawley, Western Australia (Australia); Wilce, Jackie A. [Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria (Australia); Weightman, Andrew J. [School of Biosciences, Cardiff University, Cardiff,Wales (United Kingdom); Wilce, Matthew C. J., E-mail: matthew.wilce@med.monash.edu.au [Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria (Australia); School of Pharmacology and Medicine, University of Western Australia, Crawley, Western Australia (Australia)

    2008-07-01

    The α-haloacid dehalogenase DehI from P. putida strain PP3 was cloned into a vector with an N-terminal His tag and expressed in E. coli Nova Blue strain. Purified protein was crystallized in a primitive monoclinic form and a complete native data set was collected and analysed. Pseudomonas putida strain PP3 produces two dehalogenases, DehI and DehII, which belong to the group I and II α-haloacid dehalogenases, respectively. Group I dehalogenases catalyse the removal of halides from d-haloalkanoic acids and in some cases also the l-enantiomers, both substituted at their chiral centres. Studies of members of this group have resulted in the proposal of general catalytic mechanisms, although no structural information is available in order to better characterize their function. This work presents the initial stages of the structural investigation of the group I α-haloacid dehalogenase DehI. The DehI gene was cloned into a pET15b vector with an N-terminal His tag and expressed in Escherichia coli Nova Blue strain. Purified protein was crystallized in 25% PEG 3350, 0.4 M lithium sulfate and 0.1 M bis-tris buffer pH 6.0. The crystals were primitive monoclinic (space group P2{sub 1}), with unit-cell parameters a = 68.32, b = 111.86, c = 75.13 Å, α = 90, β = 93.7, γ = 90°, and a complete native data set was collected. Molecular replacement is not an option for structure determination, so further experimental phasing methods will be necessary.

  1. Purification, crystallization and preliminary crystallographic analysis of DehI, a group I α-haloacid dehalogenase from Pseudomonas putida strain PP3

    International Nuclear Information System (INIS)

    Schmidberger, Jason W.; Wilce, Jackie A.; Weightman, Andrew J.; Wilce, Matthew C. J.

    2008-01-01

    The α-haloacid dehalogenase DehI from P. putida strain PP3 was cloned into a vector with an N-terminal His tag and expressed in E. coli Nova Blue strain. Purified protein was crystallized in a primitive monoclinic form and a complete native data set was collected and analysed. Pseudomonas putida strain PP3 produces two dehalogenases, DehI and DehII, which belong to the group I and II α-haloacid dehalogenases, respectively. Group I dehalogenases catalyse the removal of halides from d-haloalkanoic acids and in some cases also the l-enantiomers, both substituted at their chiral centres. Studies of members of this group have resulted in the proposal of general catalytic mechanisms, although no structural information is available in order to better characterize their function. This work presents the initial stages of the structural investigation of the group I α-haloacid dehalogenase DehI. The DehI gene was cloned into a pET15b vector with an N-terminal His tag and expressed in Escherichia coli Nova Blue strain. Purified protein was crystallized in 25% PEG 3350, 0.4 M lithium sulfate and 0.1 M bis-tris buffer pH 6.0. The crystals were primitive monoclinic (space group P2 1 ), with unit-cell parameters a = 68.32, b = 111.86, c = 75.13 Å, α = 90, β = 93.7, γ = 90°, and a complete native data set was collected. Molecular replacement is not an option for structure determination, so further experimental phasing methods will be necessary

  2. Synthesis of Diblock copolymer poly-3-hydroxybutyrate -block-poly-3-hydroxyhexanoate [PHB-b-PHHx] by a β-oxidation weakened Pseudomonas putida KT2442.

