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SUMMARY Pausing by RNA polymerase (RNAp) facilitates the recruitment of regulatory factors, RNA folding, and other related processes. While backtracking and intra-structural isomerization have been proposed to trigger pausing, the mechanisms of pause formation and recovery remain debated. Using high-throughput magnetic tweezers with single-molecule sensitivity, we have examined the full temporal spectrum of Escherichia coli RNAp transcription dynamics. Together with probabilistic dwell-time analysis and modelling, this has identified three distinct states that compete with elongation: a short-lived elemental pause and two long-lived backtracked pause states. We demonstrate that recovery from backtracking is not governed by diffusional Brownian motion, but rather by intrinsic RNA cleavage, whereby RNAp conformational changes hinder cleavage in long-lived pause states. We further show that state switching underlies stochastic alterations in the frequency of short pauses. Based on these findings, we propose a consensus model of intrinsic pausing that unifies all key findings while resolving earlier contradictions.

Agradecimentos: R.J., P.K.S. and D.S.O. acknowledge scholarships from FAPESP and A.M.S. and D.B.A. from CAPES/CNPq. Access to confocal microscopy was granted by INFABIC/UNICAMP (FAPESP 08/57906-3, CNPq 573913/2008-0). We acknowledge access to electron microscopy (LME) and microfabrication (LMF) facilities at the National Nanotechnology Laboratory (LNNano, Brazil) and clean room facilities at both Semiconductor Component Center (CCSNano, UNICAMP) and Device Research Laboratory (IFGW, UNICAMP). Xf.XadA2 antibodies were provided by Dr. A. A. de Souza (IAC, Cordeirópolis, São Paulo, Brazil). The authors are greatly indebted to A. Gobbi and M.H. Piazzetta from LMF/LNNano for PDMS microfluidics processing, Dr. A. Kisner from IQ/UNICAMP and P .C. Galvão, from IQ/UNESP, for technical assistance during this work Abstract: Electrically active field-effect transistors (FET) based biosensors are of paramount importance in life science applications, as they offer direct, fast, and highly sensitive label-free detection capabilities of several biomolecules of specific interest. In this work, we report a detailed investigation on surface functionalization and covalent immobilization of biomarkers using biocompatible ethanolamine and poly(ethylene glycol) derivate coatings, as compared to the conventional approaches using silica monoliths, in order to substantially increase both the sensitivity and molecular selectivity of nanowire-based FET biosensor platforms. Quantitative fluorescence, atomic and Kelvin probe force microscopy allowed detailed investigation of the homogeneity and density of immobilized biomarkers on different biofunctionalized surfaces. Significantly enhanced binding specificity, biomarker density, and target biomolecule capture efficiency were thus achieved for DNA as well as for proteins from pathogens. This optimized functionalization methodology was applied to InP nanowires that due to their low surface recombination rates were used as new active transducers for biosensors. The developed devices provide ultrahigh label-free detection sensitivities similar to 1 fM for specific DNA sequences, measured via the net change in device electrical resistance. Similar levels of ultrasensitive detection of similar to 6 fM were achieved for a Chagas Disease protein marker (IBMP8-1). The developed InP nanowire biosensor provides thus a qualified tool for detection of the chronic infection stage of this disease, leading to improved diagnosis and control of spread. These methodological developments are expected to substantially enhance the chemical robustness, diagnostic reliability, detection sensitivity, and biomarker selectivity for current and future biosensing devices FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES Fechado

Agradecimentos: The authors acknowledge J. H. Clerici, V. B. Pelegati, and M. O. Baratti for technical assistance and C. B. Santos and Prof. A. P. de Souza, from CBMEG/UNICAMP for providing the XadA1 protein. A.C. and E.P.A.M.B. would like to acknowledge Rene Van Veldhoven for the exceptional work and care for the MOVPE systems and the technical support from the Nano-Lab@TU/e cleanroom. This work was financially supported by FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo, Brazil), through grants 2010/51748-7 and 2013/02300-1, and CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico, Brazil), grant number 479486/2012-3. D.M.M. and M.P.M. acknowledge scholarships from CAPES (Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior, Brazil); P.K.S. and R.J. acknowledge FAPESP for funding their scholarships. M.A.C., A.A.S., and H.F.C. acknowledge CNPq fellowships. The authors thank the access to confocal microscopy equipment provided by the National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC) at the State University of Campinas; INFABIC is cofunded by FAPESP (08/57906-3) and CNPq (573913/2008-0). We also acknowledge the National Nano technology Laboratory (LNNano) at CNPEM, Brazil, for granting access to their electron microscopy facilities. A.C. and E.P.A.M.B. acknowledge the funding of the Dutch Technology Foundation STW (Project 11826), which is part of The Netherlands Organisation for Scientific Research (NWO), and which is partly funded by the Dutch Ministry of Economic Affairs Abstract: Surface attachment of a planktonic bacteria, mediated by adhesins and extracellular polymeric substances (EPS), is a crucial step for biofilm formation. Some pathogens can modulate cell adhesiveness, impacting host colonization and virulence. A framework able to quantify cell-surface interaction forces and their dependence on chemical surface composition may unveil adhesiveness control mechanisms as new targets for intervention and disease control. Here we employed InP nanowire arrays to dissect factors involved in the early stage biofilm formation of the phytopathogen Xylella fastidiosa. Ex vivo experiments demonstrate single-cell adhesion forces up to 45 nN, depending on the cell orientation with respect to the surface. Larger adhesion forces occur at the cell poles; secreted EPS layers and filaments provide additional mechanical support. Significant adhesion force enhancements were observed for single cells anchoring a biofilm and particularly on XadA1 adhesincoated surfaces, evidencing molecular mechanisms developed by bacterial pathogens to create a stronger holdfast to specific host tissues FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ COORDENAÇÃO DE APERFEIÇOAMENTO DE PESSOAL DE NÍVEL SUPERIOR - CAPES Aberto

