Lilian T. Costa

Cidofovir Inhibits Genome Encapsidation and Affects Morphogenesis during the Replication of Vaccinia Virus

ABSTRACT Cidofovir (CDV) is one of the most effective antiorthopoxvirus drugs, and it is widely accepted that viral DNA replication is the main target of its activity. In the present study, we report a detailed analysis of CDV effects on the replicative cycles of distinct vaccinia virus (VACV) strains: Cantagalo virus, VACV-IOC, and VACV-WR. We show that despite the approximately 90% inhibition of production of virus progeny, virus DNA accumulation was reduced only 30%, and late gene expression and genome resolution were unaltered. The level of proteolytic cleavage of the major core proteins was diminished in CDV-treated cells. Electron microscopic analysis of virus-infected cells in the presence of CDV revealed reductions as great as 3.5-fold in the number of mature forms of virus particles, along with a 3.2-fold increase in the number of spherical immature particles. A detailed analysis of purified virions recovered from CDV-treated cells demonstrated the accumulation of unprocessed p4a and p4b and nearly 67% inhibition of DNA encapsidation. However, these effects of CDV on virus morphogenesis resulted from a primary effect on virus DNA synthesis, which led to later defects in genome encapsidation and virus assembly. Analysis of virus DNA by atomic force microscopy revealed that viral cytoplasmic DNA synthesized in the presence of CDV had an altered structure, forming aggregates with increased strand overlapping not observed in the absence of the drug. These aberrant DNA aggregations were not encapsidated into virus particles.

Measuring the Strength of Interaction between the Ebola Fusion Peptide and Lipid Rafts: Implications for Membrane Fusion and Virus Infection

The Ebola fusion peptide (EBO16) is a hydrophobic domain that belongs to the GP2 membrane fusion protein of the Ebola virus. It adopts a helical structure in the presence of mimetic membranes that is stabilized by the presence of an aromatic-aromatic interaction established by Trp8 and Phe12. In spite of its infectious cycle becoming better understood recently, several steps still remain unclear, a lacuna that makes it difficult to develop strategies to block infection. In order to gain insight into the mechanism of membrane fusion, we probed the structure, function and energetics of EBO16 and its mutant W8A, in the absence or presence of different lipid membranes, including isolated domain-resistant membranes (DRM), a good experimental model for lipid rafts. The depletion of cholesterol from living mammalian cells reduced the ability of EBO16 to induce lipid mixing. On the other hand, EBO16 was structurally sensitive to interaction with lipid rafts (DRMs), but the same was not observed for W8A mutant. In agreement with these data, W8A showed a poor ability to promote membrane aggregation in comparison to EBO16. Single molecule AFM experiments showed a high affinity force pattern for the interaction of EBO16 and DRM, which seems to be a complex energetic event as observed by the calorimetric profile. Our study is the first to show a strong correlation between the initial step of Ebola virus infection and cholesterol, thus providing a rationale for Ebola virus proteins being co-localized with lipid-raft domains. In all, the results show how small fusion peptide sequences have evolved to adopt highly specific and strong interactions with membrane domains. Such features suggest these processes are excellent targets for therapeutic and vaccine approaches to viral diseases.

Sugarcane cell wall structure and lignin distribution investigated by confocal and electron microscopy

Lignocellulosic plant cell wall is considered a potential source for second generation biofuels. The plant cell wall is a highly complex structure mainly composed of cellulose, hemicelluloses, and lignin that form a network of crosslinked fibers. The structural organization of the sugarcane cell wall has not been previously analyzed in detail, and this analysis is a prerequisite for further studies on the recalcitrance and deconstruction of its biomass. In this work, cellulose and lignin localization were investigated by confocal laser scanning microscopy. In addition, the internode sugarcane cell wall structural organization was analyzed by electron microscopy. Internode stem anatomy showed a typical monocot structure consisting of epidermis, hypoderm, and vascular bundles scattered throughout ground parenchyma tissue and surrounded by sclerenchyma fibers. Confocal images of safranin labeled sugarcane showed that lignin distribution was predominant in the vessel elements, cell wall corners (CC), and middle lamella (ML), while cellulose‐rich cell walls were randomly distributed in the ML and organized in the other cell wall layers. KMnO4 cytochemistry revealed that lignin was predominantly distributed in secondary cell walls, ML and CC. Cell wall sublayers (S1, S2, and S3) were identified and measured by transmission electron microscopy. Our results provide insights that may help further understanding of sugarcane cell wall organization, which is crucial for the research and technology of plant‐based biofuel production. Microsc. Res. Tech. 76:829–834, 2013.

