Time Series Transcriptomic Analysis of Bronchoalveolar Lavage Cells from Piglets Infected with Virulent or Low-Virulent Porcine Reproductive and Respiratory Syndrome Virus 1.

Porcine reproductive and respiratory syndrome virus (PRRSV) has evolved to escape the immune surveillance for a survival advantage leading to a strong modulation of host's immune responses and favoring secondary bacterial infections. However, limited data are available on how the immunological and transcriptional responses elicited by virulent and low-virulent PRRSV-1 strains are comparable and how they are conserved during the infection. To explore the kinetic transcriptional signature associated with the modulation of host immune response at lung level, a time-series transcriptomic analysis was performed in bronchoalveolar lavage cells upon experimental in vivo infection with two PRRSV-1 strains of different virulence, virulent subtype 3 Lena strain or the low-virulent subtype 1 3249 strain. The time-series analysis revealed overlapping patterns of dysregulated genes enriched in T-cell signaling pathways among both virulent and low-virulent strains, highlighting an upregulation of co-stimulatory and co-inhibitory immune checkpoints that were disclosed as Hub genes. On the other hand, virulent Lena infection induced an early and more marked "negative regulation of immune system process" with an overexpression of co-inhibitory receptors genes related to T-cell and NK cell functions, in association with more severe lung lesion, lung viral load, and BAL cell kinetics. These results underline a complex network of molecular mechanisms governing PRRSV-1 immunopathogenesis at lung level, revealing a pivotal role of co-inhibitory and co-stimulatory immune checkpoints in the pulmonary disease, which may have an impact on T-cell activation and related pathways. These immune checkpoints, together with the regulation of cytokine-signaling pathways, modulated in a virulence-dependent fashion, orchestrate an interplay among pro- and anti-inflammatory responses. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the major threats to swine health and global production, causing substantial economic losses. We explore the mechanisms involved in the modulation of host immune response at lung level performing a time-series transcriptomic analysis upon experimental infection with two PRRSV-1 strains of different virulence. A complex network of molecular mechanisms was revealed to control the immunopathogenesis of PRRSV-1 infection, highlighting an interplay among pro- and anti-inflammatory responses as a potential mechanism to restrict inflammation-induced lung injury. Moreover, a pivotal role of co-inhibitory and co-stimulatory immune checkpoints was evidenced, which may lead to progressive dysfunction of T cells, impairing viral clearance and leading to persistent infection, favoring as well secondary bacterial infections or viral rebound. However, further studies should be conducted to evaluate the functional role of immune checkpoints in advanced stages of PRRSV infection and explore a possible T-cell exhaustion state.

Comprehensive Kinetics of Hydrolysis of Organotriethoxysilanes by 29Si NMR.

The kinetics of several representative hybrid precursors were studied via 29Si NMR: three alkyl precursors, methyltriethoxysilane, ethyltriethoxysilane, and propyltriethoxysilane; as well as two unsaturated radicals, vinyltriethoxysilane and phenyltriethoxysilane. The reaction rate is related to the chemical shift of 29Si in the NMR spectra, which gives information about the electronic density of the Si atoms and the inductive effects of substituents. The concentration of the precursors decreased exponentially with time, and the intermediate products of hydrolysis and the beginning of the condensation reactions showed curves characteristic of sequential reactions, with a similar distribution of the species as a function of the fractional conversion. For all of the precursors, condensation started when the most hydrolyzed species reached a maximum concentration of 0.30 M, when the precursor had run out. A prediction following the developed mathematical model fits the experimental results in line with a common pathway described by eight parameters.

Comprehensive analysis of pig feces metabolome by chromatographic techniques coupled to mass spectrometry in high resolution mode: Influence of sample preparation on the identification coverage.

Pig feces is an interesting biological sample to be implemented in metabolomics experiments by virtue of the information that can be deduced from the interaction between host and microbiome. However, pig fecal samples have received scant attention, especially in untargeted metabolomic studies. In this research, an analytical strategy was planned to maximize the identification coverage of metabolites found in pig fecal samples. For this purpose, two complementary platforms such as LC-QTOF MS/MS and GC-TOF/MS were used. Concerning sample preparation six extractant solvents with different polarity grade were tested to evaluate the extraction performance and, in the particular case of GC-MS, two derivatization protocols were compared. A total number of 303 compounds by combination of all the extractants and analytical platforms were tentatively identified. The main identified families were amino acids, fatty acids and derivatives, carbohydrates and carboxylic acids. For GC-TOF/MS analysis, the recommended extractant is methanol, while methoxymation was required in the derivatization protocol since this step allows detecting the α-keto acids, which are direct markers of the microbiome status. Concerning LC-QTOF MS/MS analysis, a dual extraction approach with methanol (MeOH) or MeOH/water and ethyl acetate is proposed to enhance the detection of polar and non-polar metabolites.

Hsp90 activity is necessary to acquire a proper neuronal polarization.

