FRET monitoring of transcription factor activities in living bacteria

Bacteria adapt to the constantly changing environments largely by transcriptional regulation through the activities of various transcription factors (TFs). However, techniques that monitor the in situ TF-promoter interactions in living bacteria are lacking. Herein, we developed a whole-cell TF-promoter binding assay based on the intermolecular Förster resonance energy transfer (FRET) between a fluorescent unnatural amino acid CouA which is genetically encoded into defined sites in TFs and the live cell fluorescent nucleic acid stain SYTO 9. We show that this new FRET pair monitors the intricate TF-promoter interactions elicited by various types of signal transduction systems with specificity and sensitivity. Furthermore, the assay is applicable to identify novel modulators of the regulatory systems of interest and monitor TF activities in bacteria colonized in C. elegans. In conclusion, we established a tractable and sensitive TF-promoter binding assay in living bacteria which not only complements currently available approaches for DNA-protein interactions but also provides novel opportunities for functional annotation of bacterial signal transduction systems and studies of the bacteria-host interface.

NEUTROPHIL EXTRACELLULAR TRAPS RELEASE IN GOUT AND PSEUDOGOUT DEPENDS ON THE NUMBER OF CRYSTALS REGARDLESS OF LEUKOCYTE COUNT

Objectives Microcrystal-induced arthritis is still an unresolved paradigm for medicine. Overt inflammation may be absent even when crystals occur in synovial fluid. Recently, the production of neutrophil extracellular traps (NETs) embedding monosodium urate crystals (MSU) has been proposed as a possible mechanism of the auto-resolution of the inflammatory phase during gout. We aimed to verify and quantify the release of NETs in synovial fluids during gout and pseudogout attacks and to compare any differences with respect to crystals and neutrophils number, and to analyze activation of necroptosis pathway in synovial fluid from crystal-induced arthritis. Methods Synovial fluid samples were obtained by arthrocentesis from 22 patients presenting acute crystal-induced arthritis, gout or pseudogout (n = 11 each group), and from 10 patients with acute non-crystal arthritis as controls. NETosis was quantified in synovial fluid by nucleic acid stain and by quantification of human neutrophil elastase. Activation of p-MLKL was assessed by western blot. Results We observed that synovial fluid neutrophils encountering MSU and CPPD crystals during episodes of gout and pseudogout release NETs in relation to the number of crystals in synovial fluid and irrespective of neutrophil density and type of crystal. This release was accompanied by necroptosis through the activation of the MLKL pathway. Conclusions Our findings suggest that a role of NETs in crystal-induced arthritis is to “trap extracellular particles”, including microcrystals. Embedding crystals in aggregates of NETs may be the basis of tophi and CPPD deposition and may have implications for disease evolution, rather than for spontaneous resolution of the acute attack.

Enhancement Effect of Zn-Arsenazo III Complex for G-quadruplex DNA Stability of Proto-oncogene Promoter Telomeres

Background: Controlling the structure of proto-oncogene telomeres is very important in antitumor therapy. There are relationships between G-quadruplex DNA and the growth of tumor cell. Methods: In this study, spectroscopic, cyclic voltammetry and viscosity methods were employed to investigate the interaction between Zn-Arsenazo Ⅲ complex and G-quadruplex DNA by using 4S Green Plus Nucleic Acid Stain as a spectral probe in PBS buffer. The binding ratios were n Arsenazo Ⅲ : n Zn(Ⅱ) = 5:1 for Zn-Arsenazo Ⅲ complex and n Zn- Arsenazo Ⅲ : n G-quadruplex DNA = 8:1 for Zn-Arsenazo Ⅲ-G-quadruplex DNA. The bonding constants (Kθ 298.15K=4.44x105 L·mol-1, Kθ 308.15K= 1.00x105 L·mol-1, Kθ 318.15K= 1.04x106 L·mol-1) were obtained by double reciprocal method at different temperatures, Which was found that the interaction between Zn-Arsenazo Ⅲ complex and Gquadruplex DNA was driven by enthalpy. Furthermore, the research further confirmed that the interaction mode between Zn-Arsenazo Ⅲ complex and G-quadruplex DNA was a mixed binding which involved intercalation and non-intercalation interaction. Results and Conclusion: Together these findings also have corroborated the application of stabilizing ligands and intervening with their function for target G-quadruplexes in a cellular context.