Vitamins K interact with N-terminus α-synuclein and modulate the protein fibrillization in vitro. Exploring the interaction between quinones and α-synuclein.

In the last decades, a series of compounds, including quinones and polyphenols, has been described as having anti-fibrillogenic action on α-synuclein (α-syn) whose aggregation is associated to the pathogenesis of Parkinsons disease (PD). Most of these molecules act as promiscuous anti-amyloidogenic agents, interacting with the diverse amyloidogenic proteins (mostly unfolded) through non-specific hydrophobic interactions. Herein we investigated the effect of the vitamins K (phylloquinone, menaquinone and menadione), which are 1,4-naphthoquinone (1,4-NQ) derivatives, on α-syn aggregation, comparing them with other anti-fibrillogenic molecules such as quinones, polyphenols and lipophilic vitamins. Vitamins K delayed α-syn fibrillization in substoichiometric concentrations, leading to the formation of short, sheared fibrils and amorphous aggregates, which are less prone to produce leakage of synthetic vesicles. In seeding conditions, menadione and 1,4-NQ significantly inhibited fibrils elongation, which could be explained by their ability to destabilize preformed fibrils of α-syn. Bidimensional NMR experiments indicate that a specific site at the N-terminal α-syn (Gly31/Lys32) is involved in the interaction with vitamins K, which is corroborated by previous studies suggesting that Lys is a key residue in the interaction with quinones. Together, our data suggest that 1,4-NQ, recently showed up by our group as a potential scaffold for designing new monoamine oxidase inhibitors, is also capable to modulate α-syn fibrillization in vitro.

The structure of the kinetoplast DNA network of Crithidia fasciculata revealed by atomic force microscopy.

DNA is the biopolymer most studied by scanning probe methods, and it is now possible to obtain reliable and reproducible images of DNA using atomic force microscopy (AFM). AFM has been extensively used to elucidate morphological changes to DNA structure, such as the formation of knots, nicks, supercoiling and bends. The mitochondrial or kinetoplast DNA (kDNA) of trypanosomatids is the most unusual DNA found in nature, being unique in organization and replication. The kDNA is composed of thousands of topologically interlocked DNA circles that form a giant network. To understand the biological significance of the kinetoplast DNA, it is necessary to learn more about its structure. In the present work, we used two procedures to prepare kDNA networks of Crithidia fasciculata for observation by AFM. Because AFM allows for the examination of kDNA at high resolution, we were able to identify regions of overlapping kDNA molecules and sites where several molecules cross. This found support the earlier described kDNA structural organization as composed by interlocked circles. We also observed an intricate high-density height pattern around the periphery of the network of C. fasciculata, which appears to be a bundle of DNA fibers that organizes the border of the network. Our present data confirm that AFM is a powerful tool to study the structural organization of biological samples, including complex arrays of DNA such as kDNA, and can be useful in revealing new details of structures previously visualized by other means.

Catalytic properties of lipases immobilized onto ultrasound-treated chitosan supports

