Antibacterial isoeugenol coating on stainless steel and polyethylene surfaces prevents biofilm growth.

AIMS: Pathogenic bacteria can spread between individuals or between food items via the surfaces they share. Limiting the survival of pathogens on surfaces, therefore, presents an opportunity to limit at least one route of how pathogens spread. In this study, we propose that a simple coating with the essential oil isoeugenol can be used to circumvent the problem of bacterial transfer via surfaces. METHODS AND RESULTS: Two commonly used materials, stainless steel and polyethylene, were coated by physical adsorption, and the coatings were characterized by Raman spectroscopy, atomic force microscopy and water contact angle measurements. We quantified and visualized the colonization of coated and uncoated surfaces by three bacteria: Staphylococcus aureus, Listeria monocytogenes and Pseudomonas fluorescens. No viable cells were detected on surfaces coated with isoeugenol. CONCLUSIONS: The isoeugenol coating prepared with simple adsorption proved effective in preventing biofilm formation on stainless steel and polyethylene surfaces. The result was caused by the antibacterial effect of isoeugenol, as the coating did not diminish the adhesive properties of the surface. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study demonstrates that a simple isoeugenol coating can prevent biofilm formation of S. aureus, L. monocytogenes and P. fluorescens on two commonly used surfaces.

Directing HER4 mRNA expression towards the CYT2 isoform by antisense oligonucleotide decreases growth of breast cancer cells in vitro and in vivo.

BACKGROUND: The tyrosine kinase receptor HER4 is a member of the epidermal growth factor receptor (EGFR) family. It plays diverse roles in cancer development and cancer progression and can both exert oncogenic and tumour-suppressive activities. Alternatively spliced isoforms of HER4 are critical to the different signalling possibilities of HER4. METHODS: We use a splice-switching oligonucleotide (SSO) to direct the alternative splicing of HER4 from the CYT1 to the CYT2 isoform in HER4-expressing breast cancer cells. RESULTS: Treatment with a target-specific SSO was accompanied by a decreased growth of the cells (P<0.0001). In addition, the SSO treatment induced a decreased activity of Akt. We confirmed the SSO-dependent switching of the HER4 isoform CYT1 to CYT2 expression in a xenografted mouse tumour model driven by subcutaneously injected MCF7 cells. We hence demonstrated the feasibility of SSO-directed splice-switching activity in vivo. Furthermore, the SSO treatment efficiently decreased the growth of the xenografted tumour (P=0.0014). CONCLUSION: An SSO directing the splicing of HER4 towards the CYT2 isoform has an inhibitory effect of cancer cell growth in vitro and in vivo. These results may pave the way for the development of new anticancer drugs in HER4-deregulated cancers in humans.

The miR-143/-145 cluster regulates plasminogen activator inhibitor-1 in bladder cancer.

BACKGROUND: Upregulation of the proto-oncogene plasminogen activator inhibitor-1 (PAI-1) is a common hallmark of various solid tumours, but the mechanisms controlling its expression are not fully understood. METHODS: We investigate microRNAs (miRNAs) regulating PAI-1 in a panel of normal bladder urothelial biopsies, superficial Ta bladder tumours and invasive T1-T4 tumours using expression microarrays and qRT-PCR. The prognostic implications of PAI-1 deregulation are established by tissue microarray staining of non-muscle-invasive bladder tumours. MicroRNA repression of PAI-1 is assayed by ectopic miRNA expression, argonaute immunoprecipitation and luciferase assays. RESULTS: We found that the miR-143/-145 cluster is downregulated in all stages of bladder cancer and inversely correlated with PAI-1 expression. Mature miR-143 and miR-145 are coordinately expressed, and both directly target the PAI-1 3'UTR, leading to reduced PAI-1 mRNA and protein levels. Furthermore, we show that PAI-1 and miR-145 levels may serve as useful prognostic markers for non-muscle-invasive bladder tumours for which accurate progressive outcome is currently difficult to predict. CONCLUSION: This report provides the first evidence for direct miRNA regulation of PAI-1 in bladder cancer. We also demonstrate mRNA co-targeting by a cluster of non-family miRNAs, and suggest miR-145 and PAI-1 as clinically relevant biomarkers in bladder cancer.

Interaction of hnRNP A1 with telomere DNA G-quadruplex structures studied at the single molecule level.

