Background:Bladder cancer is the tenth most common cancer worldwide.Considering its high prevalence(vulnerability to multiple recurrences and progression despite local therapy),which leads to a substantial health serv...Background:Bladder cancer is the tenth most common cancer worldwide.Considering its high prevalence(vulnerability to multiple recurrences and progression despite local therapy),which leads to a substantial health service burden,it becomes necessary to develop new strategies to increase the effectiveness of bladder tumor therapy.Natural compounds with antiproliferative effect on cancer cells could be a good choice for co-adjuvant chemotherapy.Microorganisms are one of the main sources for natural compounds.Pigments extracted from the cold-adapted microorganisms can contribute to the development of a broader range of applications in biotechnology.Violacein is a purple pigment commonly produced by many bacterial strains.We have previously shown that very low concentrations of violacein extracted from Janthinobacterium sp.produced an antiproliferative effect on HeLa cells.Objective:With the aim to determine if violacein has an antiproliferative activity on bladder cancer cells,as well as to test if it has synergistic effects on cisplatin treated cells in vitro,T24 and 253J cell lines(derived bladder cancer cells from carcinoma in situ and retroperitoneal metastasis,respectively)were exposed to different concentrations of violacein in the presence or absence of cisplatin.Methods:i)Resazurin assay and flow cytometry were performed in two bladder cancer-derived cell lines,namely T24 and 253J,to see if violacein affects cell viability and induce cell death.ii)To find out whether violacein sensitizes bladder cancer cells to cisplatin,the drug interaction among different doses of cisplatin and violacein was analyzed,as well their combination index was determined.iii)The effect of violacein to induce primary genetic damage was determined through the analysis of induced micronuclei frequency and𝛾H2AX foci,as well as performing the comet assay.Results:The half-maximal inhibitory concentration of violacein at 24 h for both cell lines were around 500 nM,and decreased below 400 nM in combination with 10μM of cisplatin,indicating antiproliferative and sensitizing effects of violacein to cisplatin in both cell lines tested.A clear cell cycle delay,as well as an increase in the percentage of cell death was observed by flow cytometry at 300 nM of violacein,either alone or in combination with cisplatin.On the other hand,the analysis of the micronucleus frequency did not evidence an increase in genetic damage.Moreover,in combined treatments with cisplatin there was a slight decrease on micronucleus induction.Besides,the induction of genetic damage was not observed through comet assay when cells were treated with violacein alone,however,when cells were treated with violacein in the presence of cisplatin(10μM).The production of genetic damage was diminished in T24 or 253J cells.By the same token,increase in the frequency of𝛾H2AX foci by violacein was not observed at any tested dose in both cell lines.Conclusion:It was shown that violacein has an in vitro antiproliferative effect in bladder cancer cell lines,sensitizing them to cisplatin.Interestingly,at doses tested,violacein did not induce genotoxicity and reduce the genotoxic effect produced by cisplatin.展开更多
Violacein,a therapeutic pigment synthesised naturally in specific bacterial systems,is commercially unexplored owing to low titers.The current study aimed to formulate a suitable medium and to develop a fed-batch stra...Violacein,a therapeutic pigment synthesised naturally in specific bacterial systems,is commercially unexplored owing to low titers.The current study aimed to formulate a suitable medium and to develop a fed-batch strategy for improving the violacein productivity in a natural producer-Chromobacterium violaceum MTCC2656.The carbon and the nitrogen sources were extensively screened and their levels were optimised for maximal violacein production.The micronutrients in the medium were subjected to a two-level statistical optimisation using design of experiment approach and the final media formulation was validated.As a fed-batch approach,a combination of pulse feeding of glucose and tryptophan with optimised broth harvest of 60%(v/v)was attempted that achieved a titer of 1046±16 mg/L with productivity of 26.12±0.64 mg/L/h in each progressive cycle of fed-batch.The strategic sequential step of media formulation and fed-batch fermentation improved the violacein titer by~5 folds.Kinetic modelling was used to understand the enhancement in fermentation performance in both the batch and fed-batch processes.The findings from the current study would enable to understand and correlate the patterns of substrate uptake and violacein formation to establish strategies for enhancing overall productivity in the fermentation processes.展开更多
