PILZ Protein Structure and Interactions with PILB and the FIMX EAL Domain: Implications for Control of Type IV Pilus Biogenesis

The PilZ protein was originally identified as necessary for type IV pilus (T4P) biogenesis. Since then, a large and diverse family of bacterial PilZ homology domains have been identified, some of which have been implicated in signaling pathways that control important processes, including motility, virulence and biofilm formation. Furthermore, many PilZ homology domains, though not PilZ itself, have been shown to bind the important bacterial second messenger bis(3`-> 5`)cyclic diGMP (c-diGMP). The crystal structures of the PilZ orthologs from Xanthomonas axonopodis pv Citri (PilZ(XAC1133), this work) and from Xanthomonas campestris pv campestris (XC1028) present significant structural differences to other PilZ homologs that explain its failure to bind c-diGMP. NMR analysis of PilZ(XAC1133) shows that these structural differences are maintained in solution. In spite of their emerging importance in bacterial signaling, the means by which NZ proteins regulate specific processes is not clear. In this study, we show that PilZ(XAC1133) binds to PilB, an ATPase required for TV polymerization, and to the EAL domain of FiMX(XAC2398), which regulates TV biogenesis and localization in other bacterial species. These interactions were confirmed in NMR, two-hybrid and far-Western blot assays and are the first interactions observed between any PilZ domain and a target protein. While we were unable to detect phosphodiesterase activity for FimXX(AC2398) in vitro, we show that it binds c-diGMP both in the presence and in the absence of PilZ(XAC1133). Site-directed mutagenesis studies for conserved and exposed residues suggest that PilZ(XAC1133) interactions with FimX(XAC2398) and PilB(XAC3239) are mediated through a hydrophobic surface and an unstructured C-terminal extension conserved only in PilZ orthologs. The FimX-PilZ-PilB interactions involve a full set of ""degenerate"" GGDEF, EAL and PilZ domains and provide the first evidence of the means by which PilZ orthologs and FimX interact directly with the TP4 machinery. (C) 2009 Elsevier Ltd. All rights reserved.

Encapsulation of single hMSCs in polyelectrolyte shells

The main objective of this Thesis was to encapsulate single viable cells within polyelectrolyte films using the Layer-by-Layer (LbL) technique. Most of the experiments used human mesenchymal stem cells (MSCs) whose characteristics (capacity of selfrenewal and potential to differentiate into several types of cells) make them particularly interesting to be used in biomedical applications. Also, most of the experiments used alginate (ALG) as the anionic polyelectrolyte and chitosan (CHI) or poly(allylamine hydrochloride) (PAH) as the cationic polyelectrolyte. Hyaluronic acid (HA) was also tested as an anionic polyelectrolyte. At the beginning of the work, the experimental conditions necessary to obtain the encapsulation of individual cells were studied and established. Through fluorescence microscopy visualization by staining the cell nucleus and using polyelectrolytes conjugated to fluorescent dyes, it was possible to prove the obtainment of capsules containing one single cell inside. Capsules aggregation was an observed problem which, despite the efforts to design an experimental process to avoid this situation (namely, by playing with cell concentration and different means of re-suspending and stirring the cells), was not completely overcome. In a second part of the project, single cells were encapsulated within polyelectrolyte layers made of CHI/ALG, PAH/ALG and PAH/HA and their viability was evaluated through the resazurin reduction assay and the Live/Dead assay. In these experiments, during the LbL process, polyelectrolyte solutions were used at a concentration of 1mg/mL based on literature. In general, the viability of the encapsulated cells was shown to be very low/absent. Then, as a consequence of the lack of viability of cells encapsulated within polyelectrolyte layers, the LbL technique was applied in cells growing adherent to the surface of cell culture plates. The cells were cultured like in a sandwich, between the surface of the cell culture dish and the polyelectrolyte layers. Also here, the polyelectrolyte solutions were used at a concentration of 1mg/mL during the LbL process. Surprisingly, cell viability was also absent in these systems. A systematic study (dose-effect study) was performed to evaluate the effect of the concentration of the individual polyelectrolytes (ALG, CHI and PAH were studied) in cell viability. Experiments were performed using cells growing adherent to the surface of cell culture plates. The results pointed out that a very high (cytotoxic) concentration of polyelectrolytes had been in use. Also, in general, PAH was much more cytotoxic than CHI, whereas ALG was the less cytotoxic polyelectrolyte. Finally, using alginate and chitosan solutions with adequate concentrations (low concentrations: 50ng/mL and 1μg/mL), the encapsulation of single viable cells was again attempted. Once again, the encapsulated cells were not shown to be viable. In conclusion, the viability of the encapsulated cells is not only dependent on the cytotoxic characteristics (or combined cytotoxic characteristics) of the polyelectrolytes but it seems that, when detached from the culture plates, the cells become too fragile and lose their viability very easily.

