72,503 research outputs found
Multiplex sorting of foodborne pathogens by on-chip free-flow magnetophoresis
This study reports multiplex sorting of Salmonella typhimurium and Escherichia coli 0157, from broth cultures and from pathogen-spiked skinned chicken breast enrichment broths by employing microfluidic free-flow magnetophoresis. Magnetic beads of different sizes and magnetite content, namely Dynabeads anti-salmonella and Hyglos-Streptavidin beads together with the corresponding pathogen-specific biotinylated recombinant phages, were utilised as affinity solid phases for the capture and concentration of viable S. typhimurium and E. coli 0157. Following optimisation, the protocol was used to demonstrate continuous magnetophoretic sorting of the two pathogen-bound magnetic bead populations from mixed cultures and from pathogen-spiked chicken pre-enrichment broths under the influence of a Halbach magnet array. For example, in the la tter case, a pure population of S. typhimurium-bound Dynabeads (72% recovery) was sorted from a 100 μL mixture containing E. coli 0157-bound Hyglos beads (67% recovery) within 1.2 min in the presence of 0.1% Tween 20. This proof-of-principle study demonstrates how more than one pathogen type can be simultaneously isolated/enriched from a single food pre-enrichment broth (e.g. Universal food enrichment broth)
Rocaglates convert DEAD-box protein eIF4A into a sequence-selective translational repressor.
Rocaglamide A (RocA) typifies a class of protein synthesis inhibitors that selectively kill aneuploid tumour cells and repress translation of specific messenger RNAs. RocA targets eukaryotic initiation factor 4A (eIF4A), an ATP-dependent DEAD-box RNA helicase; its messenger RNA selectivity is proposed to reflect highly structured 5' untranslated regions that depend strongly on eIF4A-mediated unwinding. However, rocaglate treatment may not phenocopy the loss of eIF4A activity, as these drugs actually increase the affinity between eIF4A and RNA. Here we show that secondary structure in 5' untranslated regions is only a minor determinant for RocA selectivity and that RocA does not repress translation by reducing eIF4A availability. Rather, in vitro and in cells, RocA specifically clamps eIF4A onto polypurine sequences in an ATP-independent manner. This artificially clamped eIF4A blocks 43S scanning, leading to premature, upstream translation initiation and reducing protein expression from transcripts bearing the RocA-eIF4A target sequence. In elucidating the mechanism of selective translation repression by this lead anti-cancer compound, we provide an example of a drug stabilizing sequence-selective RNA-protein interactions
Identification of G-quadruplex DNA/RNA binders: Structure-based virtual screening and biophysical characterization
Background
Recent findings demonstrated that, in mammalian cells, telomere DNA (Tel) is transcribed into telomeric repeat-containing RNA (TERRA), which is involved in fundamental biological processes, thus representing a promising anticancer target. For this reason, the discovery of dual (as well as selective) Tel/TERRA G-quadruplex (G4) binders could represent an innovative strategy to enhance telomerase inhibition.
Methods
Initially, docking simulations of known Tel and TERRA active ligands were performed on the 3D coordinates of bimolecular G4 Tel DNA (Tel2) and TERRA (TERRA2). Structure-based pharmacophore models were generated on the best complexes and employed for the virtual screening of ~ 257,000 natural compounds. The 20 best candidates were submitted to biophysical assays, which included circular dichroism and mass spectrometry at different K+ concentrations.
Results
Three hits were here identified and characterized by biophysical assays. Compound 7 acts as dual Tel2/TERRA2 G4-ligand at physiological KCl concentration, while hits 15 and 17 show preferential thermal stabilization for Tel2 DNA. The different molecular recognition against the two targets was also discussed.
Conclusions
Our successful results pave the way to further lead optimization to achieve both increased selectivity and stabilizing effect against TERRA and Tel DNA G4s.
General significance
The current study combines for the first time molecular modelling and biophysical assays applied to bimolecular DNA and RNA G4s, leading to the identification of innovative ligand chemical scaffolds with a promising anticancer profile. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio
Structural and functional glycosphingolipidomics by glycoblotting with aminooxy-functionalized gold nanoparticle
Glycosphingolipids (GSLs) synthesized in Golgi apparatus by sequential transfer of sugar residues to a ceramide lipid anchor are ubiquitously distributing on vertebrate plasma membranes. Standardized method allowing for high throughput structural profiling and functional characterization of living cell surface GSLs is of growing importance because they function as crucial signal transduction molecules in various processes of dynamic cellular recognitions. However, methods are not available for amplification of GSLs, while the genomic scale PCR amplification permits large-scale mammalian proteomic analysis. Here we communicate such an approach to a novel "omics", namely glycosphingolipidomics based on the glycoblotting method. The method, which involves selective ozonolysis of the C-C double bond in ceramide moiety and subsequent enrichment of generated GSL-aldehydes by chemical ligation using aminooxy-functionalized gold nanoparticle (aoGNP) should be of widespread utility for identifying and characterizing whole GSLs present in the living cell surfaces. The present protocol using glycoblotting permitted MALDI-TOFMS-based high throughput structural profiling of mouse brain gangliosides such as GM1, GD1a/GD1b, and GT1b for adult or GD3 in case for embryonic mouse. When mouse melanoma B16 cells were subjected to this protocol, it was demonstrated that gangliosides enriched from the plasma membranes are only GM3 bearing microheteogeneity in the structure of N-acyl chain. Surface plasmon resonance analysis revealed that aoGNP displaying whole GSLs blotted from mouse B16 melanoma cell surfaces can be used directly for monitoring specific interaction with self-assembled monolayer (SAM) of Gg3Cer (gangliotriaosylceramide). Our results indicate that GSL-selective enrichment onto aoGNP from living cell surfaces allows for rapid reconstruction of plasma membrane models mimicking intact GSL-microdomain feasible for further structural and functional characterization
The bromodomain-containing protein Ibd1 links multiple chromatin related protein complexes to highly expressed genes in Tetrahymena thermophila
Background: The chromatin remodelers of the SWI/SNF family are critical
transcriptional regulators. Recognition of lysine acetylation through a
bromodomain (BRD) component is key to SWI/SNF function; in most eukaryotes,
this function is attributed to SNF2/Brg1.