Visual and fluorescence characterisation of particulate aerosols in ice cores with imaging flow cytometry

<p>Current efforts to examine and quantify so-called ‘biomarkers’ present in polar ice samples offer exciting potential as biological and biochemical proxies for past climate and ocean dynamics. Here we present a new rapid and easily replicable method to provide measurements of the microscopic particulate content of ice samples from polar environments. Using an Amnis® Imagestream® Imaging Flow Cytometer, melted snow and ice samples from Patriot Hills in the Ellsworth Mountains, Antarctica were analysed for their particulate (biological and non-biological) content. Selective use of a nucleic acid stain pre-treatment allows for a straightforward gating strategy that resolves both autofluorescent and non-autofluorescent biological material in sample replicates. In the Patriot Hills samples this method clearly identifies marine picoplankton, along with non-biological particulates such as tephra and minerogenic material. Crucially, the 60x Brightfield images provided by the Imagestream offer a significant additional capability above standard flow cytometry systems; each object identified by the machine can be visually differentiated (automatically or manually) from particulates with similar fluorescence properties. Back-trajectory analysis with the NOAA Hybrid Single-Particle Lagrangian Integrated Trajectory (HySPLIT) model indicates that these ice-bound marine organisms originate from the Weddell and Amundsen-Bellingshausen Seas. This technique, when paired with established chemical and biochemical methods, shows considerable potential in providing valuable information about the nature and origin of aerosols and biomarker signals trapped in past ice layers.</p>

Heparin Forms Polymers with Cell-free DNA Which Elongate Under Shear in Flowing Blood

Heparin is a widely used anticoagulant which inhibits factor Xa and thrombin through potentiation of antithrombin. We recently identified that the nucleic acid stain SYTOX reacts with platelet polyphosphate due to molecular similarities, some of which are shared by heparin. We attempted to study heparin in flowing blood by live-cell fluorescence microscopy, using SYTOX for heparin visualisation. Immunostaining was performed with monoclonal antibodies directed against various heparin-binding proteins. In addition, we studied modulation of heparin activity in coagulation assays, as well its effects on fibrin formation under flow in recalcified whole blood. We found that SYTOX-positive polymers appear in heparinised blood under flow. These polymers typically associate with platelet aggregates and their length (reversibly) increases with shear rate. Immunostaining revealed that of the heparin-binding proteins assessed, they only contain histones. In coagulation assays and flow studies on fibrin formation, we found that addition of exogenous histones reverses the anticoagulant effects of heparin. Furthermore, the polymers do not appear in the presence of DNase I, heparinase I/III, or the heparin antidote protamine. These findings suggest that heparin forms polymeric complexes with cell-free DNA in whole blood through a currently unidentified mechanism.

The link between yeast cell wall porosity and plasma membrane permeability after PEF treatment

An investigation of the yeast cell resealing process was performed by studying the absorption of the tetraphenylphosphonium (TPP+) ion by the yeast Saccharomyces cerevisiae. It was shown that the main barrier for the uptake of such TPP+ ions is the cell wall. An increased rate of TPP+ absorption after treatment of such cells with a pulsed electric field (PEF) was observed only in intact cells, but not in spheroplasts. The investigation of the uptake of TPP+ in PEF treated cells exposed to TPP+ for different time intervals also showed the dependence of the absorption rate on the PEF strength. The modelling of the TPP+ uptake recovery has also shown that the characteristic decay time of the non-equilibrium (PEF induced) pores was approximately a few tens of seconds and this did not depend on the PEF strength. A further investigation of such cell membrane recovery process using a florescent SYTOX Green nucleic acid stain dye also showed that such membrane resealing takes place over a time that is like that occurring in the cell wall. It was thus concluded that the similar characteristic lifetimes of the non-equilibrium pores in the cell wall and membrane after exposure to PEF indicate a strong coupling between these parts of the cell.

A simple and sensitive SYBR Gold-based assay to quantify DNA-protein interactions

A simple, accessible, and inexpensive assay to quantify the strength of DNA-protein interactions was developed. The assay relies on capturing DNA-protein complexes using an affinity resin that binds tagged, recombinant proteins. Sequential washes with filtration spin cups and centrifugation remove non-specific interactions in a gentle, uniform manner and a final elution isolates specific DNA-protein complexes. SYBR Gold nucleic acid stain is added to the eluted product and the fluorescence intensity accurately quantifies the amount of captured DNA, ultimately illustrating the relative strength of the DNA-protein interaction. The major utility of the assay resides in the versatility and quantitative nature of the SYBR Gold:nucleic acid interaction, eliminating the need for customized or labeled oligos and permitting relatively inexpensive quantification of binding capacity. The assay also employs DNA-protein complex capture by the very common purification tag, 6xHis, but other tags could likely be utilized. Further, SYBR Gold fluorescence is compatible with a wide variety of instruments, including UV transilluminators, a staple to any molecular biology laboratory. This assay was used to compare the binding capacities of different Auxin Response Factor (ARF) transcription factors to various dsDNA targets, including the classical AuxRE motif and several divergent sequences. Results from dose-response assays suggest that different ARF proteins might show distinct comparative affinities for AuxRE variants, emphasizing that specific ARF-AuxRE binding strengths likely contribute to the complex and fine-tuned cellular auxin response.

