Tobacco mosaic virus as an AFM tip calibrator

The study of high-resolution topographic surfaces of isolated single molecules is one of the applications of atomic force microscopy (AFM). Since tip-induced distortions are significant in topographic images the exact AFM tip shape must be known in order to correct dilated AFM height images using mathematical morphology operators. In this work, we present a protocol to estimate the AFM tip apex radius using tobacco mosaic virus (TMV) particles. Among the many advantages of TMV, are its non-abrasivity, thermal stability, bio-compatibility with other isolated single molecules and stability when deposited on divalent ion pretreated mica. Compared to previous calibration systems, the advantage of using TMV resides in our detailed knowledge of the atomic structure of the entire rod-shaped particle. This property makes it possible to interpret AFM height images in term of the three-dimensional structure of TMV. Results obtained in this study show that when a low imaging force is used, the tip is sensing viral protein loops whereas at higher imaging force the tip is sensing the TMV particle core. The known size of the TMV particle allowed us to develop a tip-size estimation protocol which permits the successful erosion of tip-convoluted AFM height images. Our data shows that the TMV particle is a well-adapted calibrator for AFM tips for imaging single isolated biomolecules. The procedure developed in this study is easily applicable to any other spherical viral particles.     

Single and multiple bonds in (strept)avidin-biotin interactions

Thanks to Dynamic Force Spectroscopy (DFS) and developments of massive data analysis tools, such as YieldFinder, Atomic Force Microscopy (AFM) becomes a powerful method for analyzing long lifetime ligand-receptor interactions. We have chosen the well-known system, (strept)avidin-biotin complex, as an experimental model due to the lack of consensus on interpretations of the rupture force spectrum (Walton et al., 2008). We present new measurements of force-displacement curves for the (strept)avidin-biotin complex. These data were analyzed using the YieldFinder software based on the Bell-Evans formalism. In addition, the Williams model was adopted to interpret the bonding state of the system. Our results indicate the presence of at least two energy barriers in two loading rate regimes. Combining with structural analysis, the energy barriers can be interpreted in a novel physico-chemical context as one inner barrier for H-bond ruptures (?<1 ?), and one outer barrier for escaping from the binding pocket which is blocked by the side chain of a symmetry-related Trp120 in the streptavidin tetramer. In each loading rate regime, the presence of multiple parallel bonds was implied by the Williams model. Interestingly, we found that in literature different terms created for addressing the apparent discrepancies in the results of avidin-biotin interactions can be reconciled by taking into account multiple parallel bonds.     

Tree–Grass Coexistence in the Everglades Freshwater System

Mosaic freshwater landscapes exhibit tree-dominated patches —or tree islands—interspersed in a background of marshes and wet prairies. In the Florida Everglades, these patterned landscapes provide habitat for a variety of plant and animal species and are hotspots of biodiversity. Even though the emergence of patchy freshwater systems has been associated with climate histories, fluctuating hydrologic conditions, and internal feedbacks, a process-based quantitative understanding of the underlying dynamics is still missing. Here, we develop a mechanistic framework that relates the dynamics of vegetation, nutrients and soil accretion/loss through ecogeomorphic feedbacks and interactions with hydrologic drivers. We show that the stable coexistence of tree islands and marshes results as an effect of their both being (meta-) stable states of the system. However, tree islands are found to have only a limited resilience, in that changes in hydrologic conditions or vegetation cover may cause an abrupt shift to a stable marsh state. The inherent non-linear and discontinuous dynamics determining the stability and resilience of tree islands should be accounted for in efforts aiming at the management, conservation and restoration of these features.     

Synthesis and cytotoxic activity of new acridine-thiazolidine derivatives

Although their exact role in controlling tumour growth and apoptosis in humans remains undefined, acridine and thiazolidine compounds have been shown to act as tumour suppressors in most cancers. Based on this finding, a series of novel hybrid 5-acridin-9-ylmethylene-3-benzyl-thiazolidine-2,4-diones were synthesised via N-alkylation and Michael reaction. The cell viability was analysed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and DNA interaction assays were performed using electrochemical techniques.     

Cytotoxic activity of naphthoquinones with special emphasis on juglone and its 5-O-methyl derivative

The cytotoxicity of nine naphthoquinones (NQ) was assayed against HL-60 (leukaemia), MDA-MB-435 (melanoma), SF-295 (brain) and HCT-8 (colon), all human cancer cell lines, and peripheral blood mononuclear cells (PBMC), as representatives of normal cells, after 72 h of incubation. 5-Methoxy-1,4-naphthoquinone was the most active compound, showing IC₅₀ values in the range of 0.31 (1.7 μM) in HL-60 to 0.88 μg/mL (4.7 μM) in SF-295 and IC₅₀ of 0.69 μg/mL (3.7 μM) against PBMC. With the introduction of a bromo-substituent in position 2 or 3 of juglone, the IC₅₀ significantly decreased, regardless of the position on the NQ moiety. However, compared with juglone methyl ether, the halogen substitution decreased the activity. To further understand the mechanism underlying the cytotoxicity of 5-methoxy-1,4-naphthoquinone, studies involving DNA fragmentation, cell cycle analysis, phosphatidyl serine externalization, mitochondrial depolarization and activation of caspases 8 and 3/7 were performed in HL-60 cell line, using doxorubicin as a positive control. The results indicate that the cytotoxic 5-methoxy-1,4-naphthoquinone activates caspases 8 and 3/7 and thus induces apoptosis independent of mitochondria.     