Results: Using affinity purification coupled to mass spectrometry (AP-MS) we
identified members of a SWI/SNF complex (SWI/SNFTt) in Tetrahymena thermophila.
SWI/SNFTt is composed of 11 proteins, Snf5Tt, Swi1Tt, Swi3Tt, Snf12Tt, Brg1Tt,
two proteins with potential chromatin interacting domains and four proteins
without orthologs to SWI/SNF proteins in yeast or mammals. SWI/SNFTt subunits
localize exclusively to the transcriptionally active macronucleus (MAC) during
growth and development, consistent with a role in transcription. While
Tetrahymena Brg1 does not contain a BRD, our AP-MS results identified a
BRD-containing SWI/SNFTt component, Ibd1 that associates with SWI/SNFTt during
growth but not development. AP-MS analysis of epitope-tagged Ibd1 revealed it
to be a subunit of several additional protein complexes, including putative
SWRTt, and SAGATt complexes as well as a putative H3K4-specific histone methyl
transferase complex. Recombinant Ibd1 recognizes acetyl-lysine marks on
histones correlated with active transcription. Consistent with our AP-MS and
histone array data suggesting a role in regulation of gene expression, ChIP-Seq
analysis of Ibd1 indicated that it primarily binds near promoters and within
gene bodies of highly expressed genes during growth.
Conclusions: Our results suggest that through recognizing specific histones
marks, Ibd1 targets active chromatin regions of highly expressed genes in
Tetrahymena where it subsequently might coordinate the recruitment of several
chromatin remodeling complexes to regulate the transcriptional landscape of
vegetatively growing Tetrahymena cells.Comment: Published on BMC Epigenetics & Chromati
Aptamer-based therapeutics and their potential in radiopharmaceutical design
Aptamers, short, single stranded oligonucleotide entities, have been developed in the past 15 years against a plethora of targets and for a variety of applications. These range from inhibition of receptors and enzymes to the identification of small molecules in sensor applications, and from the development of targeted therapeutic to the design of novel diagnostic and imaging agents. Furthermore, aptamers have been designed for targets that cover a wide range of diseases, from HIV to tropical diseases, cancer and inflammation. Their easy development and flexibility of use and manipulation, offers further potential. In this paper we review their selection and consider some of the recent applications of aptamers in the design of radiopharmaceuticals for the targeted radiotherapy and medical imaging of disease
Drive, filter, and stick: A protein sorting conspiracy in photoreceptors.
The sorting of proteins into different functional compartments is a fundamental cellular task. In this issue, Maza et al. (2019. J. Cell Biol https://doi.org/10.1083/jcb.201906024) demonstrate that distinct protein populations are dynamically generated in specialized regions of photoreceptors via an interplay of protein-membrane affinity, impeded diffusion, and driven transport
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A DNA aptamer for binding and inhibition of DNA methyltransferase 1.
DNA methyltransferases (DNMTs) are enzymes responsible for establishing and maintaining DNA methylation in cells. DNMT inhibition is actively pursued in cancer treatment, dominantly through the formation of irreversible covalent complexes between small molecular compounds and DNMTs that suffers from low efficacy and high cytotoxicity, as well as no selectivity towards different DNMTs. Herein, we discover aptamers against the maintenance DNA methyltransferase, DNMT1, by coupling Asymmetrical Flow Field-Flow Fractionation (AF4) with Systematic Evolution of Ligands by EXponential enrichment (SELEX). One of the identified aptamers, Apt. #9, contains a stem-loop structure, and can displace the hemi-methylated DNA duplex, the native substrate of DNMT1, off the protein on sub-micromolar scale, leading for effective enzymatic inhibition. Apt. #9 shows no inhibition nor binding activity towards two de novo DNMTs, DNMT3A and DNMT3B. Intriguingly, it can enter cancer cells with over-expression of DNMT1, colocalize with DNMT1 inside the nuclei, and inhibit the activity of DNMT1 in cells. This study opens the possibility of exploring the aptameric DNMT inhibitors being a new cancer therapeutic approach, by modulating DNMT activity selectively through reversible interaction. The aptamers could also be valuable tools for study of the functions of DNMTs and the related epigenetic mechanisms
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