23 Viability staining techniques for cryopreserved spermatozoa in 3 caudata species

Salamanders are the most threatened vertebrate taxa; thus, conservation-based research including spermatozoa cryopreservation and other assisted reproductive technologies is essential to their survival. To determine the effectiveness of sperm cryopreservation, methods for evaluating sperm quality are necessary but underdeveloped in caudate research. Evaluating motility has been the primary analysis for sperm viability but is difficult to perform due to the scythe-like morphology, slow rotating progression, and minute undulations of the tail membrane. Estimating apoptosis is a new approach to evaluating caudate spermatozoa survival through cryostress. Fluorescent dyes, such as SYBR-14, annexin-V, and propidium iodide (PI), are valuable tools for identifying degrees of cell viability, apoptosis, and necrosis. Annexin-V marks the externalization of phosphatidylserine on the cell membrane indicating early steps in the apoptosis signalling cascade. Compromised membranes allow PI, a nucleic acid stain, access to DNA, marking cellular necrosis. The SYBR-14 is a nucleic acid stain that permeatesssss intact membranes, labelling live cells. These fluorescent stains were assessed for marking viability and stages of cell death in post-thaw spermatozoa across 3 caudate species: the Eastern tiger salamander (Ambystoma tigrinum), Kweichow Emperor newt (Tylototriton kweichowensis), and black-spotted newt (Notophthalmus meridionalis). For each species, spermic urine samples were acquired by hormone treatment and frozen based on protocols developed in A. tigrinum, yielding an average of 18.2% relative motility recovered at thaw. Straws were thawed for 5min at 20°C. Viability was tested by staining 5μL 1:1 with a 1:50 dilution of SYBR-14 and 2μL of PI. Stages of cell death were evaluated by staining 10μL with 2μL of annexin-V and 2μL of PI. Cell viability was assessed immediately under a fluorescence microscope. For each of the 3 species, 2 samples were stained with both assays in triplicate. Sperm stained with SYBR-14 alone were considered viable, and sperm stained with any annexin-V or PI were considered not viable. Visible dynamic shifting from SYBR-14 to PI staining was observed in real time, indicating rapid necrosis. Morphological abnormalities, not observed in unstained samples, were prevalent across all species following staining, signifying a possible cytotoxic effect of the dyes. High mortality and abnormality rates suggest that fluorescent dyes have elevated toxicity and permeability in caudate sperm. Caudate spermic urine has a very low osmolality, implying high permeability, which could lead to rapid staining and toxicity effects. Shorter incubation times may be required for accurate staining. Results may also indicate that cryopreservation protocols need to be species specific and do not transfer well across taxa. This is one of the earliest studies to evaluate the use of fluorescent stain protocols on measuring cell viability in caudate sperm and indicates that further refinement is required.

Changes in the Expression of Biofilm-Associated Surface Proteins inStaphylococcus aureusFood-Environmental Isolates Subjected to Sublethal Concentrations of Disinfectants

Sublethal concentrations (sub-MICs) of certain disinfectants are no longer effective in removing biofilms from abiotic surfaces and can even promote the formation of biofilms. Bacterial cells can probably adapt to these low concentrations of disinfectants and defend themselves by way of biofilm formation. In this paper, we report on threeStaphylococcus aureusbiofilm formers (strong B+++, moderate B++, and weak B+) that were cultivated with sub-MICs of commonly used disinfectants, ethanol or chloramine T, and quantified using Syto9 green fluorogenic nucleic acid stain. We demonstrate that 1.25–2.5% ethanol and 2500 μg/mL chloramine T significantly enhancedS. aureusbiofilm formation. To visualize differences in biofilm compactness betweenS. aureusbiofilms in control medium, 1.25% ethanol, or 2500 μg/mL chloramine T, scanning electron microscopy was used. To describe changes in abundance of surface-exposed proteins in ethanol- or chloramine T-treated biofilms, surface proteins were prepared using a novel trypsin shaving approach and quantified after dimethyl labeling by LC-LTQ/Orbitrap MS. Our data show that some proteins with adhesive functions and others with cell maintenance functions and virulence factor EsxA were significantly upregulated by both treatments. In contrast, immunoglobulin-binding protein A was significantly downregulated for both disinfectants. Significant differences were observed in the effect of the two disinfectants on the expression of surface proteins including some adhesins, foldase protein PrsA, and two virulence factors.