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.

Titanium dioxide (TiO2) exhibits exceptional properties such as a rapid electron transport, high dielectric constant, wide band gap, high refractive index, and large birefringence. Further, it is a photocatalyst and highly biocompatible. It is widely implemented in electronic and photonic applications like solar cells, sensors, transistors, memory devices, and force transducers. However, its use at the nanoscale has been limited by challenges in fabrication. The most common bottom-up fabrication methods lack fine dimensional control and structural uniformity. To overcome these limitations, plasma etching-based top-down fabrication is preferred in general but TiO2 suffers from the difficulty in etching [1]. To address this challenge, we have developed a novel fabrication method utilizing fluorine-based dry etching to controllably fabricate TiO2 nanostructures, and chemically modify their surfaces employing readily accessible surface functionalization methods. In our fabrication protocol (Fig 1), a chromium (Cr) etch mask provides increased resistance to dry etching and allows a wider range of aspect ratios and sidewall angles through its high etch selectivity. The patterning process using electron-beam (e-beam) lithography allows high dimensional uniformity of nanostructures. Additionally, free nanoparticles can be generated from them through mechanical cleaving (Fig 1f, Fig 2). We have optimized TiO2 etching parameters for two types of fluorine-based plasma etching configurations. First, for the trifluoromethane (CHF3)-based plasma etching of TiO2 with a reactive ion etching (RIE) machine, we obtain high etch selectivity (~15:1) as well as controlled sidewall angles. Depending on the amount of added oxygen (O2) gas flow, the angles can be varied from positive to negative, including vertical sidewalls (Fig 3a). Interestingly, with higher O2 gas flow, the fabrication of hourglass-shaped nanocylinders is also possible (Fig 3b). Second, we also optimize the sulfur hexafluoride (SF6)-based plasma etching of TiO2 with an inductively coupled plasma (ICP)-RIE machine. It allows us to achieve higher etch rates of 100-200 nm/min, compared to those of 30-50 nm/min in CHF3-based RIE, possibly due to high ion density and less passivation. Increasing ICP power, hence providing higher ion density, results in more positive sidewall angles (Fig 3c) due to more rapid Cr mask etch rates which reduce etch selectivity. On the other hand, with a reduced ICP power, we can fabricate nanocylinders with nearly vertical sidewalls (Fig 3d), demonstrating sufficient etch selectivity (~12:1). As we use the rutile-phase TiO2 that is the most difficult to etch [2], our method is expected to be directly applicable to the other phases with only minor modifications of a few etching parameters. For surface functionalization of TiO2 surfaces, we utilize widely used epoxysilanes as surface linker molecule (Fig 4a) [3]. The dense, homogeneous surface coating is demonstrated by attaching fluorophores to the epoxysilane-coated surface and imaging their fluorescence emission (Fig 4b). Moreover, the functionalization can be selectively performed only on top of nanocylinders (Fig 4c) with partial PMMA coating (Fig 1e). The introduced fabrication and functionalization methods can be employed in diverse applications that exploit the unique physical and chemical properties of TiO2 at the nanoscale, including biomolecular sensing.

Agradecimentos: This study was supported by grants from the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2012/51580-4 and 2001/07533-7). C.A.S. was the recipient of a Ph.D. fellowship from FAPESP (Process 2008/55690-3); R.J. acknowledges postdoctoral FAPESP scholarship (2009/53021-0). A.P.S. and M.A.C. are the recipients of a research fellowship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). We gratefully acknowledge the Laboratório Nacional de Luz Síncrotron (LNLS, Campinas, Brazil) for providing beamline time. We thank Alessandra P. de Souza of the APTA Citrus ‘Sylvio Moreira’ Center, Agronomic Institute of Campinas, Cordeirópolis, SP, Brazil for the assistance with X. fastidiosa biofilms and Prof. Dr. Carlos H. I. Ramos of the Institute of Chemistry, University of Campinas, Campinas, SP, Brazil for providing anti-Hsp70/Hsc70 — Global antibody Abstract: The intriguing roles of the bacterial Tol-Pal trans-envelope protein complex range from maintenance of cell envelope integrity to potential participation in the process of cell division. In this study, we report the characterization of the XfFolB and XfPal proteins of the Tol-Pal complex of Xylella fastidiosa. X. fastidiosa is a major plant pathogen that forms biofilms inside xylem vessels, triggering the development of diseases in important cultivable plants around the word. Based on functional complementation experiments in Escherichia coli tolB and pal mutant strains, we confirmed the role of xftolB and xfpal in outer membrane integrity. In addition, we observed a dynamic and coordinated protein expression profile during the X. fastidiosa biofilm development process. Using small-angle X-ray scattering (SAXS), the low-resolution structure of the isolated XfToIB-XfPal complex in solution was solved for the first time. Finally, the localization of the XfFolB and XfPal polar ends was visualized via immunofluorescence labeling in vivo during bacterial cell growth. Our results highlight the major role of the components of the cell envelope, particularly the TolB-Pal complex, during the different phases of bacterial biofilm development FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ Aberto