A bacterium B2 isolated from the Tianshan glacier of Xinjiang could produce blue pigments.According to 16S rDNA analysis, this isolate belonged to Duganella Genus .Two compounds were separated and purified from the cu...A bacterium B2 isolated from the Tianshan glacier of Xinjiang could produce blue pigments.According to 16S rDNA analysis, this isolate belonged to Duganella Genus .Two compounds were separated and purified from the cultivated Duganella B2 , named Blue-Ⅰand Blue-Ⅱ, respectively.From the spectra data of UV, MS and NMR of the compounds, Blue-Ⅰwas confirmed to be deoxyviolacein and Blue-Ⅱ was violacein.Blue-Ⅰand Blue-Ⅱ had the respective molecular weights of 327.2 and 343.2,and showed the characteristic absorption peaks at the respective 560 and 572 nm within the visible light range in ethanol solution.These results will be useful for developing the bioprocess for producing bacterial violacein.展开更多
Chromobacterium violaceum is a gram-negative bacterium, which has been used widely in microbiology labs involved in quorum sensing(QS) research. Among the QS-regulated traits of this bacterium, violacein production ha...Chromobacterium violaceum is a gram-negative bacterium, which has been used widely in microbiology labs involved in quorum sensing(QS) research. Among the QS-regulated traits of this bacterium, violacein production has received the maximum attention. Violacein production in this organism, however is not under sole control of QS machinery, and other QSregulated traits of this bacterium also need to be investigated in better detail. Though not often involved in human infections, this bacterium is being viewed as an emerging pathogen. This review attempts to highlight the recent research advances on Chromobacterium violaceum, with respect to violacein biosynthesis, development of various applications of this bacterium and its bioactive metabolite violacein, and its pathogenicity.展开更多
目的:从海洋真菌中筛选得到新型群体感应抑制剂,并对其进行活性评价。方法:首先利用紫色杆菌CV026指示菌株对真菌发酵粗提物进行活性筛选。其次通过18S r DNA序列比对进行菌种鉴定,同时采用硅胶柱色谱、凝胶柱色谱和高效液相色谱等技术...目的:从海洋真菌中筛选得到新型群体感应抑制剂,并对其进行活性评价。方法:首先利用紫色杆菌CV026指示菌株对真菌发酵粗提物进行活性筛选。其次通过18S r DNA序列比对进行菌种鉴定,同时采用硅胶柱色谱、凝胶柱色谱和高效液相色谱等技术并结合活性追踪检测分离纯化的活性化合物,再通过核磁质谱分析确定其结构。最后利用定量测定方法检测其在亚抑菌浓度下对紫色杆菌紫色菌素产量影响以及RT-PCR检测与QS调控相关基因的m RNA表达的影响。结果:从海藻共生菌中筛选到一株具有紫色杆菌群体感应抑制活性的海洋真菌Penicillium sp.QF046,其次级代谢产物中纯化到的活性化合物根据结构鉴定为一种星形曲霉毒素(asteltoxin)。该化合物对于紫色杆菌群体感应抑制浓度低于阳性对照化合物呋喃酮C30,同时抑制了群体感应相关基因m RNA水平的表达。结论:从海洋真菌Penicillium sp.QF046代谢产物中发现了一种抑制紫色杆菌群体感应的星形曲霉毒素,为进一步通过结构改造研发新型抗菌药物提供良好的前体化合物。展开更多
The communication system known as quorum sensing (QS) in gram-negative bacteria regulates biofilm formation and many other functions. The particularity of Pseudoalteromonas ulvae TC14 seems to be the absence of a LuxI...The communication system known as quorum sensing (QS) in gram-negative bacteria regulates biofilm formation and many other functions. The particularity of Pseudoalteromonas ulvae TC14 seems to be the absence of a LuxI inducer, and therefore the lack of production of small molecules of Acyl homoserine lactone (AHL). Previous studies had shown that it does not harbor the inducer of the regulatory molecule (AHL). Nevertheless, it is able to regulate these same functions (biofilm, violacein) via AHLs. This could mean the presence of specific receptors for these induction molecules, belonging to the LuxR family. The aim of this study was to test these hypotheses using molecular analysis. Genome sequencing of the P. ulvae TC14 strain was carried out by Molecular Research LP (MR DNA), using the Illumina Hiseq 2500 method. The results revealed the presence of 2,293,242 base pairs, i.e. 100% of the genomic volume. The number of coding gene sequences was 1983, and the Guanine + Cytosine (G+C) percentage of the base number was 41.55%, revealing stability in the sequenced genome. In order to verify the identity of the sequenced genome, a phylogenetic analysis based on RNA16S was carried out. This analysis resulted in 93% homology with the previously sequenced and characterized species Pseudoalteromonas tunicata, showing that it belongs to the genus Pseudoalteromonas. To these comparative results should be added those derived from genome analysis based on nucleotide percentage using the tools available at https://img.jgi.doe.gov/. The results showed that Pseudoalteromonas tunicata D2 has the highest percentage nucleotide identity (ANI) (75.7913%), followed by Pseudoalteromonas flavipulchra NCIMB2033 (72.2736%) and Pseudoalteromonas phenolica KCTC 12086 (71.6685%). Next, the search for the various genes involved in QS was carried out using sequence comparisons via the BLAST method. For LuxI, sequence templates from the genomes of Vibrio, Chromobacterium, Shewanella or even other Pseudoalteromonas species were screened against the TC14 genome. This yielded no conclusive results synonymous with the non-presence of LuxI in TC14 as assumed by previous research. The search for LuxR, on the other hand, gave rise to the presence of eight presumptive sequences. Molecular characterization of the presumptive LuxR sequences was carried out to assess their gene expression. This characterization showed expression of these LuxR homologous sequences in a range from o.11 to 5.33 picograms (pg). Even if these sequences were not analyzed in depth, the retro-transcription technique nevertheless showed a minimum of activity, which could enable us to distinguish them from inactive sequences. The next step was to compare the LuxRs found in TC14 with the so-called Solo LuxRs, which act autonomously. This study shows that P. ulvae TC14 is a bacterium with a particular LuxR-based communication system.展开更多
基金the National Agency for Re-search and Innovation(ANII-Uruguay).
