295 research outputs found
Mechanism of SigR inhibition by the anti-sigma factor RsrA from Streptomyces coelicolor
No full textThe extracytoplasmic function (ECF) sigma factor SigR (σR) is a bacterial transcription factor involved in the regulation of cytosolic redox homeostasis in Streptomyces coelicolor. SigR activity is regulated by its cognate anti-sigma factor RsrA. RsrA is a member of the zinc-anti-sigma (ZAS) family, which in its reduced state binds a single Zn2+ ion coordinated by 3 cysteine residues and a histidine. RsrA senses oxidative stress within the cytosol and responds by the formation of intramolecular disulphide bonds between its Zn-ligating cysteine residues. This results in the loss of the metal ion, a conformational rearrangement, and dissociation of SigR by an unknown mechanism. This releases SigR to bind RNA polymerase and direct the expression of a large regulon including that of the thioredoxin pathway, thereby re-establishing redox homeostasis. No structural information is available for the SigR-RsrA complex in Streptomyces coelicolor. Moreover, little is known of the mechanism by which RsrA associates with SigR and regulates gene transcription, or the role of zinc in SigR binding. In this work, a stopped-flow fluorescence-based assay was developed for investigating the kinetics of SigR-RsrA complex formation to aid our understanding of the role of the metal ion. Ultimately, trying to understand how oxidative stress causes dissociation of the Streptomyces coelicolor complex. Through tryptophan fluorescence emission quenching experiments, the SigR region 2 tryptophan (W88) and region 4 tryptophan (W159) were found to be sensitive to RsrA binding, likely denoting the binding sites for the anti-sigma factor. Pre-steady state experiments demonstrated that the metal ion has little effect on the rate of association (Kon 8.9 and 17.3x106 M-1s-1 at 35°C in the presence and absence of zinc, respectively) but a large effect on the dissociation rate constant (>500-fold faster in the absence of zinc at 35°C). Moreover, the kinetically derived Kds (0.27 nM and 62.4 nM with and without zinc respectively) agreed with the equilibrium constant for the complex derived through isothermal titration calorimetry (0.78 nM and 101.7 nM), validating the kinetic model for binding experiments. Zinc therefore has an important role in the structure of RsrA, affecting SigR-RsrA complex affinity, the off-rate for the complex and inhibition of SigR activity. Future work will exploit these assays to determine the role of zinc in redox sensing by RsrA
International Workshop on Algorithmic Bias in Search and Recommendation (BIAS)
No full textCreating efficient and effective search and recommendation algorithms has been the main objective of industry practitioners and academic researchers over the years. However, recent research has shown how these algorithms trained on historical data lead to models that might exacerbate existing biases and generate potentially negative outcomes. Defining, assessing, and mitigating these biases throughout experimental pipelines is a primary step for devising search and recommendation algorithms that can be responsibly deployed in real-world applications. This workshop aims to collect novel contributions in this field and offer a common ground for interested researchers and practitioners. More information about the workshop is available at https://biasinrecsys.github.io/sigir2024
Resveratrol, a natural antioxidant phytoalexin, and 4 synthetic derivatives can promote expression of gamma-globin gene in vitro
No full textResveratrol (3,4′5-trihydroxystilbene) is a naturally occurring phenolic compound. It is found in the skin of red grapes and in a variety of medicinal plants. Resveratrol presents many biological activities that have been applied against cardiovascular disease and cancer. One of Resveratrol properties is inhibition of the enzyme Ribonucleotide reductase. It has been found that some other Ribonucleotide reductase inhibitors like Hydroxyurea, Didox and Trimidox are also inducers of gammaglobin gene expression. In this study we investigated whether Resveratrol and 10 newly synthesized derivatives can act as inducers of gamma-globin gene and increase HbF levels. These derivatives were synthesized by the group of Dr. L.A Stivala (Department of Chemistry, University of Modena) by modifications of the polar groups of Resveratrol structure in an effort to increase its antioxidant activity. We examined the effect of Resveratrol and 10 of its derivatives on gamma- and beta-globin gene promoter activity in a dual luciferase assay in GM979 cells. Hydroxyurea, a known Ribonucleotide reductase inhibitor and HbF inducer and propionic acid, an HbF inducer, were used as positive control substances. The results showed that Resveratrol and 4 of its derivatives increased the gamma-globin gene promoter activity more than 3 fold. The compounds which exhibited an effect were subsequently tested in human erythroid liquid cultures derived from normal individuals and thalassemic patients. HbF content was measured by HPLC. Despite their promising effects on gamma-globin gene promoter activity in MEL cells, Resveratrol and its 4 active derivative compounds did not cause significant increase in HbF production in erythroid progenitor cells (BFUe)
Colicin E3 cleavage of 16S rRNA impairs decoding and accelerates tRNA translocation on Escherichia coli ribosomes.
