The Experts below are selected from a list of 318 Experts worldwide ranked by ideXlab platform
Victor C. Yang - One of the best experts on this subject based on the ideXlab platform.
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recombinant tat gelonin fusion Toxin Synthesis and characterization of heparin protamine regulated cell transduction
Journal of Biomedical Materials Research Part A, 2015Co-Authors: Meong Cheol Shin, Jingwen Zhao, Jian Zhang, Yongzhuo Huang, Mei Wang, Kyoung Ah Min, Victor C. YangAbstract:Protein Toxins, such as gelonin, are highly desirable anti-cancer drug candidates due to their unparalleled potency and repetitive reaction mechanism in inhibiting protein translation. However, for its potential application in cancer therapy, there remains the cell membrane barrier that allows permeation of only small molecules, which must be overcome. To address this challenge, we conjugated gelonin with a protein transduction domain (PTD), the TAT peptide, via genetic recombination. The chimeric TAT-gelonin fusion protein (TAT-Gel) retained equipotent N-glycosidase activity yet displayed greater cell uptake than unmodified recombinant gelonin (rGel), thereby yielding a significantly augmented cytotoxic activity. Remarkably, TAT-Gel displayed up to 177-fold lower IC₅₀ (avg. 54.3 nM) than rGel (avg. IC₅₀ : 3640 nM) in tested cell lines. This enhanced cytotoxicity, however, also raised potential toxicity concerns due to the non-selectivity of PTD in its mediated cell transduction. To solve this problem, we investigated the plausibility of regulating the cell transduction of TAT-Gel via a reversible masking using heparin and protamine. Here, we demonstrated, both in vitro and in vivo, that the cell transduction of TAT-Gel can be completely curbed with heparin and yet this heparin block can be efficiently reversed by the addition of protamine. This reversible tight regulation of the cell transduction of TAT-Gel by heparin and protamine sheds light of possible application of TAT-Gel in achieving a highly effective yet safe drug therapy for the treatment of tumors.
Christopher J. Gobler - One of the best experts on this subject based on the ideXlab platform.
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nitrogen limitation Toxin Synthesis potential and toxicity of cyanobacterial populations in lake okeechobee and the st lucie river estuary florida during the 2016 state of emergency event
PLOS ONE, 2018Co-Authors: Benjamin J. Kramer, Timothy W. Davis, Kevin A. Meyer, Barry H. Rosen, Jennifer A. Goleski, Gregory J. Dick, Genesok Oh, Christopher J. GoblerAbstract:Lake Okeechobee, FL, USA, has been subjected to intensifying cyanobacterial blooms that can spread to the adjacent St. Lucie River and Estuary via natural and anthropogenically-induced flooding events. In July 2016, a large, toxic cyanobacterial bloom occurred in Lake Okeechobee and throughout the St. Lucie River and Estuary, leading Florida to declare a state of emergency. This study reports on measurements and nutrient amendment experiments performed in this freshwater-estuarine ecosystem (salinity 0–25 PSU) during and after the bloom. In July, all sites along the bloom exhibited dissolved inorganic nitrogen-to-phosphorus ratios 95%) phytoplankton inventories from the lake to the central part of the estuary. Chlorophyll a and microcystin concentrations peaked (100 and 34 μg L-1, respectively) within Lake Okeechobee and decreased eastwards. Metagenomic analyses indicated that genes associated with the production of microcystin (mcyE) and the algal neuroToxin saxiToxin (sxtA) originated from Microcystis and multiple diazotrophic genera, respectively. There were highly significant correlations between levels of total nitrogen, microcystin, and microcystin Synthesis gene abundance across all surveyed sites (p < 0.001), suggesting high levels of nitrogen supported the production of microcystin during this event. Consistent with this, experiments performed with low salinity water from the St. Lucie River during the event indicated that algal biomass was nitrogen-limited. In the fall, densities of Microcystis and concentrations of microcystin were significantly lower, green algae co-dominated with cyanobacteria, and multiple algal groups displayed nitrogen-limitation. These results indicate that monitoring and regulatory strategies in Lake Okeechobee and the St. Lucie River and Estuary should consider managing loads of nitrogen to control future algal and microcystin-producing cyanobacterial blooms.
