Constrained G4 structures unveil topology specificity of known and new G4 binding proteins

G-quadruplexes (G4) are non-canonical secondary structures consisting in stacked tetrads of hydrogen-bonded guanines bases. An essential feature of G4 is their intrinsic polymorphic nature, which is characterized by the equilibrium between several conformations (also called topologies) and the presence of different types of loops with variable lengths. In cells, G4 functions rely on protein or enzymatic factors that recognize and promote or resolve these structures. In order to characterize new G4-dependent mechanisms, extensive researches aimed at identifying new G4 binding proteins. Using G-rich single-stranded oligonucleotides that adopt non-controlled G4 conformations, a large number of G4-binding proteins have been identified in vitro, but their specificity towards G4 topology remained unknown. Constrained G4 structures are biomolecular objects based on the use of a rigid cyclic peptide scaffold as a template for directing the intramolecular assembly of the anchored oligonucleotides into a single and stabilized G4 topology. Here, using various constrained RNA or DNA G4 as baits in human cell extracts, we establish the topology preference of several well-known G4-interacting factors. Moreover, we identify new G4-interacting proteins such as the NELF complex involved in the RNA-Pol II pausing mechanism, and we show that it impacts the clastogenic effect of the G4-ligand pyridostatin.

Constrained G4 structures unveil topology specificity of known and new G4 binding proteins

G-quadruplexes (G4) are non-canonical secondary structures consisting in stacked tetrads of hydrogen-bonded guanines bases. An essential feature of G4 is their intrinsic polymorphic nature, which is characterized by the equilibrium between several conformations (also called topologies) and the presence of different types of loops with variable lengths. In cells, G4 functions rely on protein or enzymatic factors that recognize and promote or resolve these structures. In order to characterize new G4-dependent mechanisms, extensive researches aimed at identifying new G4 binding proteins. Using G-rich single-stranded oligonucleotides that adopt non-controlled G4 conformations, a large number of G4-binding proteins have been identified in vitro, but their specificity towards G4 topology remained unknown. Constrained G4 structures are biomolecular objects based on the use of a rigid cyclic peptide scaffold as a template for directing the intramolecular assembly of the anchored oligonucleotides into a single and stabilized G4 topology. Here, using various constrained RNA or DNA G4 as baits in human cell extracts, we establish the topology preference of several well-known G4-interacting factors. Moreover, we identify new G4-interacting proteins such as the NELF complex involved in the RNA-Pol II pausing mechanism, and we show that it impacts the clastogenic effect of the G4-ligand pyridostatin.

Can non-asbestiform amphibole fibers trigger carcinogenesis mechanisms?

<p>Minerals defined as asbestos include only the fibrous varieties (length > 5 µm, diameter < 3 µm and length/diameter ratio > 3:1) and asbestiform (high tensile strength or flexibility) of serpentine and amphibole.</p><p>However, there are also prismatic varieties of amphiboles, which despite the same chemical composition, are not classified as asbestos. Their geometric ratio would fall within the concept of fiber, but the minerals are not asbestiform.</p><p>Starting from a fairly contradictory context, the goal of this work was to analyze the variables inherent the morphological, but above all, the clastogenic effect determined by exposure of both asbestiform and non-asbestiform amphiboles.</p><p>The asbestiform fibers (F3), and the other three samples containing non-asbestiform amphiboles (P1, P2, P3) were tested in A549 cells line. Each sample of fiber was inoculated in A549 cells at a concentration of 100 µg/ml for 48h. Experiments were assayed in triplicate and repeated twice. To evaluate the micronuclei number for each sample the fixed cells were dropped onto clean microscope slides, stained and observed by optical microscopy at 100X.</p><p>Obtained results showed a statistically significant increase (P < 0.05) of micronuclei in F3 exposed cells when compared to negative controls. Similar results were reported when A549 cells were exposed with non-asbestiform amphiboles P2. No significant increase of micronucleated cells was observed after exposure of cells line at samples P1 and P3.</p><p>Moreover, to investigate the effect long-term triggered after 24h post fiber exposure of the medium inoculated cells were replaced with fresh culture medium and the cultures were grown for 72h. Albeit a prolonged contact of the F3 and P3 fibers resulted in a statistically significant increase (P < 0.01) of the micronuclei, no increase was reported for P2.</p><p>These results indicate that the contact of non-asbestiform amphiboles in vitro, can determine a genetic disorder, a necessary step in the cancer development. However, the kinematic of processes and the bearing of results are to be further clarified. For this purpose, the same starting materials are presently tested to determinate the transformation efficiency in no-tumoral cells and will be analyzed the different pathway involved in the etiopathogeneses of diseases trigger by inhalation of fiber.</p>