文摘Background:Bladder cancer is the tenth most common cancer worldwide.Considering its high prevalence(vulnerability to multiple recurrences and progression despite local therapy),which leads to a substantial health service burden,it becomes necessary to develop new strategies to increase the effectiveness of bladder tumor therapy.Natural compounds with antiproliferative effect on cancer cells could be a good choice for co-adjuvant chemotherapy.Microorganisms are one of the main sources for natural compounds.Pigments extracted from the cold-adapted microorganisms can contribute to the development of a broader range of applications in biotechnology.Violacein is a purple pigment commonly produced by many bacterial strains.We have previously shown that very low concentrations of violacein extracted from Janthinobacterium sp.produced an antiproliferative effect on HeLa cells.Objective:With the aim to determine if violacein has an antiproliferative activity on bladder cancer cells,as well as to test if it has synergistic effects on cisplatin treated cells in vitro,T24 and 253J cell lines(derived bladder cancer cells from carcinoma in situ and retroperitoneal metastasis,respectively)were exposed to different concentrations of violacein in the presence or absence of cisplatin.Methods:i)Resazurin assay and flow cytometry were performed in two bladder cancer-derived cell lines,namely T24 and 253J,to see if violacein affects cell viability and induce cell death.ii)To find out whether violacein sensitizes bladder cancer cells to cisplatin,the drug interaction among different doses of cisplatin and violacein was analyzed,as well their combination index was determined.iii)The effect of violacein to induce primary genetic damage was determined through the analysis of induced micronuclei frequency and𝛾H2AX foci,as well as performing the comet assay.Results:The half-maximal inhibitory concentration of violacein at 24 h for both cell lines were around 500 nM,and decreased below 400 nM in combination with 10μM of cisplatin,indicating antiproliferative and sensitizing effects of violacein to cisplatin in both cell lines tested.A clear cell cycle delay,as well as an increase in the percentage of cell death was observed by flow cytometry at 300 nM of violacein,either alone or in combination with cisplatin.On the other hand,the analysis of the micronucleus frequency did not evidence an increase in genetic damage.Moreover,in combined treatments with cisplatin there was a slight decrease on micronucleus induction.Besides,the induction of genetic damage was not observed through comet assay when cells were treated with violacein alone,however,when cells were treated with violacein in the presence of cisplatin(10μM).The production of genetic damage was diminished in T24 or 253J cells.By the same token,increase in the frequency of𝛾H2AX foci by violacein was not observed at any tested dose in both cell lines.Conclusion:It was shown that violacein has an in vitro antiproliferative effect in bladder cancer cell lines,sensitizing them to cisplatin.Interestingly,at doses tested,violacein did not induce genotoxicity and reduce the genotoxic effect produced by cisplatin.