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Nitrogen limitation, Toxin Synthesis potential, and toxicity of cyanobacterial populations in Lake Okeechobee and the St. Lucie River Estuary, Florida, during the 2016 state of emergency event
2018Co-Authors: Benjamin J. Kramer, Timothy W. Davis, Kevin A. Meyer, Barry H. Rosen, Jennifer A. Goleski, Gregory J. Dick, Christopher J. GoblerAbstract:Lake Okeechobee, FL, USA, has been subjected to intensifying cyanobacterial blooms that can spread to the adjacent St. Lucie River and Estuary via natural and anthropogenically-induced flooding events. In July 2016, a large, toxic cyanobacterial bloom occurred in Lake Okeechobee and throughout the St. Lucie River and Estuary, leading Florida to declare a state of emergency. This study reports on measurements and nutrient amendment experiments performed in this freshwater-estuarine ecosystem (salinity 0–25 PSU) during and after the bloom. In July, all sites along the bloom exhibited dissolved inorganic nitrogen-to-phosphorus ratios < 6, while Microcystis dominated (> 95%) phytoplankton inventories from the lake to the central part of the estuary. Chlorophyll a and microcystin concentrations peaked (100 and 34 μg L-1, respectively) within Lake Okeechobee and decreased eastwards. Metagenomic analyses indicated that genes associated with the production of microcystin (mcyE) and the algal neuroToxin saxiToxin (sxtA) originated from Microcystis and multiple diazotrophic genera, respectively. There were highly significant correlations between levels of total nitrogen, microcystin, and microcystin Synthesis gene abundance across all surveyed sites (p < 0.001), suggesting high levels of nitrogen supported the production of microcystin during this event. Consistent with this, experiments performed with low salinity water from the St. Lucie River during the event indicated that algal biomass was nitrogen-limited. In the fall, densities of Microcystis and concentrations of microcystin were significantly lower, green algae co-dominated with cyanobacteria, and multiple algal groups displayed nitrogen-limitation. These results indicate that monitoring and regulatory strategies in Lake Okeechobee and the St. Lucie River and Estuary should consider managing loads of nitrogen to control future algal and microcystin-producing cyanobacterial blooms.
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daily transcriptome changes reveal the role of nitrogen in controlling microcystin Synthesis and nutrient transport in the toxic cyanobacterium microcystis aeruginosa
BMC Genomics, 2015Co-Authors: Matthew J Harke, Christopher J. GoblerAbstract:While transcriptomics have become a valuable tool for linking physiology and ecology in aquatic microbes, the temporal dynamics of global transcriptomic patterns in Microcystis have rarely been assessed. Furthermore, while many microbial studies have explored expression of nutrient transporter genes, few studies have concurrently measured nutrient assimilation rates. Here, we considered how the global transcriptomic patterns and physiology of the cyanobacterium, Microcystis aeruginosa, changed daily as cells were grown from replete to deficient nitrogen (N) conditions and then back to replete conditions. During N deprivation, Microcystis downregulated genes involved in photoSynthesis and respiration, carbon acquisition, lipid metabolism, and amino acid bioSynthesis while upregulating genes involved in N acquisition and transport. With increasing N stress, both the strength of expression and number of genes being differentially expressed increased, until N was restored at which point these patterns reversed. Uptake of 15N-labeled nitrate, ammonium and urea reflected differential expression of genes encoding transporters for these nutrients, with Microcystis appearing to preferentially increase transcription of ammonium and urea transporters and uptake of these compounds during N deprivation. Nitrate uptake and nitrate transporter expression were correlated for one set of transporters but not another, indicating these were high and low affinity nitrate transporters, respectively. Concentrations of microcystin per cell decreased during N deprivation and increased upon N restoration. However, the transcript abundance of genes involved in the Synthesis of this compound was complex, as microcystin synthetase genes involved in peptide Synthesis were downregulated under N deprivation while genes involved in tailoring and transport were upregulated, suggesting modification of the microcystin molecule under N stress as well as potential alternative functions for these genes and/or this Toxin. Collectively, this study highlights the complex choreography of gene expression, cell physiology, and Toxin Synthesis that dynamic N levels can elicit in this ecologically important cyanobacterium. Differing expression patterns of genes within the microcystin synthetase operon in response to changing N levels revealed the potential limitations drawing conclusions based on only one gene in this operon.