G-rich telomeric DNA sequences can form G-quadruplex structures. The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and a shortened derivative (UP1) are active in telomere length regulation, and it has been reported that UP1 can unwind G-quadruplex structures. Here, we investigate the interaction of hnRNP A1 with G-quadruplex DNA structures containing the human telomere repeat (TTAGGG) by gel retardation assays, ensemble fluorescence energy transfer (FRET) spectroscopy, and single molecule FRET microscopy. Our biochemical experiments show that hnRNP A1 binds well to the G-quadruplex telomeric DNA. Ensemble and single molecule FRET measurements provide further insight into molecular conformation: the telomeric DNA overhang is found to be in a folded state in the absence of hnRNP A1 and to remain predominantly in a compact state when complexed with hnRNP A1. This finding is in contrast to the previously reported crystal structures of UP1-telomere DNA complexes where the DNA oligo within the protein-DNA complex is in a fully open conformation.

Archaeal rRNA operons.

Ribosomal RNA (rRNA) operons of the archaea reflect both the unity and the diversity of this third primary taxon. They have proven to be a rich source of both molecular biological and phylogenetic information.

Investigation of particle-functionalized tissue engineering scaffolds using X-ray tomographic microscopy.

A low-density, porous chitosan/poly-(dl-lactide-co-glycolide) (PLGA) microparticle composite scaffold was produced by thermally induced phase separation followed by lyophilization, to provide a bicontinuous microstructure potentially suitable for tissue engineering and locally controlled drug release. PLGA particles were mixed into the chitosan solution and subsequent phase separation during chitosan solidification forced PLGA particles into chitosan phase (Plateau borders). The distributions of volume, surface area, and elongation of 15,422 inclusions of agglomerated PLGA particles were calculated and approximated with log-normal distribution functions from nanotomography reconstructions. Cluster analysis revealed a homogenous inclusion distribution throughout the scaffold. The spatial location and orientation of individual inclusions within the Plateau borders of the scaffold were determined and from these the nearest-neighbor inter-inclusion distance distribution calculated, showing a mean of 2.5 microm. The depth of the inclusions in Plateau borders peaks at 700 or 125 nm, respectively, indicating a step-wise drug release from inclusions successively exposed during scaffold decomposition. Particle diameter ranged from 400 nm to 3 microm and inclusion Feret lengths ranged from 800 nm to 12 microm. These findings on composite morphology and distribution of inclusions are fundamental for predicting scaffold deterioration and particle-mediated drug release during ex vivo and in vivo cell cultivation.

Circular RNAs in cancer: opportunities and challenges in the field.

Circular RNA (circRNA) is a novel member of the noncoding cancer genome with distinct properties and diverse cellular functions, which is being explored at a steadily increasing pace. The list of endogenous circRNAs involved in cancer continues to grow; however, the functional relevance of the vast majority is yet to be discovered. In general, circRNAs are exceptionally stable molecules and some have been shown to function as efficient microRNA sponges with gene-regulatory potential. Many circRNAs are highly conserved and have tissue-specific expression patterns, which often do not correlate well with host gene expression. Here we review the current knowledge on circRNAs in relation to their implications in tumorigenesis as well as their potential as diagnostic and prognostic biomarkers and as possible therapeutic targets in future personalized medicine. Finally, we discuss future directions for circRNA cancer research and current caveats, which must be addressed to facilitate the translation of basic circRNA research into clinical use.

CRM1 mediates the export of ADAR1 through a nuclear export signal within the Z-DNA binding domain.

RNA editing of specific residues by adenosine deamination is a nuclear process catalyzed by adenosine deaminases acting on RNA (ADAR). Different promoters in the ADAR1 gene give rise to two forms of the protein: a constitutive promoter expresses a transcript encoding (c)ADAR1, and an interferon-induced promoter expresses a transcript encoding an N-terminally extended form, (i)ADAR1. Here we show that (c)ADAR1 is primarily nuclear whereas (i)ADAR1 encompasses a functional nuclear export signal in the N-terminal part and is a nucleocytoplasmic shuttle protein. Mutation of the nuclear export signal or treatment with the CRM1-specific drug leptomycin B induces nuclear accumulation of (i)ADAR1 fused to the green fluorescent protein and increases the nuclear editing activity. In concurrence, CRM1 and RanGTP interact specifically with the (i)ADAR1 nuclear export signal to form a tripartite export complex in vitro. Furthermore, our data imply that nuclear import of (i)ADAR1 is mediated by at least two nuclear localization sequences. These results suggest that the nuclear editing activity of (i)ADAR1 is modulated by nuclear export.