Ultrasound sonication has been utilized to produce fragmentation of chitosan polymer and hence increase the chitosan surface area, making it more accessible to interactions with proteins. In this context, we have investigated the catalytic properties of lipases from different sources immobilized onto ultrasound-treated chitosan (ChiS) pre-activated with glutaraldehyde (ChiS-G). Atomic force microscopy indicated that ChiS-G displays a more cohesive frame without the presence of sheared/fragmented structures when compared with ChiS, which might be attributed to the cross-linking of the polysaccharide chains. The immobilization efficiency onto ChiS-G and ChiS were remarkably higher than using conventional beads. In comparison with the free enzymes, lipases immobilized onto ChiS show a slight increase of apparent Km and decrease of apparent Vmax. On the other hand, immobilization onto ChiS-G resulted in an increase of Vmax, even though a slight increase of Km was also observed. These data suggest that the activation of chitosan with glutaraldehyde has beneficial effects on the activity of the immobilized lipases. In addition, the immobilization of the lipases onto ChiS-G displayed the best reusability results: enzymes retained more than 50% of its initial activity after four reuses, which might be attributed to the covalent attachment of enzyme to activated chitosan. Overall, our findings demonstrate that the immobilization of lipases onto ultrasound-treated chitosan supports is an effective and low-cost procedure for the generation of active immobilized lipase systems, being an interesting alternative to conventional chitosan beads.

Direct immobilization of avidin protein on AFM tip functionalized by acrylic acid vapor at RF plasma

The atomic force microscopy (AFM) has been used as a force sensor to measure unbinding forces of single bound complexes in the nanonewton and piconewton range. Force spectroscopy measurements can be applied to study both intermolecular and intramolecular interactions of complex biological and synthetic macromolecules. Although the AFM has been extensively used as a nano force sensor, the commercially available cantilever is limited to silicon and silicon nitride. Those materials reduce the adhesion sensitivity with specific surface and/or molecule. Here, we functionalized the AFM tip with carboxylic groups by applying acrylic acid (AA) vapor at radio frequency plasma treatment at 100 W for 5 min. This method provides a remarkable sensitivity enhancement on the functional group interaction specificity. The functionalized tip was characterized by scanning electron microscopy. The electron beam high resolution images have not shown significant tip sharpness modification. Silicon wafers (1 0 0)—no treated and functionalized by AA plasma treatment—were characterized by Auger electron spectroscopy to elucidate the silicon surface sputtering and demonstrate functionalization. The Fourier transform‐infrared spectroscopy spectrum shows a high absorbance of avidin protein over the silicon surface functionalized by AA plasma treatment.We carried out force spectroscopy assay to measure the unbinding force between the well‐established pair biotin–avidin. At pulling speed of 2 µm/s, we measured the unbinding force of 106 ± 23 pN, which is in good agreement with the literature, demonstrating the effectiveness of the tip functionalization by AA plasma treatment in biological studies. Copyright

TORSIONAL RESONANCE MODE ATOMIC FORCE MICROSCOPY OF A PROTEIN-DNA COMPLEX

Precise mapping of protein-binding sites on DNA is an important application of atomic force microscope (AFM) imaging. For a reliable measurement of distances on curved DNA molecules, an image-processing algorithm is required, which extracts the DNA contour from topographic AFM data. To this end we implemented an image analysis method providing an efficient way to obtain the contour together with a physical map of single and multiple protein-binding sites. This method relies on a calculation of the height profile along the DNA fragment, allowing one to determine the DNA length and the relative position of the binding site occupied by a protein. As a first test, complexes of the LexA repressor protein from the Escherichia coli SOS system and DNA fragments containing a specific LexA binding site (recA operator) were imaged by the torsional resonance mode (TR mode) and analyzed using the specialized algorithm. A topographic height of less than 0.5 nm of the DNA molecules indicates repulsive imaging conditions.

Automated Object Length Measurement Applied to AFM, STM and TEM Images Based on the Snake Model

This work describes the development of an automated method to measure the length of filament-shaped objects from Atomic Force Microscopy (AFM), Scanning Tunneling Microscopy (STM) and Transmition Electron Microscopy (TEM) images. The proposed methodology can determine the length of the object(s) of interest using image segmentation, where the Parametric Deformable Model (PDM) (especially the Snake Model - SM) was applied. The measurement procedure starts from the segmentation step, where an improved erosion is applied, resulting in a thin curve representation with only two endpoints (skeleton). The piecewise linear approximation concept was also employed to measure the total length of an object, based on the dimension of the pixel (unitary length) that composes the skeleton. The accuracy of the algorithm was evaluated using a high precision STM 7x7 silicon image and a sort of DNA filaments (AFM).