Agradecimentos: The authors gratefully acknowledge the financial support of the Fundacao de Amparo a Pesquisa do Estado de Sao Paulo - FAPESP (Sao Paulo, Brazil, Grant 2007/58323-9) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico - CNPq (Brazil, Grant 471971/2011-1). We also thank the Laboratorio de Espectroscopia e Calorimetria (LEC), Laboratorio Nacional de Biociencias - LNBio (Campinas, Brazil), and the National Institute of Science and Technology on Photonics Applied to Cell Biology (INFABIC), UNICAMP, for the support for the analysis of recombinant T-Rp3. Finally, we thank Professor Maricilda Palandi de Mello, CBMEG, UNICAMP (Campinas, Brazil), for support for the HeLa cell culture Abstract: Gene therapy and DNA vaccination trials are limited by the lack of gene delivery vectors that combine efficiency and safety. Hence, the development of modular recombinant proteins able to mimic mechanisms used by viruses for intracellular trafficking and nuclear delivery is an important strategy. We designed a modular protein (named T-Rp3) composed of the recombinant human dynein light chain Rp3 fused to an N-terminal DNA-binding domain and a C-terminal membrane active peptide, TAT. The T-Rp3 protein was successfully expressed in Escherichia coli and interacted with the dynein intermediate chain in vitro. It was also proven to efficiently interact and condense plasmid DNA, forming a stable, small (similar to 100 nm) and positively charged (+28.6 mV) complex. Transfection of HeLa cells using T-Rp3 revealed that the vector is highly dependent on microtubule polarization, being 400 times more efficient than protamine, and only 13 times less efficient than Lipofectamine 2000 (TM), but with a lower cytotoxicity. Confocal laser scanning microcopy studies revealed perinuclear accumulation of the vector, most likely as a result of transport via microtubules. This study contributes to the development of more efficient and less cytotoxic proteins for non-viral gene delivery FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ Fechado

Agradecimentos: This work was financially supported by FAPESP (Fundação de Amparo a Pesquisa do Estado de Sao Paulo) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). A.L.D.M. and R.J. acknowledge scholarships from CNPq and FAPESP, respectively Abstract: The development of highly-sensitive and label-free operating semiconductor-based, biomaterial detecting sensors has important applications in areas such as environmental science, biomedical research and medical diagnostics. In the present study, we developed an Indium Phosphide (InP) semiconductor-based resistive biosensor using the change of its electronic properties upon biomaterial adsorption as sensing element. To detect biomaterial at low concentrations, the procedure of functionalization and covalent biomolecule immobilization was also optimized to guarantee high molecule density and high reproducibility which are prerequisite for reliable results. The characterization, such as biomolecular conjugation efficiency, detection concentration limits, recepton:ligand specificity and concentration detection range was analyzed by using three different biological systems: i) synthetic dsDNA and two phytopathogenic diseases, ii) the severe CB-form of Citrus Tristeza Virus (CIV) and iii) Xylella fastidiosa, both causing great economic loss worldwide. The experimental results show a sensitivity of 1 pM for specific ssDNA detection and about 2 nM for the specific detection of surface proteins of CTV and X fastidiosa phytopathogens. A brief comparison with other semiconductor based biosensors and other methodological approaches is discussed and confirms the high sensitivity and reproducibility of our InP based biosensor which could be suitable for reliable early infection diagnosis in environmental and life sciences FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQ Fechado

Agradecimentos: The authors gratefully acknowledge the financial support of the Fundação de Amparo à Pesquisa do Estado de São Paulo – FAPESP (São Paulo, Brazil). We also thank the Laboratório de Espectroscopia e Calorimetria (LEC), Laboratório Nacional de Biociências – LNBio (Campinas, Brazil), for the support on the circular dichroism studies and Professor Maricilda Palandi de Mello, CBMEG, UNICAMP (Campinas, Brazil), for the support on the HeLa cells culture. Finally, the authors thank Professor Ricardo Aparício, IQ, UNICAMP, for the help with the preliminary studies of SAXS and Professor Lucimara Gaziola de la Torre, FEQ, UNICAMP, for the help with potential zeta and particle size measurements Abstract: 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 FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP Fechado