Cell-responsive nanogels for anticancer drug delivery

One of the main goals in Nanomedicine is to create innovative drug delivery systems (DDS) capable of delivering drugs into a specific location with high efficiency. In the development of DDS, some essential properties are desired, such as biocompatibility and biodegradability. Furthermore, an ideal DDS should be able to deliver a drug in a controlled manner and minimize its side effects. These two objectives are still a challenge for researchers all around the world. Nanogels are an excellent vehicle to use in drug delivery and several other applications due to their biocompatibility. They are polymer-based networks, chemically or physically crosslinked, with at least 80-90% water in their composition. Their properties can be tuned, like the nanogel size, multifunctionality and degradability. Nanogels are capable of carrying in their interior bioactive molecules and deliver them into cells. The main objective of this project was to produce nanogels for the delivery of anticancer drugs with the ability of responding to existent stimuli inside cells (cellresponsiveness nanogels) and/or of controlled drug delivery. The nanogels were mainly based on alginate (AG), a natural biopolymer, and prepared using emulsion approaches. After their synthesis, they were used to encapsulate doxorubicin (Dox) which was chosen as a model drug. In the first part of the experimental work, disulfide-linked AG nanogels were prepared and, as expected, were redox-sensitive to a reducing environment like the intracellular medium. In the second part, AG nanogels crosslinked with both calcium ions and cationic poly(amidoamine) dendrimers were developed with improved sustained drug delivery. The prepared nanogels were characterized in terms of size, chemical composition, morphology, and drug delivery behavior (under redox/pH stimuli). The in vitro cytotoxicity of the nanogels was also tested against CAL-72 cells (an osteosarcoma cell line).

Gene delivery using dendrimer/pDNA complexes immobilized in electrospun fibers using the layer-by-layer technique

Tissue engineering is an important branch of regenerative medicine that uses cells, materials (scaffolds), and suitable biochemical and physicochemical factors to improve or replace specific biological functions. In particular, the control of cell behavior (namely, of cell adhesion, proliferation and differentiation) is a key aspect for the design of successful therapeutical approaches. In this study, poly(lactic-co-glycolic acid) (PLGA) fiber mats were prepared using the electrospinning technology (the fiber diameters were in the micrometer range). Furthermore, the electrospun fiber mats thus formed were functionalized using the layer-by- layer (LbL) technique with chitosan and alginate (natural and biodegradable polyelectrolytes having opposite charges) as a mean for the immobilization of pDNA/dendrimer complexes. The polyelectrolyte multilayer deposition was confirmed by fluorescence spectroscopy using fluorescent-labeled polyelectrolytes. The electrospun fiber mats coated with chitosan and alginate were successfully loaded with complexes of pDNA and poly(amidoamine) (PAMAM) dendrimers (generation 5) and were able of releasing them in a controlled manner along time. In addition, these mats supported the adhesion and proliferation of NIH 3T3 cells and of human mesenchymal stem cells (hMSCs) in their surface. Transfection experiments using a pDNA encoding for luciferase showed the ability of the electrospun fiber mats to efficiently serve as gene delivery systems. When a pDNA encoding for bone morphogenetic protein-2 (BMP-2) was used, the osteoblastic differentiation of hMSCs cultured on the surface of the mats was promoted. Taken together, the results revealed that merging the electrospinning technique with the LbL technique, can be a suitable methodology for the creation of biological active matrices for bone tissue engineering.

Extração, caracterização estrutural e atividades farmacológicas do olginato obtido da alga marrom Lobophora variegata (Lamouroux) Oliveira Filho, 1977

The alginic acid or alginates are acidic polysaccharides found in brown seaweed widely used in food, cosmetic, medical and pharmaceutical industry. This paper proposes the extraction, chemical characterization and verification of the pharmacological activities of brown seaweed variegata Lobophora . The alginate was extracted from the seaweed Lobophora variegata and part was sulphated for comparative purposes. The native extract showed 42% total sugar, 65% uronic acid, 0,36 % protein and 0% of sulfate, while the sulfate showed 39% , 60%, 0.36% and 27,92 % respectively. The presence of a sulfate group may be observed by the metachromasia with toluidine blue in electrophoresis system and characteristic vibration 1262,34 cm-1 in infrared spectroscopy connections assigned to S = O. We observed the formation of films and beads of native alginate, where more concentrated solution 6% resulted in a thicker and more consistent film. Native alginate showed proliferative activity at concentrations (25 and 50 mcg), (50 mg) and (100 mg) in 3T3 cell line in 24h, 48h and 72h, respectively , as the sulfated (100 mg) in 24 . Also showed antiproliferative or cytotoxic activity in HeLa cells of strain, (25 and 100 mg), (25 and 100 mg) and (25, 50 and 100 mg), to native, now for the sulfate concentrations (100 mg) in 24 (25, 50 and 100 mg) in 48 hours, and (50 and 100 mg ) 72h. For their antioxidant activity, the sulfated alginates have better total antioxidant activity reaching 29 % of the native activity while 7.5 % of activity . For the hydroxyl radical AS showed high inhibition ( between 77-83 % ) in concentrations, but the AN surpassed these numbers in the order of 78-92 % inhibition. The reducing power of AN and AS ranged between 39-82 % . In the method of ferric chelation NA reached 100 % chelating while the AS remained at a plateau oscillating 6.5%. However, in this study , we found alginates with promising pharmacological activities, to use in various industries as an antioxidant / anti-tumor compound