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interactive influences of nutrient loading zooplankton grazing and microcystin synthetase gene expression on cyanobacterial bloom dynamics in a eutrophic new york lake
Harmful Algae, 2007Co-Authors: Christopher J. Gobler, Timothy Walter Davis, Kathryn J. Coyne, Gregory L BoyerAbstract:We investigated the dynamics and toxicity of cyanobacteria populations in Lake Agawam, a eutrophic New York lake, and concurrently conducted experiments to evaluate the contrasting effects of zooplankton (mesozooplankton and microzooplankton) grazing and nutrient loading on the abundance and Toxin content of cyanobacteria populations. Molecular techniques were used to assess the presence and expression of the microcystin synthetase gene. Lake Agawam hosted dense blooms (> 105 cells mL(-1)) of Microcystis sp. and Anabaena sp. with consistently elevated levels of microcystin (1.0-25 mu g L-1) and lower levels of anaToxin-a (similar to 1.0 mu g L-1 during late summer only). Polymerase chain reaction (PCR) analysis targeting the microcystin synthetase gene (mcyE) indicated that Microcystis sp., and not Anabaena sp., was responsible for microcystin production in this system. Moreover, reverse transcriptase PCR (RT-PCR) indicated that the Microcystis population expressed the mcyE gene during summer months, but that gene expression declined to undetectable levels during the fall as in situ cell densities in the lake declined. During summer, when there was elevated Microcystis densities (> 8 x 10(4) cells mL(-1)) expressing meyE, experimental Daphnia sp. enrichment did not alter algal biomass (100% of experiments; n = 6). However, during fall months when the mcyE gene expression was not detected and Microcystis densities were lower and declining (4 x 10(3) to 5 x 10(4) Cell S mL(-1)), zooplankton enrichment yielded significantly reduced (p < 0.05) cyanobacteria biomass relative to control treatments in most experiments (80%; n = 4). In contrast to mesozooplankton, microzooplankton actively grazed algal biomass at significant rates (1.2 +/- 0.3 day(-1)) throughout the study. Microcystis and other cyanobacterial populations did not respond to nutrient amendments during early summer but experienced significantly increased growth rates and Toxin concentrations (microcystin and anaToxin-a) during late summer and early fall nitrogen enrichment experiments. As such, the dominance of Microcystis sp. blooms during the summer was linked to nutrient replete conditions and the suppression of mesozooplankton (but not microzooplankton) grazing which itself appeared to be influenced by cellular Toxin Synthesis by Microcystis. The demise of the bloom was associated with N-limitation which reduced growth rates and Toxin production by Microcystis and in turn may have permitted zooplankton to graze cells.
Meong Cheol Shin - One of the best experts on this subject based on the ideXlab platform.
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recombinant tat gelonin fusion Toxin Synthesis and characterization of heparin protamine regulated cell transduction
Journal of Biomedical Materials Research Part A, 2015Co-Authors: Meong Cheol Shin, Jingwen Zhao, Jian Zhang, Yongzhuo Huang, Mei Wang, Kyoung Ah Min, Victor C. YangAbstract:Protein Toxins, such as gelonin, are highly desirable anti-cancer drug candidates due to their unparalleled potency and repetitive reaction mechanism in inhibiting protein translation. However, for its potential application in cancer therapy, there remains the cell membrane barrier that allows permeation of only small molecules, which must be overcome. To address this challenge, we conjugated gelonin with a protein transduction domain (PTD), the TAT peptide, via genetic recombination. The chimeric TAT-gelonin fusion protein (TAT-Gel) retained equipotent N-glycosidase activity yet displayed greater cell uptake than unmodified recombinant gelonin (rGel), thereby yielding a significantly augmented cytotoxic activity. Remarkably, TAT-Gel displayed up to 177-fold lower IC₅₀ (avg. 54.3 nM) than rGel (avg. IC₅₀ : 3640 nM) in tested cell lines. This enhanced cytotoxicity, however, also raised potential toxicity concerns due to the non-selectivity of PTD in its mediated cell transduction. To solve this problem, we investigated the plausibility of regulating the cell transduction of TAT-Gel via a reversible masking using heparin and protamine. Here, we demonstrated, both in vitro and in vivo, that the cell transduction of TAT-Gel can be completely curbed with heparin and yet this heparin block can be efficiently reversed by the addition of protamine. This reversible tight regulation of the cell transduction of TAT-Gel by heparin and protamine sheds light of possible application of TAT-Gel in achieving a highly effective yet safe drug therapy for the treatment of tumors.