基金funded by the All-India Council of Technical Education,New Delhi,India。
文摘Violacein,a therapeutic pigment synthesised naturally in specific bacterial systems,is commercially unexplored owing to low titers.The current study aimed to formulate a suitable medium and to develop a fed-batch strategy for improving the violacein productivity in a natural producer-Chromobacterium violaceum MTCC2656.The carbon and the nitrogen sources were extensively screened and their levels were optimised for maximal violacein production.The micronutrients in the medium were subjected to a two-level statistical optimisation using design of experiment approach and the final media formulation was validated.As a fed-batch approach,a combination of pulse feeding of glucose and tryptophan with optimised broth harvest of 60%(v/v)was attempted that achieved a titer of 1046±16 mg/L with productivity of 26.12±0.64 mg/L/h in each progressive cycle of fed-batch.The strategic sequential step of media formulation and fed-batch fermentation improved the violacein titer by~5 folds.Kinetic modelling was used to understand the enhancement in fermentation performance in both the batch and fed-batch processes.The findings from the current study would enable to understand and correlate the patterns of substrate uptake and violacein formation to establish strategies for enhancing overall productivity in the fermentation processes.
文摘A bacterium B2 isolated from the Tianshan glacier of Xinjiang could produce blue pigments.According to 16S rDNA analysis, this isolate belonged to Duganella Genus .Two compounds were separated and purified from the cultivated Duganella B2 , named Blue-Ⅰand Blue-Ⅱ, respectively.From the spectra data of UV, MS and NMR of the compounds, Blue-Ⅰwas confirmed to be deoxyviolacein and Blue-Ⅱ was violacein.Blue-Ⅰand Blue-Ⅱ had the respective molecular weights of 327.2 and 343.2,and showed the characteristic absorption peaks at the respective 560 and 572 nm within the visible light range in ethanol solution.These results will be useful for developing the bioprocess for producing bacterial violacein.
文摘Chromobacterium violaceum is a gram-negative bacterium, which has been used widely in microbiology labs involved in quorum sensing(QS) research. Among the QS-regulated traits of this bacterium, violacein production has received the maximum attention. Violacein production in this organism, however is not under sole control of QS machinery, and other QSregulated traits of this bacterium also need to be investigated in better detail. Though not often involved in human infections, this bacterium is being viewed as an emerging pathogen. This review attempts to highlight the recent research advances on Chromobacterium violaceum, with respect to violacein biosynthesis, development of various applications of this bacterium and its bioactive metabolite violacein, and its pathogenicity.
文摘The communication system known as quorum sensing (QS) in gram-negative bacteria regulates biofilm formation and many other functions. The particularity of Pseudoalteromonas ulvae TC14 seems to be the absence of a LuxI inducer, and therefore the lack of production of small molecules of Acyl homoserine lactone (AHL). Previous studies had shown that it does not harbor the inducer of the regulatory molecule (AHL). Nevertheless, it is able to regulate these same functions (biofilm, violacein) via AHLs. This could mean the presence of specific receptors for these induction molecules, belonging to the LuxR family. The aim of this study was to test these hypotheses using molecular analysis. Genome sequencing of the P. ulvae TC14 strain was carried out by Molecular Research LP (MR DNA), using the Illumina Hiseq 2500 method. The results revealed the presence of 2,293,242 base pairs, i.e. 100% of the genomic volume. The number of coding gene sequences was 1983, and the Guanine + Cytosine (G+C) percentage of the base number was 41.55%, revealing stability in the sequenced genome. In order to verify the identity of the sequenced genome, a phylogenetic analysis based on RNA16S was carried out. This analysis resulted in 93% homology with the previously sequenced and characterized species Pseudoalteromonas tunicata, showing that it belongs to the genus Pseudoalteromonas. To these comparative results should be added those derived from genome analysis based on nucleotide percentage using the tools available at https://img.jgi.doe.gov/. The results showed that Pseudoalteromonas tunicata D2 has the highest percentage nucleotide identity (ANI) (75.7913%), followed by Pseudoalteromonas flavipulchra NCIMB2033 (72.2736%) and Pseudoalteromonas phenolica KCTC 12086 (71.6685%). Next, the search for the various genes involved in QS was carried out using sequence comparisons via the BLAST method. For LuxI, sequence templates from the genomes of Vibrio, Chromobacterium, Shewanella or even other Pseudoalteromonas species were screened against the TC14 genome. This yielded no conclusive results synonymous with the non-presence of LuxI in TC14 as assumed by previous research. The search for LuxR, on the other hand, gave rise to the presence of eight presumptive sequences. Molecular characterization of the presumptive LuxR sequences was carried out to assess their gene expression. This characterization showed expression of these LuxR homologous sequences in a range from o.11 to 5.33 picograms (pg). Even if these sequences were not analyzed in depth, the retro-transcription technique nevertheless showed a minimum of activity, which could enable us to distinguish them from inactive sequences. The next step was to compare the LuxRs found in TC14 with the so-called Solo LuxRs, which act autonomously. This study shows that P. ulvae TC14 is a bacterium with a particular LuxR-based communication system.