An Evaluation of Anti-Clastogenic Activities for Garlic (Allium Sativum), Turmeric (Curcuma Longa) and Tulsi (Ocimum Tenuiflorum) with Clastogen (Mitomycin) in Human Lymphocytes – A Case Study

Chromosomal aberrations based human syndrome are very critical and sometimes leads to lethality. Such syndrome or disorders are often irreversible. In this present study, we evaluated the preventing effect of herbal extracts from Ocimum (tulsi), Curcuma (turmeric) and Allium (garlic) in developing chromosomal aberrations in whole blood human lymphocytes. The results showed that the clastogenic effect was minimised by the combination of all three herbal extracts compared to the individual extract effects. The spontaneous chromosomal aberrations caused in lymphoma cells were also minimized by the herbal extracts.

Assessment of Potential Clastogenic Effect of the Insecticide Mospilan® 20SP in Human Peripheral Blood Lymphocytes After in Vitro Exposure

Acetamiprid, that is known as the commercial formulation Mospilan® 20SP is the part of the neonicotinoid insecticide group and is widely used against various pests. In our study we assessed the potential clastogenic effects of Mospilan® in human peripheral blood lymphocytes in vitro using a chromosome aberration test. The lymphocytes were treated with acetamiprid in the concentration range of 5, 10, 25 and 50 µg.ml−1 for 24 and 48 h. After 24 h exposure, the insecticide induced statistically significant higher levels of chromosome aberrations from the concentration of 10 µg.ml−1 (P < 0.05 and P < 0.001) and a significant decrease in mitotic index (MI) at the concentrations of 25 and 50 µg.ml−1 (P < 0.05 and P < 0.01), respectively. After a 48 h exposure, we found a dose dependent increase in the percentage of chromosome aberrations at all concentrations (P < 0.05; P < 0.01 and P < 0.001) and a decrease in MI at concentrations of 25 and 50 µg.ml−1 (P < 0.05 and P < 0.01). Our results indicated that neonicotinoid insecticide formulations containing acetamiprid may have potential cytotoxic and genotoxic effects.

The repeated cytogenetic analysis of subjects occupationally exposed to nanoparticles: a pilot study

The application of nanomaterials has been rapidly increasing during recent years. Inhalation exposure to nanoparticles (NP) may result in negative toxic effects but there is a critical lack of human studies, especially those related to possible DNA alterations. We analyzed pre-shift and post-shift a group of nanocomposite researchers with a long-term working background (17.8 ± 10.0 years) and matched controls. The study group consisted of 73.2% males and 26.8% females. Aerosol exposure monitoring during a working shift (involving welding, smelting, machining) to assess the differences in exposure to particulate matter (PM) including nanosized fractions <25–100 nm, and their chemical analysis, was carried out. A micronucleus assay using Human Pan Centromeric probes, was applied to distinguish between the frequency of centromere positive (CEN+) and centromere negative (CEN−) micronuclei (MN) in the binucleated cells. This approach allowed recognition of the types of chromosomal damage: losses and breaks. The monitoring data revealed differences in the exposure to NP related to individual working processes, and in the chemical composition of nanofraction. The cytogenetic results of this pilot study demonstrated a lack of effect of long-term (years) exposure to NP (total frequency of MN, P = 0.743), although this exposure may be responsible for DNA damage pattern changes (12% increase of chromosomal breaks—clastogenic effect). Moreover, short-term (daily shift) exposure could be a reason for the increase of chromosomal breaks in a subgroup of researchers involved in welding and smelting processes (clastogenic effect, P = 0.037). The gender and/or gender ratio of the study participants was also an important factor for the interpretation of the results. As this type of human study is unique, further research is needed to understand the effects of long-term and short-term exposure to NP.