Michael L Brosnahan - One of the best experts on this subject based on the ideXlab platform.
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evaluation of sxta and rdna qpcr assays through monitoring of an inshore bloom of alexandrium catenella group 1
Scientific Reports, 2019Co-Authors: Shauna A. Murray, Donald M. Anderson, Gurjeet S Kohli, Rendy Ruvindy, Michael L BrosnahanAbstract:Alexandrium catenella (formerly A. tamarense Group 1, or A. fundyense) is the leading cause of Paralytic Shellfish Poisoning in North and South America, Europe, Africa, Australia and Asia. The quantification of A.catenella via sxtA, a gene involved in Paralytic Shellfish Toxin Synthesis, may be a promising approach, but has not been evaluated in situ on blooms of A. catenella, in which cell abundances may vary from not detectable to in the order of 106 cells L−1. In this study, we compared sxtA assay performance to a qPCR assay targeted to a species-specific region of ribosomal DNA (rDNA) and an established fluorescent in situ hybridization (FISH) microscopy method. Passing-Bablok regression analyses revealed the sxtA assay to overestimate abundances when <5 cell equivalents A. catenella DNA were analysed, but otherwise was closer to microscopy estimates than the rDNA assay, which overestimated abundance across the full range of concentrations analysed, indicative of a copy number difference between the bloom population and a culture used for assay calibration a priori. In contrast, the sxtA assay performed more consistently, indicating less copy number variation. The sxtA assay was generally reliable, fast and effective in quantifying A. catenella and was predictive of PST contamination of shellfish.
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Evaluation of sxtA and rDNA qPCR assays through monitoring of an inshore bloom of Alexandrium catenella Group 1
'Springer Science and Business Media LLC', 2019Co-Authors: Sa Murray, Ruvindy R, Gs Kohli, Dm Anderson, Michael L BrosnahanAbstract:© 2019, The Author(s). Alexandrium catenella (formerly A. tamarense Group 1, or A. fundyense) is the leading cause of Paralytic Shellfish Poisoning in North and South America, Europe, Africa, Australia and Asia. The quantification of A.catenella via sxtA, a gene involved in Paralytic Shellfish Toxin Synthesis, may be a promising approach, but has not been evaluated in situ on blooms of A. catenella, in which cell abundances may vary from not detectable to in the order of 106 cells L−1. In this study, we compared sxtA assay performance to a qPCR assay targeted to a species-specific region of ribosomal DNA (rDNA) and an established fluorescent in situ hybridization (FISH) microscopy method. Passing-Bablok regression analyses revealed the sxtA assay to overestimate abundances when
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formal revision of the alexandrium tamarense species complex dinophyceae taxonomy the introduction of five species with emphasis on molecular based rdna classification
Protist, 2014Co-Authors: Uwe John, Michael L Brosnahan, Shauna A. Murray, Wayne R Litaker, Marina Montresor, Donald M. AndersonAbstract:The Alexandrium tamarense species complex is one of the most studied marine dinoflagellate groups due to its ecological, toxicological and economic importance. Several members of this complex produce saxiToxin and its congeners - potent neuroToxins that cause paralytic shellfish poisoning. Isolates from this complex are assigned to A. tamarense, A. fundyense, or A. catenella based on two main morphological characters: the ability to form chains and the presence/absence of a ventral pore between Plates 1′ and 4′. However, studies have shown that these characters are not consistent and/or distinctive. Further, phylogenies based on multiple regions in the rDNA operon indicate that the sequences from morphologically indistinguishable isolates partition into five clades. These clades were initially named based on their presumed geographic distribution, but recently were renamed as Groups I-V following the discovery of sympatry among some groups. In this study we present data on morphology, ITS/5.8S genetic distances, ITS2 compensatory base changes, mating incompatibilities, toxicity, the sxtA Toxin Synthesis gene, and rDNA phylogenies. All results were consistent with each group representing a distinct cryptic species. Accordingly, the groups were assigned species names as follows: Group I, A. fundyense; Group II, A. mediterraneum; Group III, A. tamarense; Group IV, A. pacificum; Group V, A. australiense.