Efeito da associação do herbicida clomazone a nanoesferas de alginato/quitosana na sorção em solos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Nanopartículas de alginato como sistema de liberação para o herbicida clomazone

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Controle de nematóides parasitos gastrintestinais por Arthrobotrys musiformis em ovinos naturalmente infestados mantidos em pastagens

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Susceptibility of adult and larval stages of the horn fly, Haematobia irritans, to the entomopathogenic fungus Metarhizium anisopliae under field conditions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeitos do tratamento com Hidrogel na cicatrização de úlceras venosas de membros inferiores: revisão sistemática

Chronic venous disease (CVD) is evident among the chronic diseases and affects the elderly population and primarily is responsible for leg ulcers in this population. The use of dressings in the care of a venous ulcer is a fundamental part of the treatment for healing, however, evidence to assist in choosing the best dressing is scarce. The main objective of this study was to evaluate the effectiveness of treatment with hydrogel in the healing of venous ulcers using search methods, synthesis of information and statistical research through a systematic review and meta-analysis. Randomized controlled trials were selected in the following databases: CENTRAL; DARE; NHS EED; MEDLINE; EMBASE; CINAHL. Beyond these databases three websites were consulted to identify ongoing studies: ClinicalTrials.gov, OMS ICTRP e ISRCTN. The primary outcomes were analyzed: complete wound healing, incidence of wound infection and the secondary were: changes in ulcer size, time to ulcer healing, recurrence of ulcer, quality of life of participants, pain and costs of treatment. Four studies are currently included in the review with a total of 250 participants. The use of hydrogel appears to be superior to conventional dressing, gauze soaked in saline, for the healing of venous leg ulcers; 16/30 patients showed complete healing of ulcers (RR 5,33, 95%CI [1,73,16,42]). The alginate gel was shown to be more effective when compared to the hydrogel dressing in reduction of the wound area; 61,2% (± 26,2%) with alginate e 19,4% (± 24,3%) with hydrogel at the end of four weeks of treatment. Manuka honey has shown to be similar to the hydrogel dressings in percentage of area reduction. This review demonstrated that there is no evidence available about the effectiveness of the hydrogel compared to other types of dressings on the healing of venous leg ulcers of the lower limbs, thus demonstrating the need of future studies to assist health professionals in choosing the correct dressing.

Matrizes poliméricas puras e modificadas para adsorção do gás sulfeto de hidrogênio

The underground natural gas found associated or not with oil is characterized by a mixture of hydrocarbons and residual components such as carbon dioxide (CO2), nitrogen gas (N2) and hydrogen sulfide (H2S), called contaminants. The H2S especially promotes itself as a contaminant of natural gas to be associated with corrosion of pipelines, to human toxicity and final applications of Natural Gas (NG). The sulfur present in the GN must be fully or partially removed in order to meet the market specifications, security, transport or further processing. There are distinct and varied methods of desulfurization of natural gas processing units used in Natural Gas (UPGN). In order to solve these problems have for example the caustic washing, absorption, the use of membranes and adsorption processes is costly and great expenditure of energy. Arises on such findings, the need for research to active processes of economic feasibility and efficiency. This work promoted the study of the adsorption of sulfide gas in polymer matrices hydrogen pure and modified. The substrates of Poly(vinyl chloride) (PVC), poly(methyl methacrylate) (PMMA) and sodium alginate (NaALG) were coated with vanadyl phosphate compounds (VOPO4.2H2O), vanadium pentoxide (V2O5), rhodamine B (C28H31N2O3Cl) and ions Co2+ and Cu2+, aiming to the adsorption of hydrogen sulfide gas (H2S). The adsorption tests were through a continuous flow of H2S in a column system (fixed bed reactor) adsorption on a laboratory scale. The techniques used to characterize the adsorbents were Infrared spectroscopy (FTIR), thermogravimetry analysis (TGA), X-ray fluorescence (XRF), the X-ray diffraction (XRD) electron microscopy (SEM). Such work indicates, the results obtained, the adsorbents modified PMMA, PVC and NaALG have a significant adsorptive capacity. The matrix that stood out and had the best adsorption capacity, was to ALG modified Co2+ with a score of 12.79 mg H2S / g matrix