RanGTP-regulated interactions of CRM1 with nucleoporins and a shuttling DEAD-box helicase.

CRM1 is an export receptor mediating rapid nuclear exit of proteins and RNAs to the cytoplasm. CRM1 export cargoes include proteins with a leucine-rich nuclear export signal (NES) that bind directly to CRM1 in a trimeric complex with RanGTP. Using a quantitative CRM1-NES cargo binding assay, significant differences in affinity for CRM1 among natural NESs are demonstrated, suggesting that the steady-state nucleocytoplasmic distribution of shuttling proteins could be determined by the relative strengths of their NESs. We also show that a trimeric CRM1-NES-RanGTP complex is disassembled by RanBP1 in the presence of RanGAP, even though RanBP1 itself contains a leucine-rich NES. Selection of CRM1-binding proteins from Xenopus egg extract leads to the identification of an NES-containing DEAD-box helicase, An3, that continuously shuttles between the nucleus and the cytoplasm. In addition, we identify the Xenopus homologue of the nucleoporin CAN/Nup214 as a RanGTP- and NES cargo-specific binding site for CRM1, suggesting that this nucleoporin plays a role in export complex disassembly and/or CRM1 recycling.

Strategies to identify potential therapeutic target sites in RNA.

Antisense agents are powerful tools to inhibit gene expression in a sequence-specific manner. They are used for functional genomics, as diagnostic tools and for therapeutic purposes. Three classes of antisense agents can be distinguished by their mode of action: single-stranded antisense oligodeoxynucleotides; catalytic active RNA/DNA such as ribozymes, DNA- or locked nucleic acid (LNA)zymes; and small interfering RNA molecules known as siRNA. The selection of target sites in highly structured RNA molecules is crucial for their successful application. This is a difficult task, since RNA is assembled into nucleoprotein complexes and forms stable secondary structures in vivo, rendering most of the molecule inaccessible to intermolecular base pairing with complementary nucleic acids. In this review, we discuss several selection strategies to identify potential target sites in RNA molecules. In particular, we focus on combinatorial library approaches that allow high throughput screening of sequences for the design of antisense agents.

Mucin-mediated nanocarrier disassembly for triggered uptake of oligonucleotides as a delivery strategy for the potential treatment of mucosal tumours.

This work demonstrates gastric mucin-triggered nanocarrier disassembly for release of antisense oligonucleotides and consequent unassisted cellular entry as a novel oral delivery strategy. A fluorescence activation-based reporter system was used to investigate the interaction and mucin-mediated disassembly of chitosan-based nanocarriers containing a 13-mer DNA oligonucleotide with a flanked locked RNA nucleic acid gapmer design. Gastric mucins were shown to trigger gapmer release from nanocarriers that was dependent on the interaction time, mucin concentration and N : P ratio with a maximal release at N : P 10. In contrast to siRNA, naked gapmers exhibited uptake into mucus producing HT-MTX mono-cultures and HT-MTX co-cultured with the carcinoma epithelial cell line Caco-2. Importantly, in vivo gapmer uptake was observed in epithelial tissue 30 min post-injection in murine intestinal loops. The findings present a mucosal design-based system tailored for local delivery of oligonucleotides that may maximize the effectiveness of gene silencing therapeutics within tumours at mucosal sites.

SF2/ASF binds to a splicing enhancer in the third HIV-1 tat exon and stimulates U2AF binding independently of the RS domain.

Splicing of a single HIV-1 primary transcript into more than 30 different mRNAs is regulated by a combination of suboptimal splice sites, cis-acting RNA splicing enhancers and silencers, and trans-acting factors. We have studied the splicing of the second tat intron (SD4 to SA7) and find that activation of splicing by SF2/ASF is mediated by a degenerate exon splicing enhancer (ESE3), consisting of at least three functionally independent sub-elements. One of these sub-elements appears to have both enhancing and silencing properties, depending on the context. SF2/ASF stimulates U2AF65 binding to the suboptimal tat polypyrimidine tract in an ESE3-dependent manner, whereas the exon splicing silencer (ESS3) that is located downstream of the ESE3 inhibits this step. Truncated SF2/ASF protein without the RS domain binds specifically to the ESE3 and retains almost full capacity to stimulate U2AF65 binding and activate splicing. This suggests that SF2/ASF can stimulate the recruitment of U2AF65 by an RS domain-independent mechanism.

