Single-cell characterization of autotransporter mediated Escherichia coli surface display of disulfide-bond containing proteins

Autotransporters (ATs) are a family of bacterial proteins containing a C-terminal ?-barrel-forming domain that facilitates the translocation of N-terminal passenger domain whose functions range from adhesion to proteolysis. Genetic replacement of the native passenger domain with heterologous proteins is an attractive strategy not only for applications such as biocatalysis, live-cell vaccines, and protein engineering but also for gaining mechanistic insights toward understanding AT translocation. The ability of ATs to efficiently display functional recombinant proteins containing multiple disulfides has remained largely controversial. By employing high-throughput single-cell flow cytometry, we have systematically investigated the ability of the Escherichia coli AT Antigen 43 (Ag43) to display two different recombinant reporter proteins, a single-chain antibody (M18 scFv) that contains two disulfides and chymotrypsin that contains four disulfides, by varying the signal peptide and deleting the different domains of the native protein. Our results indicate that only the C-terminal ?-barrel and the threaded ?-helix are essential for efficient surface display of functional recombinant proteins containing multiple disulfides. These results imply that there are no inherent constraints for functional translocation and display of disulfide bond-containing proteins mediated by the AT system and should open new avenues for protein display and engineering

Sequential release of TNFα and phospholipase A2 in a rat model of LPS-induced pleurisy

The levels of extracellular phospholipase A2 (sPLA2) and TNFα, and cell accumulation were measured in the pleural washings obtained at different times following the induction of Escherichia coli lipopolysaccharide (LPS, 100 μg/cavity) pleurisy in rats. TNFα peaked at 2 hours (3036 ± 160.3 units/ml) and decreased thereafter. Conversely, levels of sPLA2 peaked at 48 hours (1.97 ± 0.64 ng/ml) and were increased further (14.02 ± 4.16 ng/ml) by pretreatment with anti-TNFα antibody. Cell accumulation was not affected by antibody pretreatment. These data indicate that the sPLA2 enzyme is involved in LPS-induced pleurisy. The enzyme seems not to be stimulated by TNFα which may be involved in the downregulation of sPLA2 in this model of inflammation

Peripheral relaxant activity of apomorphine and of a D1 selective receptor agonist on human corpus cavernosum strips

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Hydrogen sulfide-induced dual vascular effect involves arachidonic acid cascade in rat mesenteric arterial bed.

Hydrogen sulfide (H(2)S), a novel gaseous transmitter, is considered a physiological regulator of vascular homeostasis. Recent evidence suggests H(2)S as an endothelium-hyperpolarizing factor (EDHF) candidate. To address this issue, we evaluated the vascular effect of sodium hydrogen sulfide (NaHS), an H(2)S donor, on the rat mesenteric arterial bed. NaHS concentration-response curve was performed on preconstricted mesenteric arterial bed. To assess the contribution of EDHF, we performed a pharmacologic dissection using indomethacin, N(G)-nitro-L-arginine methyl ester (L-NAME), or apamin and charybdotoxin as cyclooxygenase, nitric-oxide synthase, and calcium-dependent potassium channel inhibitors, respectively. In another set of experiments, we used 4-(4-octadecylphenyl)-4-oxobutenoic acid, baicalein, or proadifen as phospholipase A(2) (PLA(2)), lipoxygenase, and cytochrome P450 inhibitors, respectively. Finally, an immunofluorescence study was performed to support the involvement of PLA(2) in mesenteric artery challenged by NaHS. NaHS promoted a dual vascular effect (i.e., vasoconstriction and vasodilation). L-NAME or baicalein administration affected neither NaHS-mediated vasodilation nor vasoconstriction, whereas apamin and charybdotoxin significantly inhibited NaHS-induced relaxation. Pretreatment with PLA(2) inhibitor abolished both the contracting and the relaxant effect, whereas P450 cytochrome blocker significantly reduced NaHS-mediated relaxation. The immunofluorescence study showed that NaHS caused a migration of cytosolic PLA(2) close to the nucleus, which implicates activation of this enzyme. Our data indicate that H(2)S could activate PLA(2), which in turn releases arachidonic acid leading, initially, to vasoconstriction followed by vasodilation mediated by cytochrome P450-derived metabolites. Because EDHF has been presumed to be a cytochrome P450 derivative of the arachidonic acid, our results suggest that H(2)S acts through EHDF release

The Timing and Strength of Regional Brain Activation Associated with Word Recognition in Children with Reading Difficulties

The study investigates the relative degree and timing of cortical activation across parietal, temporal, and frontal regions during performance of a continuous visual-word recognition task in children who experience reading difficulties (N = 44, RD) and typical readers (N = 40, NI). Minimum norm estimates of regional neurophysiological activity were obtained from magnetoencephalographic recordings. Children with RD showed bilaterally reduced neurophysiological activity in the superior and middle temporal gyri, and increased activity in rostral middle frontal and ventral occipitotemporal cortices, bilaterally. The temporal profile of activity in the RD group, featured near-simultaneous activity peaks in temporal, inferior parietal, and prefrontal regions, in contrast to a clear temporal progression of activity among these areas in the NI group. These results replicate and extend previous MEG and fMRI results demonstrating atypical, latency-dependent attributes of the brain circuit involved in word reading in children with reading difficulties

Comparative study between wet and dry etching of silicon for microchannels fabrication

FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOIn this work we present a comparative study of two processes for the fabrication of an array of microchannels for microfluidics applications, based on integrated-circuit technology process steps, such as lithography and dry etching. Two different methods were investigated in order to study the resulting microstructures: wet and dry deep etching of silicon substrate. The typical etching depth necessary to the target application is 50 mu m.1093015FAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULOFAPESP - FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO2016/09509-112. Conference on Advanced Fabrication Technologies for Micro/Nano Optics and Photonics3 a 5 de Fevereiro de 2019San Francisco, CA, Estados UnidosSPIE; Nanoscribe Gmb

Essential requirement for sphingosine kinase activity in eNOS-dependent NO release and vasorelaxation

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts both as an extracellular ligand for endothelial differentiation gene receptor family and as an intracellular second messenger. Cellular levels of S1P are low and tightly regulated by sphingosine kinase (SPK). Recent studies have suggested that eNOS pathway may function as a downstream target for the biological effects of receptor-mediated S1P. Here we have studied the possible interplay between intracellular SIP generation and the eNOS activation pathway. S1P causes an endothelium-dependent vasorelaxation in rat aorta that is PTX sensitive, inhibited by L-NAME that involves eNOS phosphorylation, and mainly dependent on hsp90. When rat aorta rings were incubated with the SPK inhibitor DL-threo-dihydrosphingosine (DTD), there was a concentration-dependent reduction of Ach-induced vasorelaxation, implying a consistent contribution of sphingolipid pathway through intracellular sphingosine release and phosphorylation. Co-immunoprecipitation experiments consistently showed increased association of hsp90 with eNOS after exposure of cells to S1P as well to BK or calcium ionophore A-23187. Interestingly, as opposite to A-23187, BK and S1P effect were significantly inhibited by pretreatment with the SPK inhibitor DTD. In conclusion, our data demonstrate that an interplay exists among eNOS, hsp90, and intracellularly generated S1P where eNOS coupling to hsp90 is a major determinant for NO release as confirmed by our functional and molecular studies

Comportamento Sazonal do Fluxo de Co2 em Ecossistema de Manguezal na Amazônia

Seasonal behavior of CO2 flux in mangrove ecosystems in the Amazo