    Science.gov (United States)

    Tripathi, Lakshmi; Wu, Lin-Ping; Chen, Jinchun; Chen, Guo-Qiang

    2012-04-05

    Block polyhydroxyalkanoates (PHA) were reported to be resistant against polymer aging that negatively affects polymer properties. Recently, more and more attempts have been directed to make PHA block copolymers. Diblock copolymers PHB-b-PHHx consisting of poly-3-hydroxybutyrate (PHB) block covalently bonded with poly-3-hydroxyhexanoate (PHHx) block were for the first time produced successfully by a recombinant Pseudomonas putida KT2442 with its β-oxidation cycle deleted to its maximum. The chloroform extracted polymers were characterized by nuclear magnetic resonance (NMR), thermo- and mechanical analysis. NMR confirmed the existence of diblock copolymers consisting of 58 mol% PHB as the short chain length block with 42 mol% PHHx as the medium chain length block. The block copolymers had two glass transition temperatures (Tg) at 2.7°C and -16.4°C, one melting temperature (Tm) at 172.1°C and one cool crystallization temperature (Tc) at 69.1°C as revealed by differential scanning calorimetry (DSC), respectively. This is the first microbial short-chain-length (scl) and medium-chain-length (mcl) PHA block copolymer reported. It is possible to produce PHA block copolymers of various kinds using the recombinant Pseudomonas putida KT2442 with its β-oxidation cycle deleted to its maximum. In comparison to a random copolymer poly-3-hydroxybutyrate-co-3-hydroxyhexanoate (P(HB-co-HHx)) and a blend sample of PHB and PHHx, the PHB-b-PHHx showed improved structural related mechanical properties.

  3. Display of a thermostable lipase on the surface of a solvent-resistant bacterium, Pseudomonas putida GM730, and its applications in whole-cell biocatalysis

    Directory of Open Access Journals (Sweden)

    Kwon Seok-Joon

    2006-04-01

    Full Text Available Abstract Background Whole-cell biocatalysis in organic solvents has been widely applied to industrial bioprocesses. In two-phase water-solvent processes, substrate conversion yields and volumetric productivities can be limited by the toxicity of solvents to host cells and by the low mass transfer rates of the substrates from the solvent phase to the whole-cell biocatalysts in water. Results To solve the problem of solvent toxicity, we immobilized a thermostable lipase (TliA from Pseudomonas fluorescens on the cell surface of a solvent-resistant bacterium, Pseudomonas putida GM730. Surface immobilization of enzymes eliminates the mass-transfer limitation imposed by the cell wall and membranes. TliA was successfully immobilized on the surface of P. putida cells using the ice-nucleation protein (INP anchoring motif from Pseudomonas syrinage. The surface location was confirmed by flow cytometry, protease accessibility and whole-cell enzyme activity using a membrane-impermeable substrate. Three hundred and fifty units of whole-cell hydrolytic activity per gram dry cell mass were obtained when the enzyme was immobilized with a shorter INP anchoring motif (INPNC. The surface-immobilized TliA retained full enzyme activity in a two-phase water-isooctane reaction system after incubation at 37°C for 12 h, while the activity of the free form enzyme decreased to 65% of its initial value. Whole cells presenting immobilized TliA were shown to catalyze three representative lipase reactions: hydrolysis of olive oil, synthesis of triacylglycerol and chiral resolution. Conclusion In vivo surface immobilization of enzymes on solvent-resistant bacteria was demonstrated, and appears to be useful for a variety of whole-cell bioconversions in the presence of organic solvents.

  4. Molecular level biodegradation of phenol and its derivatives through dmp operon of Pseudomonas putida: A bio-molecular modeling and docking analysis.

    Science.gov (United States)

    Ray, Sujay; Banerjee, Arundhati

    2015-10-01

    Participation of Pseudomonas putida-derived methyl phenol (dmp) operon and DmpR protein in the biodegradation of phenol or other harmful, organic, toxic pollutants was investigated at a molecular level. Documentation documents that P. putida has DmpR protein which positively regulates dmp operon in the presence of inducers; like phenols. From the operon, phenol hydroxylase encoded by dmpN gene, participates in degrading phenols after dmp operon is expressed. For the purpose, the 3-D models of the four domains from DmpR protein and of the DNA sequences from the two Upstream Activation Sequences (UAS) present at the promoter region of the operon were demonstrated using discrete molecular modeling techniques. The best modeled structures satisfying their stereo-chemical properties were selected in each of the cases. To stabilize the individual structures, energy optimization was performed. In the presence of inducers, probable interactions among domains and then the two independent DNA structures with the fourth domain were perused by manifold molecular docking simulations. The complex structures were made to be stable by minimizing their overall energy. Responsible amino acid residues, nucleotide bases and binding patterns for the biodegradation, were examined. In the presence of the inducers, the biodegradation process is initiated by the interaction of phe50 from the first protein domain with the inducers. Only after the interaction of the last domain with the DNA sequences individually, the operon is expressed. This novel residue level study is paramount for initiating transcription in the operon; thereby leading to expression of phenol hydroxylase followed by phenol biodegradation. Copyright © 2015. Published by Elsevier B.V.