Confecção e caracterização físico-química de filmes compósitos autossustentáveis NaAlg-TiO2-WO3

A new self-sustainable film was prepared through the sol-gel modified method, previously employed in our research group; sodium alginate was used as the polymer matrix, along with plasticizer glycerol, doped with titanium dioxide (TiO2) and tungsten trioxide (WO3). By varying WO3 concentration (0,8, 1,6, 2,4 and 3,2 μmol) and keeping TiO2 concentration constant (059 mmol), it was possible to study the contribution of these oxides on the obtained films morphological and electrical properties. Self-sustainable films have analyzed by Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffraction (XDR), Scanning Electron Microscope (SEM), Energy Dispersive X-ray Spectroscopy (EDS) and Electrochemical Impedance Spectroscopy (EIS). By the IR specters, it was possible identify the TiO2, and posteriorly WO3, addition has provided dislocation of alginate characteristics bands to smaller vibrations frequencies indicating an electrostatic interaction between the oxides and the polymer matrix. Diffractograms show predominance of the amorphous phase in the films. SEM, along with EDX, analysis revealed self-sustainable films showed surface with no cracks and relative dispersion of the oxides throughout the polymer matrix. From Impedance analysis, it was observe increasing WO3 concentration to 2,4 μmol provided a reduction of films resistive properties and consequent improvement of conductive properties

Obtenção e caracterização do sistema compósito alginato de sódio-dióxido de titânio em formas de pó e de membrana

The alginates are copolymers of 1→4-linked β-D-mannuronic acid (M) and α-Lguluronic acid (G) residues that are arranjed in a block structure along a linear chain. Titanium dioxide, TiO2, is a ceramic material and can exist in three distinct crystallography forms: anatase, brookite and rutile. composites of organic and inorganic materials have better properties than the components alone. Thus, this study aims to synthesize, characterize and analyze the composite NaAlg-TiO2 in the form of powder and film. The synthesis of composite powders was performed using the sol-gel process and obtain the composite film was performed using the slow evaporation process, then the composites were analyzed by infrared spectroscopy, fluorescence x ray, thermal analysis, attenuated total reflection (ATR), x ray diffraction and impedance spectroscopy. The X ray diffraction patterns of composite powders show that with increasing calcination temperature, there were no complete transition of rutile-anatase crystalline phase, since at all temperatures studied (300, 500, 700, 900 and 1100ºC) were observed peaks of anatase phase. Thermal analysis shows that at 400°C caused the decomposition of sodium alginate in sodium carbonate and above 600°C, we observe an exothermic peak related to the decomposition of sodium carbonate and in the presence of titanium dioxide becomes sodium titanate. The XRD results confirm the formation of sodium carbonate at 700ºC and the formation sodium titanate in the temperature range 900-1100ºC. The sodium titanate influenced the electrical properties of the material, because with increasing temperature there was a decrease in conductivity, probably due to the creation of Ti vacancies, since the sodium can induce the reduction of surface Ti4+ ions into Ti3+ species. The infrared spectra of the composites in the form of powder and film showed a small shift in the bands compared to the spectrum of pure alginate, indicating that these shifts, even small ones, have evidence of miscibility between the polymer and ceramic material

Cultura de condrócitos em arcabouço tridimensional: hidrogel de alginato

OBJETIVOS: O presente estudo teve como objetivo cultivar condrócitos retirados da articulação do joelho de coelhos encapsulados em hidrogel de alginato (HA) e caracterizar a produção de matriz extracelular (ECM). MÉTODOS: A cartilagem articular foi removida do joelho de coelhos, com três a seis meses, fragmentada em pedaços de 1mm e submetida à digestão enzimática. Uma concentração de 1x106 céls/mL foram ressuspensas em uma solução de alginato de sódio a 1,5% (w/v), em seguida fez-se o processo de gelatinização em CaCl2 (102 mM), permitindo a formação do HA e cultivo em meio DMEM-F12 durante quatro semanas. A distribuição das células e a ECM foram acessadas através das secções histológicas coradas com e azul de toluidina hematoxilina e eosina (HE). RESULTADOS: Houve um aumento no número e na viabilidade dos condrócitos durante as quatro semanas de cultura. Através das análises histológicas dos HAs corados com azul de toluidina e HE foi possível observar a distribuição definida dos condrócitos no hidrogel, assemelhando-se a grupos isógenos e formação de matriz territorial. CONCLUSÃO: Este estudo demonstrou a eficiência do HA como arcabouço para ser usado na cultura de condrócitos, constituindo uma alternativa no reparo de lesões na cartilagem articular.

Efficacy of Duddingtonia flagrans and Arthrobotrys robusta in controlling sheep parasitic gastroenteritis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)