No full textThe cytotoxin colicin E3 targets the 30S subunit of bacterial ribosomes and specifically cleaves 16S rRNA at the decoding centre, thereby inhibiting translation. Although the cleavage site is well known, it is not clear which step of translation is inhibited. We studied the effects of colicin E3 cleavage on ribosome functions by analysing individual steps of protein synthesis. We find that the cleavage affects predominantly the elongation step. The inhibitory effect of colicin E3 cleavage originates from the accumulation of sequential impaired decoding events, each of which results in low occupancy of the A site and, consequently, decreasing yield of elongating peptide. The accumulation leads to an almost complete halt of translation after reading of a few codons. The cleavage of 16S rRNA does not impair monitoring of codon-anticodon complexes or GTPase activation during elongation-factor Tu-dependent binding of aminoacyl-tRNA, but decreases the stability of the codon-recognition complex and slows down aminoacyl-tRNA accommodation in the A site. The tRNA-mRNA translocation is faster on colicin E3-cleaved than on intact ribosomes and is less sensitive to inhibition by the antibiotic viomycin
Ferredoxin containing bacteriocins suggest a novel mechanism of iron uptake in <i>Pectobacterium spp</i>
No full textIn order to kill competing strains of the same or closely related bacterial species, many bacteria produce potent narrow-spectrum protein antibiotics known as bacteriocins. Two sequenced strains of the phytopathogenic bacterium <i>Pectobacterium carotovorum</i> carry genes encoding putative bacteriocins which have seemingly evolved through a recombination event to encode proteins containing an N-terminal domain with extensive similarity to a [2Fe-2S] plant ferredoxin and a C-terminal colicin M-like catalytic domain. In this work, we show that these genes encode active bacteriocins, pectocin M1 and M2, which target strains of <i>Pectobacterium carotovorum</i> and <i>Pectobacterium atrosepticum</i> with increased potency under iron limiting conditions. The activity of pectocin M1 and M2 can be inhibited by the addition of spinach ferredoxin, indicating that the ferredoxin domain of these proteins acts as a receptor binding domain. This effect is not observed with the mammalian ferredoxin protein adrenodoxin, indicating that <i>Pectobacterium spp.</i> carries a specific receptor for plant ferredoxins and that these plant pathogens may acquire iron from the host through the uptake of ferredoxin. In further support of this hypothesis we show that the growth of strains of <i>Pectobacterium carotovorum</i> and <i>atrosepticum</i> that are not sensitive to the cytotoxic effects of pectocin M1 is enhanced in the presence of pectocin M1 and M2 under iron limiting conditions. A similar growth enhancement under iron limiting conditions is observed with spinach ferrodoxin, but not with adrenodoxin. Our data indicate that pectocin M1 and M2 have evolved to parasitise an existing iron uptake pathway by using a ferredoxin-containing receptor binding domain as a Trojan horse to gain entry into susceptible cells
Augmented Collaborative Spaces for Collective Sense Making: The Dicode Approach.