  5. Analysis of aromatic catabolic pathways in Pseudomonas putida KT 2440 using a combined proteomic approach: 2-DE/MS and cleavable isotope-coded affinity tag analysis.

    Science.gov (United States)

    Kim, Young Hwan; Cho, Kun; Yun, Sung-Ho; Kim, Jin Young; Kwon, Kyung-Hoon; Yoo, Jong Shin; Kim, Seung Il

    2006-02-01

    Proteomic analysis of Pseudomonas putida KT2440 cultured in monocyclic aromatic compounds was performed using 2-DE/MS and cleavable isotope-coded affinity tag (ICAT) to determine whether proteins involved in aromatic compound degradation pathways were altered as predicted by genomic analysis (Jiménez et al., Environ Microbiol. 2002, 4, 824-841). Eighty unique proteins were identified by 2-DE/MS or MS/MS analysis from P. putida KT2440 cultured in the presence of six different organic compounds. Benzoate dioxygenase (BenA, BenD) and catechol 1,2-dioxygenase (CatA) were induced by benzoate. Protocatechuate 3,4-dixoygenase (PcaGH) was induced by p-hydroxybenzoate and vanilline. beta-Ketoadipyl CoA thiolase (PcaF) and 3-oxoadipate enol-lactone hydrolase (PcaD) were induced by benzoate, p-hydroxybenzoate and vanilline, suggesting that benzoate, p-hydroxybenzoate and vanilline were degraded by different dioxygenases and then converged in the same beta-ketoadipate degradation pathway. An additional 110 proteins, including 19 proteins from 2-DE analysis, were identified by cleavable ICAT analysis for benzoate-induced proteomes, which complemented the 2-DE results. Phenylethylamine exposure induced beta-ketoacyl CoA thiolase (PhaD) and ring-opening enzyme (PhaL), both enzymes of the phenylacetate (pha) biodegradation pathway. Phenylalanine induced 4-hydroxyphenyl-pyruvate dioxygenase (Hpd) and homogentisate 1,2-dioxygenase (HmgA), key enzymes in the homogentisate degradation pathway. Alkyl hydroperoxide reductase (AphC) was induced under all aromatic compounds conditions. These results suggest that proteome analysis complements and supports predictive information obtained by genomic sequence analysis.

  6. The oxidation of alkylaryl sulfides and benzo[b]thiophenes by Escherichia coli cells expressing wild-type and engineered styrene monooxygenase from Pseudomonas putida CA-3.

    Science.gov (United States)

    Nikodinovic-Runic, Jasmina; Coulombel, Lydie; Francuski, Djordje; Sharma, Narain D; Boyd, Derek R; Ferrall, Rory Moore O; O'Connor, Kevin E

