In vivo analysis of staphylococcus aureus-infected mice reveals differential temporal and spatial expression patterns of fhuD2

Staphylococcus aureus is an opportunistic human pathogen and a major cause of invasive infections such as bacteremia, endocarditis, pneumonia and wound infections. FhuD2 is a staphylococcal lipoprotein involved in the uptake of iron-hydroxymate and is under the control of the iron uptake regulator Fur. The protein is part of an investigational multi-component vaccine formulation that has shown protective efficacy in several murine models of infection. Even though fhuD2 expression was shown to be upregulated in murine kidneys infected with S. aureus, it is unknown whether the bacterium undergoes increased iron deprivation during prolonged infection. Furthermore, different infection niches of S. aureus might provide different environments and iron availability resulting in different fhuD2 expression pattern within different host organs. To address these questions, we characterized the in vitro expression of the fhuD2 gene and confirmed Fur-dependent iron-regulation of its expression. We further investigated its expression in mice infected with a bioluminescent reporter strain of S. aureus expressing the luciferase operon under the control of the fhuD2 promoter. The emission of bioluminescence in different organs was followed over a seven-day time course, as well as quantitative real-time PCR analysis of the RNA transcribed from the endogenous fhuD2 gene. Using this approach, we could show that fhuD2 expression was induced during infection in all organs analyzed and that differences in expression were observed in the temporal expression profiles, and between infected organs. Our data suggest that S. aureus undergoes increased iron deprivation during progression of infection in diverse host organs and accordingly induces dedicated iron acquisition mechanisms. Since FhuD2 plays a central role in providing the pathogen with the required iron, further knowledge of the patterns of fhuD2 expression in vivo during infection is instrumental in better defining the role of this antigen in S. aureus pathogenesis and as a vaccine antigen

Temperature Dependence of Steady-State and Presteady-State Kinetics of a Type IIb Na+/Pi Cotransporter

The temperature dependence of the transport kinetics of flounder Na+-coupled inorganic phosphate (Pi) cotransporters (NaPi-IIb) expressed in Xenopus oocytes was investigated using radiotracer and electrophysiological assays. 32Pi uptake was strongly temperature-dependent and decreased by ∼80% at a temperature change from 25°C to 5°C. The corresponding activation energy (E a) was ∼14 kcalmol−1 for the cotransport mode. The temperature dependence of the cotransport and leak modes was determined from electrogenic responses to 1 mM Pi and phosphonoformic acid (PFA), respectively, under voltage clamp. The magnitude of the Pi- and PFA-induced changes in holding current decreased with temperature. E a at −100 mV for the cotransport and leak modes was ∼16 kcalmol−1 and ∼11 kcalmol−1, respectively, which suggested that the leak is mediated by a carrier, rather than a channel, mechanism. Moreover, E a for cotransport was voltage-independent, suggesting that a major conformational change in the transport cycle is electroneutral. To identify partial reactions that confer temperature dependence, we acquired presteady-state currents at different temperatures with 0 mM Pi over a range of external Na+. The relaxation time constants increased, and the peak time constant shifted toward more positive potentials with decreasing temperature. Likewise, there was a depolarizing shift of the charge distribution, whereas the total available charge and apparent valency predicted from single Boltzmann fits were temperature-independent. These effects were explained by an increased temperature sensitivity of the Na+-debinding rate compared with the other voltage-dependent rate constant

Functionally Important Residues in the Predicted 3rd Transmembrane Domain of the Type IIa Sodium-phosphate Cotransporter (NaPi-IIa)

The type IIa Na+/Pi, cotransporter (NaPi-IIa) mediates electrogenic transport of three Na+ and one divalent Pi ion (and one net positive charge) across the cell membrane. Sequence comparison of electrogenic NaPi-IIa and IIb isoforms with the electroneutral NaPi-IIc isoform pointed to the third transmembrane domain (TMD-3) as a possibly significant determinant of substrate binding. To elucidate the role of TMD-3 in the topology and mechanism underlying NaPi-IIa function we subjected it to cysteine scanning mutagenesis. The constructs were expressed in Xenopus oocytes and Pi transport kinetics were assayed by electrophysiology and radiotracer uptake. Cys substitution resulted in only marginally altered kinetics of Pi transport in those mutants providing sufficient current for analysis. Only one site, at the extracellular end of TMD-3, appeared to be accessible to methanethiosulfonate reagents. However, additional mutations carried out at D224 (replaced by E, G or N) and N227 (replaced by D or Q) resulted in markedly altered voltage and substrate dependencies of the Pi-dependent currents. Replacing Asp-224 (highly conserved in electrogenic a and b isoforms) with Gly (the residue found in the electroneutral c isoform) resulted in a mutant that mediated electroneutral Na+-dependent Pi transport. Since electrogenic NaPi-II transports 3 Na+/transport cycle, whereas electroneutral NaPi-IIc only transports 2, we speculate that this loss of electrogenicity might result from the loss of one of the three Na+ binding sites in NaPi-II