A role for the basic patch and the C terminus of RanGTP in regulating the dynamic interactions with importin beta, CRM1 and RanBP1.

Transport of macromolecules between the nucleus and cytoplasm involves the recognition of intrinsic localization signals by either import or export receptors. The interaction of the receptors with their cargo is regulated by the small GTPase Ran in its GTP bound state. We have investigated the interaction of RanGTP with the import factor, importin beta, the export factor, CRM1, and the Ran binding protein, RanBP1, in solution. Importin beta specifically protected residues in the switch regions and basic patch region of Ran against proteolytic cleavage, whereas RanBP1 protected the C terminus. Moreover, the binding of importin beta induced a conformational change in the structure of Ran leading to an exposure of the C terminus and stimulated the binding of RanBP1. Mutating the basic patch (HRKK(142)) of Ran resulted in an increased binding of RanBP1 and weakened importin beta binding. In contrast to wild-type Ran, the mutant Ran could be released from importin beta independently of importin alpha. These data provide experimental support for a model in which the accessibility of the C terminus of Ran is influenced by an intramolecular interaction between the basic patch and the C-terminal acidic DEDDDL(216) motif. Binding of importin beta probably disrupts this interaction causing an exposure of the C-terminal extension, which is favorable for RanBP1 binding. Interestingly, basic patch mutations abolish CRM1 interaction, indicating that the determinants for RanGTP binding to the export factor, CRM1, is different from the import factor, importin beta.

HIV-1 rev nuclear export signal binding peptides isolated by phage display.

The human immunodeficiency virus type 1 (HIV-1) Rev protein is absolutely essential in the viral replication cycle, where it induces the production of viral structural proteins. Rev functions in part by inducing the nuclear export of incompletely spliced mRNA species specified by the presence of an RNA element, the Rev response element (RRE). Several proteins implicated in RNA processing and nucleo-cytoplasmic transport have been shown to interact with Rev, however, their exact roles remain unknown. To map potential protein recognition sites within the Rev structure, we have screened a phage library, displaying random 15-mer peptides, and isolated clones exhibiting similar sequences that specifically interact with Rev. The binding sites on Rev of the corresponding synthetic peptides were characterised by protein footprinting, involving partial proteolysis of radioactively end-labelled Rev protein. Two of the peptides produced a significant footprint within the nuclear export signal of Rev, raising the possibility that they mimic the binding of cellular protein factors implicated in nuclear export.

Evolutionary relationships amongst archaebacteria. A comparative study of 23 S ribosomal RNAs of a sulphur-dependent extreme thermophile, an extreme halophile and a thermophilic methanogen.

The 23 S RNA genes representative of each of the main archaebacterial subkingdoms, Desulfurococcus mobilis an extreme thermophile, Halococcus morrhuae an extreme halophile and Methanobacterium thermoautotrophicum a thermophilic methanogen, were cloned and sequenced. The inferred RNA sequences were aligned with all the available 23 S-like RNAs of other archaebacteria, eubacteria/chloroplasts and the cytoplasm of eukaryotes. Universal secondary structural models containing six major structural domains were refined, and extended, using the sequence comparison approach. Much of the present structure was confirmed but six new helices were added, including one that also exists in the eukaryotic 5.8 S RNA, and extensions were made to several existing helices. The data throw doubt on whether the 5' and 3' ends of the 23 S RNA interact, since no stable helix can form in either the extreme thermophile or the methanogen RNA. A few secondary structural features, specific to the archaebacterial RNAs were identified; two of these were supported by a comparison of the archaebacterial RNA sequences, and experimentally, using chemical and ribonuclease probes. Seven tertiary structural interactions, common to all 23 S-like RNAs, were predicted within unpaired regions of the secondary structural model on the basis of co-variation of nucleotide pairs; two lie in the region of the 23 S RNA corresponding to 5.8 S RNA but they are not conserved in the latter. The flanking sequences of each of the RNAs could base-pair to form long RNA processing stems. They were not conserved in sequence but each exhibited a secondary structural feature that is common to all the archaebacterial stems for both 16 S and 23 S RNAs and constitutes a processing site. Kingdom-specific nucleotides have been identified that are associated with antibiotic binding sites at functional centres in 23 S-like RNAs: in the peptidyl transferase centre (erythromycin-domain V) the archaebacterial RNAs classify with the eukaryotic RNAs; at the elongation factor-dependent GTPase centre (thiostrepton-domain II) they fall with the eubacteria, and at the putative amino acyl tRNA site (alpha-sarcin-domain VI) they resemble eukaryotes. Two of the proposed tertiary interactions offer a structural explanation for how functional coupling of domains II and V occurs at the peptidyl transferase centre. Phylogenetic trees were constructed for the archaebacterial kingdom, and for the other two kingdoms, on the basis of the aligned 23 S-like RNA sequences.(ABSTRACT TRUNCATED AT 400 WORDS)