    2013-06-01

    Nine different sulfur-containing compounds were biotransformed to the corresponding sulfoxides by Escherichia coli Bl21(DE3) cells expressing styrene monooxygenase (SMO) from Pseudomonas putida CA-3. Thioanisole was consumed at 83.3 μmoles min(-1) g cell dry weight(-1) resulting mainly in the formation of R-thioanisole sulfoxide with an enantiomeric excess (ee) value of 45 %. The rate of 2-methyl-, 2-chloro- and 2-bromo-thioanisole consumption was 2-fold lower than that of thioanisole. Surprisingly, the 2-methylthioanisole sulfoxide product had the opposite (S) configuration to that of the other 2-substituted thioanisole derivatives and had a higher ee value (84 %). The rate of oxidation of 4-substituted thioanisoles was higher than the corresponding 2-substituted substrates but the ee values of the products were consistently lower (10-23 %). The rate of benzo[b]thiophene and 2-methylbenzo[b]thiophene sulfoxidation was approximately 10-fold lower than that of thioanisole. The ee value of the benzo[b]thiophene sulfoxide could not be determined as the product racemized rapidly. E. coli cells expressing an engineered SMO (SMOeng R3-11) oxidised 2-substituted thioanisoles between 1.8- and 2.8-fold faster compared to cells expressing the wild-type enzyme. SMOeng R3-11 oxidised benzo[b]thiophene and 2-methylbenzo[b]thiophene 10.1 and 5.6 times faster that the wild-type enzyme. The stereospecificity of the reaction catalysed by SMOeng was unchanged from that of the wild type. Using the X-ray crystal structure of the P. putida S12 SMO, it was evident that the entrance of substrates into the SMO active site is limited by the binding pocket bottleneck formed by the side chains of Val-211 and Asn-46 carboxyamide group.

  7. Cloning and mRNA expression pattern analysis under low ...

    African Journals Online (AJOL)

    This research cloned endochitinase-antifreeze protein precursor (EAPP) gene of Dong-mu 70 rye (Secale cereale) by designing special primers according to Genbank's EAPP gene sequence, and analyzing the influence of low temperature stress on the expression of mRNA with RT-PCR. The results indicated that the ...

  8. Lorlatinib in non-small-cell lung cancer with ALK or ROS1 rearrangement: an international, multicentre, open-label, single-arm first-in-man phase 1 trial.

    Science.gov (United States)

    Shaw, Alice T; Felip, Enriqueta; Bauer, Todd M; Besse, Benjamin; Navarro, Alejandro; Postel-Vinay, Sophie; Gainor, Justin F; Johnson, Melissa; Dietrich, Jorg; James, Leonard P; Clancy, Jill S; Chen, Joseph; Martini, Jean-François; Abbattista, Antonello; Solomon, Benjamin J

    2017-12-01

    Most patients with anaplastic lymphoma kinase (ALK)-rearranged or ROS proto-oncogene 1 (ROS1)-rearranged non-small-cell lung cancer (NSCLC) are sensitive to tyrosine kinase inhibitor (TKI) therapy, but resistance invariably develops, commonly within the CNS. This study aimed to analyse the safety, efficacy, and pharmacokinetic properties of lorlatinib, a novel, highly potent, selective, and brain-penetrant ALK and ROS1 TKI with preclinical activity against most known resistance mutations, in patients with advanced ALK-positive or ROS1-positive NSCLC. In this international multicentre, open-label, single-arm, first-in-man phase 1 dose-escalation study, eligible patients had advanced ALK-positive or ROS1-positive NSCLC and were older than 18 years, with an Eastern Cooperative Oncology Group performance status of 0 or 1, and adequate end-organ function. Lorlatinib was administered orally to patients at doses ranging from 10 mg to 200 mg once daily or 35 mg to 100 mg twice daily, with a minimum of three patients receiving each dose. For some patients, tumour biopsy was done before lorlatinib treatment to identify ALK resistance mutations. Safety was assessed in patients who received at least one dose of lorlatinib; efficacy was assessed in the intention-to-treat population (patients who received at least one dose of study treatment and had either ALK or ROS1 rearrangement). The primary endpoint was dose-limiting toxicities during cycle 1 according to investigator assessment; secondary endpoints included safety, pharmacokinetics, and overall response. This study is ongoing and is registered with ClinicalTrials.gov, number NCT01970865. Between Jan 22, 2014, and July 10, 2015, 54 patients received at least one dose of lorlatinib, including 41 (77%) with ALK-positive and 12 (23%) with ROS1-positive NSCLC; one patient had unconfirmed ALK and ROS1 status. 28 (52%) patients had received two or more TKIs, and 39 (72%) patients had CNS metastases. The most common treatment