Vitamina A em dietas para tilápia do Nilo

Dietary vitamin supplementation decrease stress caused by high stocking density, and boosts immunological system of farmed fish. A studied was carried out to determine vitamin A requirements of Nile tilapia (Oreochromis niloticus) in an all male group (13.8 ± 1.2 g) and a mixed sex population (9.8 ± 2.3 g). Fish stocked in 100-L plastic aquaria (26.0 ± 1.0ºC) were fed to near satiety, twice a day, seven days a week, during 75 days with vitamin A-free, semi-purified diets supplemented with 0; 600; 1,200; 1,800; 2,400; 3,000; 3,600; 4,200; 4,800 and 5,400 International Units (IU) of retinyl palmitate (30% vitamin A) per kg of diet in a completely randomized experimental design, factorial arrangement 2c10 (n = 4). Deficiency signs of vitamin A were observed in fish fed 0 to 1.200 IU vitamin A kg-1 diet; moderate signs were observed in fish fed diets with 1.800 to 3.600 IU vitamin A kg-1 diet; no interactions group*level (p < 0.05) were detected. Dietary levels of vitamin A up to 5.400 IU kg-1 influenced final weight and weight gain of fish (p < 0.05), but did not influence feed consumption (p > 0.05). A group effect was observed regarding all performance variables (p < 0.0001). Quantification of hepatic retinol (HPLC) detected vitamin A only in fish fed 5.400 IU retinol kg-1 of diet, therefore characterizing that dietary retinol was used and stored. The quantity of 5.400 IU of retinol kg-1 of diet is recommended for adequate nutrition of Nile tilapia.A suplementação de vitaminas na dieta diminui o estresse e estimula o sistema imunológico causado por altas densidades de estocagem dos peixes. Este trabalho determinou a exigência em vitamina A para a tilápia do Nilo em uma população monosexo masculina (13.8 ± 1.2 g) e em uma população original (9.8 ± 2.3 g). Os peixes foram estocados em aquários plásticos de 100 L (26.0 ± 1.0ºC) e alimentados "ad libitum", duas vezes ao dia, sete dias da semana, durante 75 dias com dieta semi-purificada suplementada com 0; 600; 1.200; 1.800; 2.400; 3.000; 3.600; 4.200; 4.800 e 5.400 UI de retinyl palmitato (30% de vitamina A) por kg de dieta, em um delineamento experimental totalmente ao acaso e arranjo fatorial 2c10 (n = 4). Deficiência nutricional severa foi observada em peixes alimentados com 0 a 1.200 UI vitamina A kg-1 de dieta; sinais moderados foram encontrados em peixes alimentados com 1.800 a 3.600 UI vitamina A kg-1 de dieta; interações grupo*nível (p < 0,05) não foram detectadas. O aumento de nível de vitamina A influenciou no peso final e no ganho de peso dos peixes (p < 0,05), mas não influenciou o consumo de ração (p > 0,05). Foi observado efeito de grupo no desempenho dos peixes (p < 0,0001). Foi detectado o retinol hepático através de HPLC somente no grupo alimentado com 5.400 UI de retinol kg-1 de dieta, caracterizando assim que o mesmo foi utilizado e armazenado. A quantidade de 5.400 IU de retinol kg-1 de dieta é a mínima recomendada para tilápia do Nilo.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

NeuraGED: A GNN estimation for Graph–Edit Distance

Graph generative models often lack a proper reconstruction loss to evaluate the distance between the generated graph and the target graph. This is particularly important for molecular graph generators based on autoencoders, which should reconstruct the input graphs as precisely as possible. Though, the distance estimation can be useful for any graph generator based on reconstruction, including sequential methods relying on Graph Neural Networks. Since graphs are discrete entities by nature, general graph spaces lack a reliable, general, and computationally affordable distance function. Graph Edit Distance is of course an exact, general, and permutation–invariant method for evaluating the difference between two graphs defined in the same graph space. Since it needs all the possible combinations of pairs of nodes from the two graphs, its exact computation is a NP-complete problem and cannot be carried out for graphs larger than ten nodes. As a consequence, a comprehensive soft–estimation method for Graph Edit Distance based on siamese Graph Neural Networks is proposed. A theoretical discussion is carried out, showing that the proposed method can provide a reliable and precise soft–estimation of the Graph Edit Distance on molecular graphs. Molecular graph generators can therefore use this distance estimation as a powerful non–permutation–invariant reconstruction loss. Moreover, the experimental results show that the distance estimation is accurate, with a very low Mean Squared Error loss value

Structure–Function Relations of the First and Fourth Extracellular Linkers of the Type IIa Na+/Pi Cotransporter: II. Substrate Interaction and Voltage Dependency of Two Functionally Important Sites

