Cell surface protein detection with immunogold labelling in ESEM: optimisation of the method and semi-quantitative analysis

In this work we used a combination of immunogold labelling (IGL) and environmental scanning electron microscopy (ESEM) to detect the presence of a protein on the cell surface. To achieve this purpose we chose as experimental system 3T3 Swiss Albino Mouse Fibroblasts and galectin-3. This protein, whose sub-cellular distribution is still under discussion, is involved in a large number of cell physiological and pathological processes. IGL technique has been utilised by many authors in combination with SEM and TEM to obtain the identification/localisation of receptors and antigens, both in cells and tissues. ESEM represents an important tool in biomedical research, since it does not require any severe processing of the sample, lowering the risk of generating artefacts and interfere with IGL procedure. The absence of metal coating could yield further advantages for our purpose as the labelling detection is based on the atomic number difference between Nanogold spheres and the biological material. Using the gaseous secondary electron detector (GSED) compositional contrast is easily revealed by the backscattered electrons component of the signal. In spite of this fact, only few published papers present a combination of ESEM and IGL. Hereby we present our method, optimised to improve the intensity and the specificity of the labelling signal, in order to obtain a semi-quantitative evaluation of the labelling signal.     

Clerocidin-mediated DNA footprinting discriminates among different G-quadruplex conformations and detects tetraplex folding in a duplex environment

Background

G-quadruplexes are polymorphic non-canonical nucleic acid conformations involved both in physiological and pathological processes. Given the high degree of folding heterogeneity and comparable conformational stabilities, different G-quadruplex forms can occur simultaneously, hence rendering the use of basic instrumental methods for structure determination, like X-ray diffraction or NMR, hardly useful. Footprinting techniques represent valuable and relatively rapid alternative to characterize DNA folding. The natural diterpenoid clerocidin is an alkylating agent that specifically reacts at single-stranded DNA regions, with different mechanisms depending on the exposed nucleotide.

Methods

Clerocidin was used to footprint G-quadruplex structures formed by telomeric and oncogene promoter sequences (c-myc, bcl-2, c-kit2), and by the thrombin binding aptamer.

Results

The easy modulability of CL reactivity towards DNA bases permitted to discriminate fully and partially protected sites, highlights stretched portions of the G-quadruplex conformation, and discriminate among topologies adopted by one sequence in different environmental conditions. Importantly, CL displayed the unique property to allow detection of G-quadruplex folding within a duplex context.

Conclusions

CL is a finely performing new tool to unveil G-quadruplex arrangements in DNA sequences under genomically relevant conditions.

General significance

Nucleic acid G-quadruplex structures are an emerging research field because of the recent indication of their involvement in a series of key biological functions, in particular in regulation of proliferation-associated gene expression. The use of clerocidin as footprinting agent to identify G-quadruplex structures under genomically relevant conditions may allow detection of new G-quadruplex-based regulatory regions.     

Smart materials as scaffolds for tissue engineering

In this review, we focused our attention on the more important natural extracellular matrix (ECM) molecules (collagen and fibrin), employed as cellular scaffolds for tissue engineering and on a class of semi-synthetic materials made from the fusion of specific oligopeptide sequences, showing biological activities, with synthetic materials. In particular, these new "intelligent" scaffolds may contain oligopeptide cleaving sequences specific for matrix metalloproteinases (MMPs), integrin binding domains, growth factors, anti-thrombin sequences, plasmin degradation sites, and morphogenetic proteins. The aim was to confer to these new "intelligent" semi-synthetic biomaterials, the advantages offered by both the synthetic materials (processability, mechanical strength) and by the natural materials (specific cell recognition, cellular invasion, and the ability to supply differentiation/proliferation signals). Due to their characteristics, these semi-synthetic biomaterials represent a new and versatile class of biomimetic hybrid materials that hold clinical promise in serving as implants to promote wound healing and tissue regeneration.     