  9. Biosynthesis of the Nylon 12 Monomer, ω-Aminododecanoic Acid with Novel CYP153A, AlkJ, and ω-TA Enzymes.

    Science.gov (United States)

    Ahsan, Md Murshidul; Jeon, Hyunwoo; P Nadarajan, Saravanan; Chung, Taeowan; Yoo, Hee-Wang; Kim, Byung-Gee; Patil, Mahesh D; Yun, Hyungdon

    2018-04-01

    Bioplastics are derived from renewable biomass sources, such as vegetable oils, cellulose, and starches. An important and high-performance member of the bioplastic family is Nylon 12. The biosynthesis of ω-amino dodecanoic acid (ω-AmDDA), the monomer of Nylon 12 from vegetable oil derivatives is considered as an alternative to petroleum-based monomer synthesis. In this study, for the production of ω-AmDDA from dodecanoic acid (DDA), the cascade of novel P450 (CYP153A), alcohol dehydrogenase (AlkJ), and ω-transaminase (ω-TA) is developed. The regioselective ω-hydroxylation of 1 mM DDA with near complete conversion (>99%) is achieved using a whole-cell biocatalyst co-expressing CYP153A, ferredoxin reductase and ferredoxin. When the consecutive biotransformation of ω-hydroxy dodecanoic acid (ω-OHDDA) is carried out using a whole-cell biocatalyst co-expressing AlkJ and ω-TA, 1.8 mM ω-OHDDA is converted into ω-AmDDA with 87% conversion in 3 h. Finally, when a one-pot reaction is carried out with 2 mM DDA using both whole-cell systems, 0.6 mM ω-AmDDA is produced after a 5 h reaction. The results demonstrated the scope of the potential cascade reaction of novel CYP153A, AlkJ, and ω-TA for the production of industrially important bioplastic monomers, amino fatty acids, from FFAs. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. APPLICATION OF PSEUDOMONAS PUTIDA AND RHODOCOCCUS SP. BY BIODEGRADATION OF PAH(S, PCB(S AND NEL SOIL SAMPLES FROM THE HAZARDOUS WASTE DUMP IN POZĎÁTKY (CZECH REPUBLIC

    Directory of Open Access Journals (Sweden)

    Radmila Kucerova

    2006-12-01

    Full Text Available The objective of the project was a laboratory check of biodegradation of soil samples contaminated by PAH(s, PCB(s and NEL from the hazardous waste dump in the Pozďátky locality. For the laboratory check, pure bacterial cultures of Rhodococcus sp. and Pseudomonas putida have been used. It is apparent from the laboratory experiments results that after one-month bacterial leaching, applying the bacterium of Rhodococcus sp. there is a 83 % removal of NEL, a 79 % removal of PAH(s and a 14 % removal of PCB(s. Applying a pure culture of Pseudomonas putida there is a 87 % removal of NEL, a 81 % removal of PAH(s and a 14 % removal of PCB(s.

  11. Proactive management strategies for potential gastrointestinal adverse reactions with ceritinib in patients with advanced ALK-positive non-small-cell lung cancer

    International Nuclear Information System (INIS)