Functionally important sites in the predicted first and fourth extracellular linkers of the type IIa Na+/Pi cotransporter (NaPi-IIa) were identified by cysteine scanning mutagenesis (Ehnes et al., 2004). Cysteine substitution or modification with impermeant and permeant methanethiosulfonate (MTS) reagents at certain sites resulted in changes to the steady-state voltage dependency of the cotransport mode (1 mM Pi, 100 mM Na+ at pH 7.4) of the mutants. At Gly-134 (ECL-1) and Met-533 (ECL-4), complementary behavior of the voltage dependency was documented with respect to the effect of cys-substitution and modification. G134C had a weak voltage dependency that became even stronger than that of the wild type (WT) after MTS incubation. M533C showed a WT-like voltage dependency that became markedly weaker after MTS incubation. To elucidate the underlying mechanism, the steady-state and presteady-state kinetics of these mutants were studied in detail. The apparent affinity constants for Pi and Na+ did not show large changes after MTS exposure. However, the dependency on external protons was changed in a complementary manner for each mutant. This suggested that cys substitution at Gly-134 or modification of Cys-533 had induced similar conformational changes to alter the proton modulation of transport kinetics. The changes in steady-state voltage dependency correlated with changes in the kinetics of presteady-state charge movements determined in the absence of Pi, which suggested that voltage-dependent transitions in the transport cycle were altered. The steady-state and presteady-state behavior was simulated using an eight-state kinetic model in which the transition rate constants of the empty carrier and translocation of the fully loaded carrier were found to be critical determinants of the transport kinetics. The simulations predict that cys substitution at Gly-134 or cys modification of Cys-533 alters the preferred orientation of the empty carrier from an inward to outward-facing conformation for hyperpolarizing voltages

Globally Convergent Adaptive Tracking of Angular Velocity and Inertia Identification for a 3-DOF Rigid Body

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/57820/1/AdaptiveTrackingTCST2006.pd

Structure–Function Relations of the First and Fourth Predicted Extracellular Linkers of the Type IIa Na+/Pi Cotransporter: I. Cysteine Scanning Mutagenesis

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693–705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V ≤ −80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport

Vitamin A in diets for Nile tilapia

A suplementação de vitaminas na dieta diminui o estresse e estimula o sistema imunológico causado por altas densidades de estocagem dos peixes. Este trabalho determinou a exigência em vitamina A para a tilápia do Nilo em uma população monosexo masculina (13.8 ± 1.2 g) e em uma população original (9.8 ± 2.3 g). Os peixes foram estocados em aquários plásticos de 100 L (26.0 ± 1.0ºC) e alimentados "ad libitum", duas vezes ao dia, sete dias da semana, durante 75 dias com dieta semi-purificada suplementada com 0; 600; 1.200; 1.800; 2.400; 3.000; 3.600; 4.200; 4.800 e 5.400 UI de retinyl palmitato (30% de vitamina A) por kg de dieta, em um delineamento experimental totalmente ao acaso e arranjo fatorial 2c10 (n = 4). Deficiência nutricional severa foi observada em peixes alimentados com 0 a 1.200 UI vitamina A kg-1 de dieta; sinais moderados foram encontrados em peixes alimentados com 1.800 a 3.600 UI vitamina A kg-1 de dieta; interações grupo*nível (p ; 0,05). Foi observado efeito de grupo no desempenho dos peixes (p ; 0.05). A group effect was observed regarding all performance variables (p < 0.0001). Quantification of hepatic retinol (HPLC) detected vitamin A only in fish fed 5.400 IU retinol kg-1 of diet, therefore characterizing that dietary retinol was used and stored. The quantity of 5.400 IU of retinol kg-1 of diet is recommended for adequate nutrition of Nile tilapia

Vitamina A em dietas para tilápia do Nilo

Dietary vitamin supplementation decrease stress caused by high stocking density, and boosts immunological system of farmed fish. A studied was carried out to determine vitamin A requirements of Nile tilapia (Oreochromis niloticus) in an all male group (13.8 ± 1.2 g) and a mixed sex population (9.8 ± 2.3 g). Fish stocked in 100-L plastic aquaria (26.0 ± 1.0ºC) were fed to near satiety, twice a day, seven days a week, during 75 days with vitamin A-free, semi-purified diets supplemented with 0; 600; 1,200; 1,800; 2,400; 3,000; 3,600; 4,200; 4,800 and 5,400 International Units (IU) of retinyl palmitate (30% vitamin A) per kg of diet in a completely randomized experimental design, factorial arrangement 2c10 (n = 4). Deficiency signs of vitamin A were observed in fish fed 0 to 1.200 IU vitamin A kg-1 diet; moderate signs were observed in fish fed diets with 1.800 to 3.600 IU vitamin A kg-1 diet; no interactions group*level (p 0.05). A group effect was observed regarding all performance variables (p 0,05). Foi observado efeito de grupo no desempenho dos peixes (p < 0,0001). Foi detectado o retinol hepático através de HPLC somente no grupo alimentado com 5.400 UI de retinol kg-1 de dieta, caracterizando assim que o mesmo foi utilizado e armazenado. A quantidade de 5.400 IU de retinol kg-1 de dieta é a mínima recomendada para tilápia do Nil