No full textSense making is at the heart of cognitively complex and data intensive decision making processes. It is often conducted in collective spaces through exchange of ideas, discussions, analysing situations, and exploring alternatives. This position paper proposes a novel approach to facilitate collective sense making via a collaboration platform which (a) offers multiple views to collaboration (including forums, mind maps, and argumentation structure), and (b) provides intelligent support to understand sense making behaviour by employing user and community modelling techniques. The work is conducted in the framework of the EU funded Dicode project, developing intelligent services for data-intensive collaboration and decision making
Thermodynamic consequences of bipartite immunity protein binding to the ribosomal ribonuclease colicin E3
No full textColicin E3 is a 60 kDa, multidomain protein antibiotic that targets its ribonuclease activity to an essential region of the 16S ribosomal RNA of Escherichia coli. To prevent suicide of the producing cell, synthesis of the toxin is accompanied by the production of a 10 kDa immunity protein (Im3) that binds strongly to the toxin and abolishes its enzymatic activity. In the present work, we study the interaction of Im3 with the isolated cytotoxic domain (E3 rRNase) and intact colicin E3 through presteady-state kinetics and thermodynamic measurements. The isolated E3 rRNase domain forms a high affinity complex with Im3 (K(d) = 10(-12) M, in 200 mM NaCl at pH 7.0 and 25 degrees C). The interaction of Im3 with full-length colicin E3 under the same conditions is however significantly stronger (K(d) = 10(-14) M). The difference in affinity arises almost wholly from a marked decrease in the dissociation rate constant for the full-length complex (8 x 10(-7) s(-1)) relative to the E3 rRNase-Im3 complex (1 x 10(-4) s(-1)), with their association rates comparable ( approximately 10(8) M(-1) s(-1)). Thermodynamic measurements show that complex formation is largely enthalpy driven. In light of the recently published crystal structure of the colicin E3-Im3 complex, the additional stabilization of the wild-type complex can be ascribed to the interaction of Im3 with the N-terminal translocation domain of the toxin. These observations suggest a mechanism whereby dissociation of the immunity protein prior to translocation into the target cell is facilitated by the loss of the Im3-translocation domain interaction
Recent trends in the gene therapy of β-thalassemia
No full textAlessia Finotti,1–3 Laura Breda,4 Carsten W Lederer,6,7 Nicoletta Bianchi,1–3 Cristina Zuccato,1–3 Marina Kleanthous,6,7 Stefano Rivella,4,5 Roberto Gambari1–3 1Laboratory for the Development of Gene and Pharmacogenomic Therapy of Thalassaemia, Biotechnology Centre of Ferrara University, Ferrara, Italy; 2Associazione Veneta per la Lotta alla Talassemia, Rovigo, Italy; 3Department of Life Sciences and Biotechnology, Section of Biochemistry and Molecular Biology, Ferrara University, Ferrara, Italy; 4Department of Pediatrics, Division of Haematology/Oncology, Weill Cornell Medical College, New York, NY, USA; 5Department of Cell and Development Biology, Weill Cornell Medical College, New York, NY, USA; 6Department of Molecular Genetics Thalassaemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus; 7Cyprus School of Molecular Medicine, Nicosia, Cyprus Abstract: The β-thalassemias are a group of hereditary hematological diseases caused by over 300 mutations of the adult β-globin gene. Together with sickle cell anemia, thalassemia syndromes are among the most impactful diseases in developing countries, in which the lack of genetic counseling and prenatal diagnosis have contributed to the maintenance of a very high frequency of these genetic diseases in the population. Gene therapy for β-thalassemia has recently seen steadily accelerating progress and has reached a crossroads in its development. Presently, data from past and ongoing clinical trials guide the design of further clinical and preclinical studies based on gene augmentation, while fundamental insights into globin switching and new technology developments have inspired the investigation of novel gene-therapy approaches. Moreover, human erythropoietic stem cells from β-thalassemia patients have been the cellular targets of choice to date whereas future gene-therapy studies might increasingly draw on induced pluripotent stem cells. Herein, we summarize the most significant developments in β-thalassemia gene therapy over the last decade, with a strong emphasis on the most recent findings, for β-thalassemia model systems; for β-, γ-, and anti-sickling β-globin gene addition and combinatorial approaches including the latest results of clinical trials; and for novel approaches, such as transgene-mediated activation of γ-globin and genome editing using designer nucleases. Keywords: Thalassemia, gene therapy, HbF induction, transcription factors, induced pluripotent stem cells, genome editing, TALEN, CRISPR, ZF
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