    Schaefer, Eric S; Baik, Christina

    2016-01-01

    Anaplastic lymphoma kinase (ALK) gene fusions occur in 3%–7% of non-small-cell lung cancer (NSCLC) cases. Ceritinib, a once-daily, oral ALK inhibitor, has activity against crizotinib-resistant and crizotinib-naïve NSCLC, including brain metastases. Ceritinib (Zykadia™) was granted accelerated approval by the US Food and Drug Administration in 2014 for treating crizotinib-resistant ALK-positive NSCLC. Adverse events (AEs), particularly gastrointestinal (GI) AEs, are commonly experienced at the recommended dose of 750 mg/d and ∼38% of patients require dose interruption or reduction for GI AEs. This case study details our experience with the use of proactive GI AE management regimens in patients treated with ceritinib (750 mg/d) across two study sites. Proactive Regimens A and B were implemented in patients with metastatic ALK-positive NSCLC treated with ceritinib to manage drug-related GI AEs. Regimen A comprised ondansetron and diphenoxylate/atropine or loperamide, taken 30 minutes prior to ceritinib dose. Regimen B included dicyclomine (taken with the first ceritinib dose), ondansetron (taken 30 minutes prior to ceritinib dose for the first seven doses), and loperamide (taken as needed with the onset of diarrhea). The proactive medications were tapered off depending on patient tolerability to ceritinib. Nine patient cases are presented. Starting Regimens A or B before the first dose of ceritinib, or as soon as GI symptoms were encountered, prevented the need for dose reduction due to GI toxicity in eight of the nine patients. Using these regimens, 78% of patients were able to remain on 750 mg/d fasting. Two patients received 23 months and 16 months of therapy and remain on ceritinib 750 mg/d and 600 mg/d, respectively. Although not currently recommended or implemented in clinical studies, based on the patients evaluated here, upfront or proactive treatment plans that address AEs early on can allow the majority of patients to remain on the approved 750 mg

  12. Evaluation of nutrients removal (NO3-N, NH3-N and PO4-P) with Chlorella vulgaris, Pseudomonas putida, Bacillus cereus and a consortium of these microorganisms in the treatment of wastewater effluents.

    Science.gov (United States)

    Gómez-Guzmán, Abril; Jiménez-Magaña, Sergio; Guerra-Rentería, A Suggey; Gómez-Hermosillo, César; Parra-Rodríguez, F Javier; Velázquez, Sergio; Aguilar-Uscanga, Blanca Rosa; Solis-Pacheco, Josue; González-Reynoso, Orfil

    2017-07-01

    In this research removal of NH 3 -N, NO 3 -N and PO 4 -P nutrients from municipal wastewater was studied, using Chlorella vulgaris, Pseudomonas putida, Bacillus cereus and an artificial consortium of them. The objective is to analyze the performance of these microorganisms and their consortium, which has not been previously studied for nutrient removal in municipal wastewater. A model wastewater was prepared simulating the physicochemical characteristics found at the wastewater plant in Chapala, Mexico. Experiments were carried out without adding an external carbon source. Results indicate that nutrient removal with Chlorella vulgaris was the most efficient with a removal of 24.03% of NO 3 -N, 80.62% of NH 3 -N and 4.30% of PO 4 -P. With Bacillus cereus the results were 8.40% of NO 3 -N, 28.80% of NH 3 -N and 3.80% of PO 4 -P. The removals with Pseudomonas putida were 2.50% of NO 3 -N, 41.80 of NH 3 -N and 4.30% of PO 4 -P. The consortium of Chlorella vulgaris-Bacillus cereus-Pseudomonas putida removed 29.40% of NO 3 -N, 4.2% of NH 3 -N and 8.4% of PO 4 -P. The highest biomass production was with Bacillus cereus (450 mg/l) followed by Pseudomonas putida (444 mg/l), the consortium (205 mg/l) and Chlorella vulgaris (88.9 mg/l). This study highlights the utility of these microorganisms for nutrient removal in wastewater treatments.

  13. Optimización de un medio de cultivo para la producción de biomasa de la cepa Pseudomonas putida UA 44 aislada del suelo bananero de Uraba –Antioquia

    OpenAIRE

    Becerra Mejía, Camilo Andrés

    2007-01-01

    En el presente trabajo se diseñó y optimizó un medio de cultivo químicamente definido para la bacteria Pseudomonas putida UA44 con potencial PGPR aislada del suelo de la zona bananera de Uraba por Ramirez (2004). Los resultados obtenidos en el medio químicamente definido (MD) se compararon con los hallados en un medio de cultivo semicomplejo (TSB: Triptic Soy Broth por sus siglas en Ingles).

  14. FDA Approval: Alectinib for the Treatment of Metastatic, ALK-Positive Non-Small Cell Lung Cancer Following Crizotinib.