Spatial arrangement of DNA-dependent RNA polymerase of Escherichia coli and DNA in the specific complex. A neutron small angle scattering study.

In this paper we demonstrate that neutron small angle scattering is a suitable method to study the spatial arrangement of large specific protein-DNA complexes. We studied the complex of DNA-dependent RNA polymerase of Escherichia coli and a 130 base-pair DNA fragment containing the strong promoter A1 of bacteriophage T7. Contrast variation of the complex with deuterium allowed us to "visualize" either RNA polymerase, or DNA, or both components in situ. From the corresponding scattering curves information was derived about: (1) Conformational changes of RNA polymerase and DNA by complex formation: comparison of the scattering profiles of the isolated and complexed components showed that by specific complex formation the cross-section of RNA polymerase decreases, while the DNA fragment does not undergo a gross conformational change. (2) The spatial arrangement of RNA polymerase and DNA in the specific complex from the cross-sectional radii of gyration of the complex the normal distance dn between the centre of gravity of the RNA polymerase and the axis of the DNA fragment was derived as 5.0 (+/- 0.3) nm. On the basis of these and footprinting data a low resolution model of the RNA polymerase-promoter complex is proposed. The main feature of this model is the positioning of RNA polymerase to only one side of the DNA.

Modern methods for probing RNA structure.

Molecular biologists have been remarkably successful in dividing large RNAs into small functional modules manageable for NMR and X-ray studies. At the same time biophysical, biochemical and genetic tools in RNA structure determination have reached a level of sophistication, at which we start to see a glimpse of molecular dynamics and the mechanism of RNA mediated catalysis.

Monitoring patterned enzymatic polymerization on DNA origami at single-molecule level.

DNA origami has been used to orchestrate reactions with nano-precision using a variety of biomolecules. Here, the dynamics of albumin-assisted, localized single-molecule DNA polymerization by terminal deoxynucleotidyl transferase on a 2D DNA origami are monitored using AFM in liquid. Direct visualization of the surface activity revealed the mechanics of growth.

Tools for the production and purification of full-length, N- or C-terminal 32P-labeled protein, applied to HIV-1 Gag and Rev.

We have constructed two new vectors for the production of foreign proteins in Escherichia coli. The vectors, pGEX-GTH and pET-HTG, produce protein fused to glutathione S-transferase (GST) at the N- and C-termini, respectively, allowing one-step purification on glutathione-Sepharose. Furthermore, they carry the recognition sequence (RRASV) for the catalytic subunit of cAMP-dependent heart muscle kinase (HMK) at the terminus distal to the GST tag, enabling specific 32P labeling in vitro. By positioning the GST and HMK sequences at opposite ends of the introduced gene, only full-length fusion protein becomes radiolabeled after purification. Avoiding the labeling of shorter fusion protein species, often observed in bacterial expression of foreign genes, is particularly important for a number of different purposes, including protein mobility shift analysis and protein footprinting technology.

Probing the structure of HIV-1 Rev by protein footprinting of multiple monoclonal antibody-binding sites.

Human immunodeficiency virus type 1 (HIV-1) Rev is a small RNA-binding protein which is essential for viral replication. To investigate the structure of Rev we have mapped the binding sites of a panel of monoclonal antibodies (mAb) by protein footprinting and identified a mAb protecting amino acids within both the N- and C-terminal parts of Rev. Our mapping results support a previously proposed structure (Auer et al., Biochemistry, 33 (1994) 2988-2996) predicting that a helix-loop-helix motif in Rev brings the termini of the protein into proximity. Furthermore, we demonstrate that the binding sites mapped by protein footprinting are in agreement with conventional epitope mapping results and that this technique provides an advantageous strategy for mapping discontinuous sites.