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Evolution of SaxiToxin Synthesis in Cyanobacteria and Dinoflagellates
Molecular Biology and Evolution, 2012Co-Authors: Jeremiah D. Hackett, Michael L Brosnahan, Donald M. Anderson, David M Kulis, Jennifer H. Wisecaver, Debashish Bhattacharya, F. Gerald Plumley, Deana L. ErdnerAbstract:Dinoflagellates produce a variety of toxic secondary metabolites that have a significant impact on marine ecosystems and fisheries. SaxiToxin (STX), the cause of paralytic shellfish poisoning, is produced by three marine dinoflagellate genera and is also made by some freshwater cyanobacteria. Genes involved in STX Synthesis have been identified in cyanobacteria but are yet to be reported in the massive genomes of dinoflagellates. We have assembled comprehensive transcriptome data sets for several STX-producing dinoflagellates and a related non-toxic species and have identified 265 putative homologs of 13 cyanobacterial STX Synthesis genes, including all of the genes directly involved in Toxin Synthesis. Putative homologs of four proteins group closely in phylogenies with cyanobacteria and are likely the functional homologs of sxtA, sxtG, and sxtB in dinoflagellates. However, the phylogenies do not support the transfer of these genes directly between toxic cyanobacteria and dinoflagellates. SxtA is split into two proteins in the dinoflagellates corresponding to the N-terminal portion containing the methyltransferase and acyl carrier protein domains and a C-terminal portion with the aminotransferase domain. Homologs of sxtB and N-terminal sxtA are present in non-toxic strains, suggesting their functions may not be limited to saxiToxin production. Only homologs of the C-terminus of sxtA and sxtG were found exclusively in toxic strains. A more thorough survey of STX+ dinoflagellates will be needed to determine if these two genes may be specific to SXT production in dinoflagellates. The A. tamarense transcriptome does not contain homologs for the remaining STX genes. Nevertheless, we identified candidate genes with similar predicted biochemical activities that account for the missing functions. These results suggest that the STX Synthesis pathway was likely assembled independently in the distantly related cyanobacteria and dinoflagellates, although using some evolutionarily related proteins. The biological role of STX is not well understood in either cyanobacteria or dinoflagellates. However, STX production in these two ecologically distinct groups of organisms suggests that this Toxin confers a benefit to producers that we do not yet fully understand.
Bruno Dupuy - One of the best experts on this subject based on the ideXlab platform.
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The Regulatory Networks That Control Clostridium difficile Toxin Synthesis
Toxins, 2016Co-Authors: Isabelle Martin-verstraete, Johann Peltier, Bruno DupuyAbstract:The pathogenic clostridia cause many human and animal diseases, which typically arise as a consequence of the production of potent exoToxins. Among the enterotoxic clostridia, Clostridium difficile is the main causative agent of nosocomial intestinal infections in adults with a compromised gut microbiota caused by antibiotic treatment. The symptoms of C. difficile infection are essentially caused by the production of two exoToxins: TcdA and TcdB. Moreover, for severe forms of disease, the spectrum of diseases caused by C. difficile has also been correlated to the levels of Toxins that are produced during host infection. This observation strengthened the idea that the regulation of Toxin Synthesis is an important part of C. difficile pathogenesis. This review summarizes our current knowledge about the regulators and sigma factors that have been reported to control Toxin gene expression in response to several environmental signals and stresses, including the availability of certain carbon sources and amino acids, or to signaling molecules, such as the autoinducing peptides of quorum sensing systems. The overlapping regulation of key metabolic pathways and Toxin Synthesis strongly suggests that Toxin production is a complex response that is triggered by bacteria in response to particular states of nutrient availability during infection.