    Science.gov (United States)

    Larkins, Erin; Blumenthal, Gideon M; Chen, Huanyu; He, Kun; Agarwal, Rajiv; Gieser, Gerlie; Stephens, Olen; Zahalka, Eias; Ringgold, Kimberly; Helms, Whitney; Shord, Stacy; Yu, Jingyu; Zhao, Hong; Davis, Gina; McKee, Amy E; Keegan, Patricia; Pazdur, Richard

    2016-11-01

    On December 11, 2015, the FDA granted accelerated approval to alectinib (Alecensa; Genentech) for the treatment of patients with anaplastic lymphoma receptor tyrosine kinase (ALK)-positive, metastatic non-small cell lung cancer (NSCLC) who have progressed on or are intolerant to crizotinib. This approval was based on two single-arm trials including 225 patients treated with alectinib 600 mg orally twice daily. The objective response rates (ORR) by an independent review committee in these studies were 38% [95% confidence interval (CI), 28-49] and 44% (95% CI, 36-53); the median durations of response (DOR) were 7.5 months and 11.2 months. In a pooled analysis of 51 patients with measurable disease in the central nervous system (CNS) at baseline, the CNS ORR was 61% (95% CI, 46-74); the CNS DOR was 9.1 months. The primary safety analysis population included 253 patients. The most common adverse reactions were fatigue (41%), constipation (34%), edema (30%), and myalgia (29%). The most common laboratory abnormalities were anemia (56%), increased aspartate aminotransferase (51%), increased alkaline phosphatase (47%), increased creatine phosphokinase (43%), hyperbilirubinemia (39%), hyperglycemia (36%), increased alanine aminotransferase (34%), and hypocalcemia (32%). Dose reductions due to adverse reactions occurred in 12% of patients, whereas 27% of patients had alectinib dosing interrupted for adverse reactions. Permanent discontinuation of alectinib due to adverse reactions occurred in only 6% of patients. With the clinically meaningful ORR and DOR as well as the safety profile observed in these trials, alectinib was determined to have a favorable benefit-risk profile for the treatment of the indicated population. Clin Cancer Res; 22(21); 5171-6. ©2016 AACR. ©2016 American Association for Cancer Research.

  15. Interleukin-21 mRNA expression during virus infections

    DEFF Research Database (Denmark)

    Holm, Christian; Nyvold, Charlotte Guldborg; Paludan, Søren Riis

    2006-01-01

    and activational effects of IL-21 on different leukocytes come into play in vivo in an immune response has so far not been fully investigated. We show here for the first time in vivo, that IL-21 mRNA is produced in the spleen when mice are challenged with herpes simplex virus type 2 (HSV-2) or lymphocytic...... choriomeningitis virus (LCMV). We show in HSV-2 challenged mice that this production takes place in CD4+ T cell fractions and is absent in CD4+ T cell-depleted fractions. We also show that the peak of IL-21 mRNA production in both the HSV-2 and LCMV-challenged mice coincides with the onset of the adaptive immune...

  16. Collagen mRNA levels changes during colorectal cancer carcinogenesis

    DEFF Research Database (Denmark)

    Skovbjerg, Hanne; Anthonsen, Dorit; Lothe, Inger M B

    2009-01-01

    BACKGROUND: Invasive growth of epithelial cancers is a complex multi-step process which involves dissolution of the basement membrane. Type IV collagen is a major component in most basement membranes. Type VII collagen is related to anchoring fibrils and is found primarily in the basement membrane...... zone of stratified epithelia. Immunohistochemical studies have previously reported changes in steady-state levels of different alpha(IV) chains in several epithelial cancer types. In the present study we aimed to quantitatively determine the mRNA levels of type IV collagen (alpha1/alpha 4/alpha 6......) and type VII collagen (alpha1) during colorectal cancer carcinogenesis. METHODS: Using quantitative RT-PCR, we have determined the mRNA levels for alpha1(IV), alpha 4(IV), alpha 6(IV), and alpha1(VII) in colorectal cancer tissue (n = 33), adenomas (n = 29) and in normal tissue from the same individuals...

  17. Peptide inhibitors of botulinum neurotoxin by mRNA display

    International Nuclear Information System (INIS)

    Yiadom, Kwabena P.A.B.; Muhie, Seid; Yang, David C.H.

    2005-01-0