Conformation and Stability of Intramolecular Telomeric G-Quadruplexes: Sequence Effects in the Loops

Telomeres are guanine-rich sequences that protect the ends of chromosomes. These regions can fold into G-quadruplex structures and their stabilization by G-quadruplex ligands has been employed as an anticancer strategy. Genetic analysis in human telomeres revealed extensive allelic variation restricted to loop bases, indicating that the variant telomeric sequences maintain the ability to fold into G-quadruplex. To assess the effect of mutations in loop bases on G-quadruplex folding and stability, we performed a comprehensive analysis of mutant telomeric sequences by spectroscopic techniques, molecular dynamics simulations and gel electrophoresis. We found that when the first position in the loop was mutated from T to C or A the resulting structure adopted a less stable antiparallel topology; when the second position was mutated to C or A, lower thermal stability and no evident conformational change were observed; in contrast, substitution of the third position from A to C induced a more stable and original hybrid conformation, while mutation to T did not significantly affect G-quadruplex topology and stability. Our results indicate that allelic variations generate G-quadruplex telomeric structures with variable conformation and stability. This aspect needs to be taken into account when designing new potential anticancer molecules.     

6-Hydroxy Derivative as New Desfluoroquinolone (DFQ): Synthesis and DNA-Binding Study

Abstract

A new 6-desfluoroquinolone derivative, characterized by the presence of a 6-hydroxyl group instead of the usual fluorine atom at the C-6 position, was synthesized with the aim to better understand the mechanistic role of the C-6 substituent in the quinolone/DNA/DNA-gyrase interaction. The antibacterial activity unambiguously shows that the hydroxyl group is a good substitute for the C-6 fluorine atom, especially against Gram-positive bacteria. On the contrary, it is a very weak inhibitor of the target DNA gyrase, displaying the highest IC₅₀ value observed for all the C-6 substituted analogues. This behaviour could be explained on the basis of its DNA binding properties.     

Preferred interaction of d-peptidyl-anthraquinones with double-stranded B-DNA

The quest for more specific drugs in antitumor chemotherapy led us to the design of anthraquinone–peptide conjugates capable of selective recognition of the nucleic acid. We present here the DNA binding characteristics, sequence specificity and geometry of interaction of a pair of enantiomers containing the lysine–glycine dipeptide in the side chains. The d enantiomer binds right handed double stranded DNA more efficiently than the l form under all conditions tested. The source of higher binding affinity is not electrostatic in nature and rests in the more favorable hydrophobic contacts of the d-lysyl side chains in the drug-DNA complex. Both derivatives exhibit preference for alternating GC base sequences and intercalate into DNA in a threading mode as suggested by chiroptical and theoretical studies. The d enantiomer, being a peptidyl derivative that contains a non-natural amino acid, has the considerable advantage of being less susceptible to enzymatic hydrolysis and could therefore represent a lead compound for further development.     

New Peptidyl-Anthraquinones: Synthesis and DNA Binding

Abstract

Aminoacyl-hydroxy-anthraquinones bearing glicyl, varyl, lysyl and tryptophanyl residues in the side-chain were synthesized as new potential DNA-directed drugs. These compounds bind very tightly to double-stranded DNA by intercalating their planar portion into the nucleic acid and further stabilizing the complex through electrostatic contacts with the backbone phosphates. All protonated groups in the side-chains participate in the latter process. The free energy of DNA-binding corrected for the electrostatic contribution is similar for the lysyl and glicyl derivatives, which points to a common geometry of intercalation.     

Adhesion and proliferation of fibroblasts on RF plasma-deposited nanostructured fluorocarbon coatings: evidence of FAK activation

We used combined plasma-deposition process to deposit smooth and nanostructured fluorocarbon coatings on polyethylenethereftalate (PET) substrates, to obtain surfaces with identical chemical composition and different roughness, and investigate the effect of surface nanostructures on adhesion and proliferation of 3T3 Swiss Albino Mouse fibroblasts. Untreated PET and polystyrene (PS) were used as controls for cell culture. We have found that the statistically significant increase of cell proliferation rate and FAK (a nonreceptor tyrosine kinase) activation detected on ROUGH fluorocarbon surfaces is due to the presence of nanostructures. Changes in cytoskeletal organization and phospho FAK (tyr 397) localization were evident after 60 min on cells adhering to ROUGH surfaces. This change was characterized by the formation of actin stress fibers along lamellar membrane protrusion instead of usual focal contacts. Also the morphology of the adhering fibroblasts (60 min) adhering on ROUGH surfaces was found quite different compared to cells adhering on smooth ones.