Aerosol delivery of star polymer-siRNA nanoparticles as a therapeutic strategy to inhibit lung tumor growth.

Lung cancer is a major contributor to cancer-related death worldwide. siRNA nanomedicines are powerful tools for cancer therapeutics. However, there are challenges to overcome to increase siRNA delivery to solid tumors, including penetration of nanoparticles into a complex microenvironment following systemic delivery while avoiding rapid clearance by the reticuloendothelial system, and limited siRNA release from endosomes once inside the cell. Here we characterized cell uptake, intracellular trafficking, and gene silencing activity of miktoarm star polymer (PDMAEMA-POEGMA) nanoparticles (star nanoparticles) complexed to siRNA in lung cancer cells. We investigated the potential of nebulized star-siRNA nanoparticles to accumulate into orthotopic mouse lung tumors to inhibit expression of two genes [ßIII-tubulin, Polo-Like Kinase 1 (PLK1)] which: 1) are upregulated in lung cancer cells; 2) promote tumor growth; and 3) are difficult to inhibit using chemical drugs. Star-siRNA nanoparticles internalized into lung cancer cells and escaped the endo-lysosomal pathway to inhibit target gene expression in lung cancer cells in vitro. Nebulized star-siRNA nanoparticles accumulated into lungs and silenced the expression of ßIII-tubulin and PLK1 in mouse lung tumors, delaying aggressive tumor growth. These results demonstrate a proof-of-concept for aerosol delivery of star-siRNA nanoparticles as a novel therapeutic strategy to inhibit lung tumor growth.

Influência do período de armazenamento na qualidade do ovo integral pasteurizado refrigerado / Influence of storage period on refrigerated pasteurized whole egg quality

Avaliaram-se o pH e a qualidade microbiológica de ovos integrais pasteurizados refrigerados obtidos de dois tipos de matéria-prima: o ovo in natura (comercial ) e o ovo galado (ovo fértil). Os tratamentos foram dispostos no delineamento inteiramente ao acaso, em parcelas subdivididas, sendo na parcela dois tipos de ovos integrais pasteurizados, o comercial e o galado, e na subparcela quatro períodos de estocagem sob temperatura de refrigeração, um, sete, 14 e 21 dias. Não foi observada a presença de Salmonella spp. em nenhuma amostra analisada, e para os ovos comerciais, o período de estocagem não contribuiu para o aumento (P>0,05) da contaminação por mesófilos aeróbios, coliformes a 35ºC, Staphylococcus spp. e bolores e leveduras. Para as amostras de ovos galados, o período de armazenamento influenciou no aumento (P<0,05) da contagem de mesófilos aeróbios, coliformes a 35ºC, bolores e leveduras, e Staphylococcus spp. Os valores de pH aumentaram durante os primeiros dias do armazenamento e depois voltaram a diminuir. Concluiu-se que os ovos integrais galados pasteurizados apresentam pior qualidade em relação aos ovos integrais comerciais pasteurizados, e que o período de validade sob refrigeração desses tipos de ovos poderia ser de sete e 14 dias, respectivamente.

The pH and microbiological quality of refrigerated pasteurized whole eggs at 4ºC obtained from two types of raw materials, in nature (commercial) egg and the fertile egg were evaluated. The treatments were arranged in a completely randomized split plot design, the two types of pasteurized whole eggs (commercial and fertile) were alloted to the plots and four periods of storage under refrigeration (one, seven, 14 and 21 days) to the split plot. The presence of Salmonella spp. was not observed in the samples and the commercial pasteurized whole egg storage period did not contribute to the increase (P>0.05) in mesophilic aerobic coliforms at 35ºC, Staphylococcus spp and mold and yeast contamination. For samples of fertile pasteurized whole eggs, the storage period influenced the increased (P<0.05) count of mesophilic aerobic, coliforms at 35ºC, mold and yeast and Staphylococcus spp. The pH values rose during the first days of storage and then decreased again. It was concluded that fertile pasteurized whole eggs have lower quality than the commercial pasteurized whole eggs, and that the shelf life of these could be seven and 14 days, respectively.

Bipartite gating in the outer membrane protein FecA.

The recent structure determination of FecA, with and without ligand, shows the existence of two gates. These are the extracellular loops closing over the binding site and the plug located inside the barrel. It indicates a process which is described as bipartite gating and allows for a rational distinction between the binding event and the transport process.

Crystal structure of the outer membrane active transporter FepA from Escherichia coli.

Integral outer membrane receptors for iron chelates and vitamin B12 carry out specific ligand transport against a concentration gradient. Energy for active transport is obtained from the proton-motive force of the inner membrane through physical interaction with TonB-ExbB-ExbD, an inner membrane complex. Here we report the crystal structure of an active transport, outer membrane receptor at 2.4 A resolution. Two distinct functional domains are revealed: (i) a 22-stranded beta-barrel that spans the outer membrane and contains large extracellular loops which appear to function in ligand binding; and (ii) a globular N-terminal domain that folds into the barrel pore, inhibiting access to the periplasm and contributing two additional loops for potential ligand binding. These loops could provide a signaling pathway between the processes of ligand recognition and TonB-mediated transport. The blockage of the pore suggests that the N-terminal domain must undergo a conformational rearrangement to allow ligand transport into the periplasm.

Synapsin I is structurally similar to ATP-utilizing enzymes.

Synapsins are abundant synaptic vesicle proteins with an essential regulatory function in the nerve terminal. We determined the crystal structure of a fragment (synC) consisting of residues 110-420 of bovine synapsin I; synC coincides with the large middle domain (C-domain), the most conserved domain of synapsins. SynC molecules are folded into compact domains and form closely associated dimers. SynC monomers are strikingly similar in structure to a family of ATP-utilizing enzymes, which includes glutathione synthetase and D-alanine:D-alanine ligase. SynC binds ATP in a Ca2+-dependent manner. The crystal structure of synC in complex with ATPgammaS and Ca2+ explains the preference of synC for Ca2+ over Mg2+. Our results suggest that synapsins may also be ATP-utilizing enzymes.

Identification, expression, and crystallization of the protease-resistant conserved domain of synapsin I.

A 35-37-kDa protease-resistant domain of synapsin Ia/ Ib, apparently produced by low levels of endogenous proteases in vapor diffusion droplets, slowly formed crystals diffracting X-rays to approximately 10 A resolution. The fragment mainly consisted of the highly conserved C domain common to the synapsin I/II family plus short N- and C-terminal flanking segments. Two constructs (SynA and SynB) of synthetic gene fragments coding for the C domain of synapsin with or without C-terminal flanking sequence were expressed in Escherichia coli as fusion proteins attached to the soluble protein glutathione-S-transferase. The fusion proteins were purified by affinity chromatography. Subsequent in situ cleavage with TEV protease resulted in the release of highly pure synapsin fragments, which were further purified by ion exchange chromatography. SynA and SynB formed crystals within three days, which diffracted to better than 3 A using a conventional X-ray source and to about 2 A using a synchrotron X-ray source. SynA crystals have the symmetry of the trigonal space groups P3(1)21 or P3(2)21 and the unit cell dimensions a = b = 77.4 A, c = 188.5 A, alpha = beta = 90 degrees, gamma = 120 degrees. SynB crystals have the symmetry of the orthorhombic space group C222(1) with the unit cell dimension a = 104.6 A, b = 113.3 A, and c = 273.8 A.