Cytogenetic biomonitoring of inhabitants of a large uranium mineralization area: the municipalities of Monte Alegre, Prainha, and Alenquer, in the State of Pará, Brazil

Uranium is a natural radioactive metallic element; its effect on the organism is cumulative, and chronic exposure to this element can induce carcinogenesis. Three cities of the Amazon region—Monte Alegre, Prainha, and Alenquer—in North Brazil, are located in one of the largest uranium mineralization areas of the world. Radon is a radioactive gas, part of uranium decay series and readily diffuses through rock. In Monte Alegre, most of the houses are built of rocks removed from the Earth’s crust in the forest, where the uranium reserves lie. The objective of the present work is to determine the presence or absence of genotoxicity and risk of carcinogenesis induced by natural exposure to uranium and radon in the populations of these three cities. The frequency of micronuclei (MN) and chromosomal aberrations (CA) showed no statistically significant differences between the control population and the three study populations (P > 0.05). MN was also analyzed using the fluorescence in situ hybridization (FISH) technique, with a centromere-specific probe. No clastogenic and/or aneugenic effects were found in the populations. Using FISH analysis, other carcinogenesis biomarkers were analyzed, but neither the presence of the IGH/BCL2 translocation nor an amplification of the MYC gene and 22q21 region was detected. Clastogenicity and DNA damage were also not found in the populations analyzed using the alkaline comet assay. The mitotic index showed no cytotoxicity in the analyzed individuals’ lymphocytes. Once we do not have data concerning radiation doses from other sources, such as cosmic rays, potassium, thorium, or anthropogenic sources, it is hard to determine if uranium emissions in this geographic region where our study population lives are too low to cause significant DNA damage. Regardless, genetic analyses suggest that the radiation in our study area is not high enough to induce DNA alterations or to interfere with mitotic apparatus formation. It is also possible that damages caused by radiation doses undergo cellular repair.     

Evaluation of the genotoxicity of piplartine, an alkamide of Piper tuberculatum, in yeast and mammalian V79 cells

The genus Piper belongs to the Piperaceae family, and includes species of commercial and medicinal importance. Chemical studies on Piper species resulted in the isolation of several biologically active molecules, including alkaloid amides, such as piplartine. This molecule, isolated from Piper tuberculatum, has significant cytotoxic activity against tumor cell lines, and presents antifungal, anti-platelet aggregation, anxiolytic, and antidepressant effects. In order to understand the biological properties of piplartine, this study investigated the genotoxicity and the induction of apoptosis by piplartine in V79 cells and its mutagenic and recombinogenic potential in Saccharomyces cerevisiae. Piplartine induced dose-dependent cytotoxicity in S. cerevisiae cultures in either stationary -- or exponential growth phase. In addition, piplartine was not mutagenic when cells were treated during exponential-growth phase and kept in buffer solution, but it increased the frequencies of point, frameshift, and forward mutations when cells were treated in medium during growth. Piplartine treatment induced DNA strand breaks in V79 cells, as detected by neutral and alkaline comet assay. In cell cycle analysis, piplartine induced G2/M cell cycle arrest, probably as a consequence of the DNA damage induced and repair. Moreover, piplartine treatment induced apoptosis in a dose-dependent manner, as observed by a decrease in mitochondrial membrane potential and an increase in internucleosomal DNA fragmentation. These data suggest that the DNA damage caused by piplartine induces G2/M cell cycle arrest, followed by apoptosis. Moreover, we suggest that cells surviving piplartine-induced DNA damage can accumulate mutations, since this alkaloid was mutagenic and recombinogenic in S. cerevisiae assays.     

Antiproliferative effects of two amides, piperine and piplartine, from Piper species

The present work evaluated the cytotoxicity of piplartine {5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-trans-2-propenyl]-2(1H)pyridinone} and piperine {1-[5-(1,3)-benzodioxol-5-yl)-1-oxo-2,4-pentadienyl]piperidine}, components obtained from Piper species. The substances were tested for their cytotoxicity on the brine shrimp lethality assay, sea urchin eggs development, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay using tumor cell lines and lytic activity on mouse erythrocytes. Piperine showed higher toxicity in brine shrimp (DL50 = 2.8 +/- 0.3 microg/ml) than piplartine (DL50 = 32.3 +/- 3.4 microg/ml). Both piplartine and piperine inhibited the sea urchin eggs development during all phases examined, first and third cleavage and blastulae, but in this assay piplartine was more potent than piperine. In the MTT assay, piplartine was the most active with IC50 values in the range of 0.7 to 1.7 microg/ml. None of the tested substances induced hemolysis of mouse erythrocytes, suggesting that the cytotoxicity of piplartine and piperine was not related to membrane damage.     

Cytotoxic activity of pisosterol, a triterpene isolated from Pisolithus tinctorius (Mich.: Pers.) Coker & Couch, 1928

Pisolithus tinctorius (Basidiomycete) is an ectomicorrhizal fungus found in the roots and soil surrounding of many species of eucalyptus and pine trees. The present work verified the cytotoxic potential of pisosterol, a triterpene isolated from P. tinctorius collected in the Northeast region of Brazil, on three different animal cell models: mouse erythrocytes, sea urchin embryos and tumor cells. Pisosterol lacked activity on mouse erythrocytes as well as on the development of sea urchin eggs, but strongly inhibited the growth of all seven tumor cell lines tested, especially the leukemia and melanoma cells (IC50 of 1.55, 1.84 and 1.65 microg/ ml for CEM, HL-60 and B16, respectively). The results found for pisosterol were compared with those of doxorubicin and etoposide.