Highly birefringent TiO2 nanocylinders : characterization and application in the optical torque wrench

Recent advances in optical tweezers have enabled the direct manipulation and measurement of optical torque using light spin momentum transferred to trapped birefringent particles. This powerful technique, termed Optical Torque Wrench (OTW), relies on trapping of quartz (SiO2) microcylinders which have become a standard and convenient probe for single-molecule studies. Here, we explore an alternative photonic probe based on rutile (TiO2) which has almost thirty-fold larger birefringence compared to quartz particles. By employing this promising material to fabricate rutile nanocylinders whose sizes can be easily tuned, we significantly enhance the accessible range of optical torques and angular frequencies in the OTW. In future, these novel photonic probes will allow us to study not only slowly processing torque-generating biological systems, as the genome processing machinery, but also fast rotating motors, including ATP-synthase and the bacterial flagellar motor

Trapping of highly birefringent rutile nanocylinders in the optical torque wrench

The optical torque wrench (OTW) is a powerful technique to measure the torsional properties of different biomolecules, including DNA, DNA- processing protein complexes and rotary motors. To date, quartz has proven to be a convenient birefringent material out of which to synthesize the micron-sized particles essential for this technique. However, the relatively low birefringence of quartz, which limits the maximal torque that can be applied in OTW, hampers the study of certain biological systems. A more attractive material is rutile, which has a thirty-fold higher birefringence. To date, however, the application of rutile in the trapping has been restricted due to its high refractive index, which results in low trapping efficiency. Here, we have employed finite element method calculations to determine the optimal dimensions of sub-micron-sized rutile cylinders for tight stable optical trapping. Using these calculations as a guideline, we have designed and devel- oped a nanofabrication protocol that allows us to produce rutile cylinders with the desired sizes at high yield. We have characterized the fabricated cylinders in the OTW setup and quantified both their linear and angular trapping proper- ties. In addition, we demonstrate full translational and rotational control of these functionalized cylinders tethered to individual DNA molecules for use in single-molecule applications

Spatiotemporal Distribution Of Different Extracellular Polymeric Substances And Filamentation Mediate Xylella Fastidiosa Adhesion And Biofilm Formation

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Microorganism pathogenicity strongly relies on the generation of multicellular assemblies, called biofilms. Understanding their organization can unveil vulnerabilities leading to potential treatments; spatially and temporally-resolved comprehensive experimental characterization can provide new details of biofilm formation, and possibly new targets for disease control. Here, biofilm formation of economically important phytopathogen Xylella fastidiosa was analyzed at single-cell resolution using nanometer-resolution spectro-microscopy techniques, addressing the role of different types of extracellular polymeric substances (EPS) at each stage of the entire bacterial life cycle. Single cell adhesion is caused by unspecific electrostatic interactions through proteins at the cell polar region, where EPS accumulation is required for more firmly-attached, irreversibly adhered cells. Subsequently, bacteria form clusters, which are embedded in secreted loosely-bound EPS, and bridged by up to ten-fold elongated cells that form the biofilm framework. During biofilm maturation, soluble EPS forms a filamentous matrix that facilitates cell adhesion and provides mechanical support, while the biofilm keeps anchored by few cells. This floating architecture maximizes nutrient distribution while allowing detachment upon larger shear stresses; it thus complies with biological requirements of the bacteria life cycle. Using new approaches, our findings provide insights regarding different aspects of the adhesion process of X. fastidiosa and biofilm formation.5Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)FAPESP [2010/51748-7, 2010/18107-8, 2010/50712-9]CNPq [479486/2012-3]CNPq [573913/2008-0]FAPESP [08/57906-3

Surface Physicochemical Properties At The Micro And Nano Length Scales: Role On Bacterial Adhesion And Xylella Fastidiosa Biofilm Development.

The phytopathogen Xylella fastidiosa grows as a biofilm causing vascular occlusion and consequently nutrient and water stress in different plant hosts by adhesion on xylem vessel surfaces composed of cellulose, hemicellulose, pectin and proteins. Understanding the factors which influence bacterial adhesion and biofilm development is a key issue in identifying mechanisms for preventing biofilm formation in infected plants. In this study, we show that X. fastidiosa biofilm development and architecture correlate well with physicochemical surface properties after interaction with the culture medium. Different biotic and abiotic substrates such as silicon (Si) and derivatized cellulose films were studied. Both biofilms and substrates were characterized at the micro- and nanoscale, which corresponds to the actual bacterial cell and membrane/ protein length scales, respectively. Our experimental results clearly indicate that the presence of surfaces with different chemical composition affect X. fastidiosa behavior from the point of view of gene expression and adhesion functionality. Bacterial adhesion is facilitated on more hydrophilic surfaces with higher surface potentials; XadA1 adhesin reveals different strengths of interaction on these surfaces. Nonetheless, despite different architectural biofilm geometries and rates of development, the colonization process occurs on all investigated surfaces. Our results univocally support the hypothesis that different adhesion mechanisms are active along the biofilm life cycle representing an adaptation mechanism for variations on the specific xylem vessel composition, which the bacterium encounters within the infected plant.8e7524

The Muonium Atom as a Probe of Physics beyond the Standard Model

The observed interactions between particles are not fully explained in the successful theoretical description of the standard model to date. Due to the close confinement of the bound state muonium ($M = \mu^+ e^-$) can be used as an ideal probe of quantum electrodynamics and weak interaction and also for a search for additional interactions between leptons. Of special interest is the lepton number violating process of sponteanous conversion of muonium to antimuonium.Comment: 15 pages,6 figure

Development of a recombinant fusion protein based on the dynein light chain LC8 for non-viral gene delivery

The low efficiency of gene transfer is a recurrent problem in DNA vaccine development and gene therapy studies using non-viral vectors such as plasmid DNA (pDNA). This is mainly due to the fact that during their traffic to the target cell's nuclei, plasmid vectors must overcome a series of physical, enzymatic and diffusional barriers. The main objective of this work is the development of recombinant proteins specifically designed for pDNA delivery, which take advantage of molecular motors like dynein, for the transport of cargos from the periphery to the centrosome of mammalian cells. A DNA binding sequence was fused to the N-terminus of the recombinant human dynein light chain LC8. Expression studies indicated that the fusion protein was correctly expressed in soluble form using E. coli BL21(DE3) strain. As expected, gel permeation assays found the purified protein mainly present as dimers, the functional oligomeric state of LC8. Gel retardation assays and atomic force microscopy proved the ability of the fusion protein to interact and condense pDNA. Zeta potential measurements indicated that LC8 with DNA binding domain (LD4) has an enhanced capacity to interact and condense pDNA, generating positively charged complexes. Transfection of cultured HeLa cells confirmed the ability of the LD4 to facilitate pDNA uptake and indicate the involvement of the retrograde transport in the intracellular trafficking of pDNA: LD4 complexes. Finally, cytotoxicity studies demonstrated a very low toxicity of the fusion protein vector, indicating the potential for in vivo applications. The study presented here is part of an effort to develop new modular shuttle proteins able to take advantage of strategies used by viruses to infect mammalian cells, aiming to provide new tools for gene therapy and DNA vaccination studies. (C) 2012 Elsevier B.V. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo - FAPESP (Sao Paulo, Brazil)Laboratorio de Espectroscopia e Calorimetria (LEC), Laboratorio Nacional de Biociencias - LNBio (Campinas, Brazil