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The key sigma factor of transition phase, SigH, controls sporulation, metabolism, and virulence factor expression in Clostridium difficile.
Journal of Bacteriology, 2011Co-Authors: Laure Saujet, Bruno Dupuy, Marc Monot, Olga Soutourina, Isabelle Martin-verstraeteAbstract:Toxin Synthesis in Clostridium difficile increases as cells enter into stationary phase. We first compared the expression profiles of strain 630E during exponential growth and at the onset of stationary phase and showed that genes involved in sporulation, cellular division, and motility, as well as carbon and amino acid metabolism, were differentially expressed under these conditions. We inactivated the sigH gene, which encodes an alternative sigma factor involved in the transition to post-exponential phase in Bacillus subtilis. Then, we compared the expression profiles of strain 630E and the sigH mutant after 10 h of growth. About 60% of the genes that were differentially expressed between exponential and stationary phases, including genes involved in motility, sporulation, and metabolism, were regulated by SigH, which thus appears to be a key regulator of the transition phase in C. difficile. SigH positively controls several genes required for sporulation. Accordingly, sigH inactivation results in an asporogeneous phenotype. The spo0A and CD2492 genes, encoding the master regulator of sporulation and one of its associated kinases, and the spoIIA operon were transcribed from a SigH-dependent promoter. The expression of tcdA and tcdB, encoding the Toxins, and of tcdR, encoding the sigma factor required for Toxin production, increased in a sigH mutant. Finally, SigH regulates the expression of genes encoding surface-associated proteins, such as the Cwp66 adhesin, the S-layer precursor, and the flagellum components. Among the 286 genes positively regulated by SigH, about 40 transcriptional units presenting a SigH consensus in their promoter regions are good candidates for direct SigH targets.
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Clostridium difficile Toxin Synthesis is negatively regulated by TcdC
Journal of Medical Microbiology, 2008Co-Authors: Bruno Dupuy, R. Govind, A. Antunes, S. MatamourosAbstract:Clostridium difficile Toxin Synthesis is growth phase-dependent and is regulated by various environmental signals. The Toxin genes tcdA and tcdB are located in a pathogenicity locus, which also includes three accessory genes, tcdR, tcdC and tcdE. TcdR has been shown to act as an alternative sigma factor that mediates positive regulation of both the Toxin genes and its own gene. The tcdA, tcdB and tcdR genes are transcribed during the stationary growth phase. The tcdC gene, however, is expressed during exponential phase. This expression pattern suggested that TcdC may act as a negative regulator of Toxin gene expression. TcdC is a small acidic protein without any conserved DNA-binding motif. It is able to form dimers and its N-terminal region includes a putative transmembrane domain. Genetic and biochemical evidence showed that TcdC negatively regulates C. difficile Toxin Synthesis by interfering with the ability of TcdR-containing RNA polymerase to recognize the tcdA and tcdB promoters. In addition, the C. difficile NAP1/027 epidemic strains that produce higher levels of Toxins have mutations in tcdC. Interestingly, a frameshift mutation at position 117 of the tcdC coding sequence seems to be, at least in part, responsible for the hypertoxigenicity phenotype of these epidemic strains.
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Regulation of Toxin Synthesis in Clostridium difficile by an alternative RNA polymerase sigma factor
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: N. Mani, Bruno DupuyAbstract:Clostridium difficile, a causative agent of antibiotic-associated diarrhea and its potentially lethal form, pseudomembranous colitis, produces two large protein Toxins that are responsible for the cellular damage associated with the disease. The level of Toxin production appears to be critical for determining the severity of the disease, but the mechanism by which Toxin Synthesis is regulated is unknown. The product of a gene, txeR, that lies just upstream of the tox gene cluster was shown to be needed for tox gene expression in vivo and to activate promoter-specific transcription of the tox genes in vitro in conjunction with RNA polymerases from C. difficile, Bacillus subtilis, or Escherichia coli. TxeR was shown to function as an alternative sigma factor for RNA polymerase. Because homologs of TxeR regulate Synthesis of Toxins and a bacteriocin in other Clostridium species, TxeR appears to be a prototype for a novel mode of regulation of Toxin genes.