Chaperones are critical for the folding and regulation of a wide array of cellular proteins. Heat Shock Proteins (Hsps) are the most representative group of chaperones. Hsp90 represents up to 1-2% of soluble protein. Although the Hsp90 role is being studied in neurodegenerative diseases, its role in neuronal differentiation remains mostly unknown. Since neuronal polarity mechanisms depend on local stability and degradation, we asked whether Hsp90 could be a regulator of axonal polarity and growth. Thus, we studied the role of Hsp90 activity in a well established model of cultured hippocampal neurons using an Hsp90 specific inhibitor, 17-AAG. Our present data shows that Hsp90 inhibition at different developmental stages disturbs neuronal polarity formation or axonal elongation. Hsp90 inhibition during the first 3h in culture promotes multiple axon morphology, while this inhibition after 3h slows down axonal elongation. Hsp90 inhibition was accompanied by decreased Akt and GSK3 expression, as well as, a reduced Akt activity. In parallel, we detected an alteration of kinesin-1 subcellular distribution. Moreover, these effects were seconded by changes in Hsp70/Hsc70 subcellular localization that seem to compensate the lack of Hsp90 activity. In conclusion, our data strongly suggests that Hsp90 activity is necessary to control the expression, activity or location of specific kinases and motor proteins during the axon specification and axon elongation processes. Even more, our data demonstrate the existence of a "time-window" for axon specification in this model of cultured neurons after which the inhibition of Hsp90 only affects axonal elongation mechanisms.

Dysfunction of the PI3K-Akt-GSK-3 pathway is a common feature in cell culture and in vivo models of prion disease.

AIMS: Transmissible spongiform encephalopathies, also called prion diseases, are characterized by the cerebral accumulation of misfolded prion protein (PrP(SC) ) and subsequent neurodegeneration. However, despite considerable research effort, the molecular mechanisms underlying prion-induced neurodegeneration are poorly understood. Here, we explore the hypothesis that prions induce dysfunction of the PI3K/Akt/GSK-3 signalling pathway. METHODS: We employed two parallel approaches. Using cell cultures derived from mouse primary neurones and from a human neuronal cell line, we identified common elements that were modified by the neurotoxic fragment of PrP(106-126) . These studies were then complemented by comparative analyses in a mouse model of prion infection. RESULTS: The presence of a polymerized fragment of the prion protein (PrP(106-126) ) or of a prion strain altered PI3K-mediated signalling, as evidenced by Akt inhibition and GSK-3 activation. PI3K activation by the addition of insulin or the expression of a constitutively active Akt mutant restored normal levels of Akt and GSK-3 activity. These changes were correlated with a reduction in caspase activity and an increase in neuronal survival. Moreover, we found that activation of caspase 3, Erk and GSK-3 are common features of PrP(106-126) -mediated neurotoxicity in cellular systems and prion infection in the mouse cerebellum, while activation of caspase 12 and JNK was observed in cellular models. CONCLUSIONS: Our findings in cell culture and in vivo models of prion disease demonstrate marked alterations to the PI3K/Akt/GSK-3 pathway and suggest that two additional pathways contribute to PrP-induced neurotoxicity as responsible of JNK and caspase 12 activation.

Proteomic analysis of intestinal mucosa responses to Salmonella enterica serovar typhimurium in naturally infected pig.

Salmonella enterica serovar typhimurium (S. typhimurium) is one of the most frequent Salmonella serotypes isolated from European pigs. Despite the advances in understanding the mechanisms involved in host-pathogen interactions and host cell responses to S. typhimurium, the global change that occurs in naturally exposed populations has been poorly characterized. Here, we present a proteomics study on intestinal mucosa of pigs naturally infected with S. typhimurium, in order to better understand the pathogenesis of salmonellosis and the pathways which might be affected after infection. Samples were analyzed by 2D-DIGE and 44 different proteins exhibited statistically significant differences. The data set was analyzed by employing the Ingenuity Pathway Analysis and the physiological function most significantly perturbed were immunological and infectious disease, cellular assembly and organization and metabolism. The pathways implicated in the porcine immune response to S. typhimurium were gluconeogenesis and Rho GDI/RhoA signaling, and our results suggest that keratins and the intermediate filaments could play an important role in the damage of the mucosa and in the success of infection. The role of these findings in salmonellosis has been discussed, as well as the importance of analyzing naturally infected animals to have a complete picture of the infection. Also, we compared the results found in this work with those obtained in a similar study using experimentally infected animals.

Oestradiol signalling through the Akt-mTORC1-S6K1.

The oestradiol plays an important role in normal brain development and exerts neuroprotective actions. Oestradiol is mainly produced in the ovary and in addition is locally synthesised in the brain. Most of the oestradiol functions have been associated with its capacity to directly bind and dimerize "classical oestrogen receptors" (ERs), alpha and beta. The ERs' actions have been classified as "genomic" and "non-genomic" depending on whether protein synthesis occurs through ER driven transcription or not. Indeed, recent evidence suggests that oestrogen may also act as a more general "trophic factor". Hence, we have studied the capacity of oestradiol to activate the PI3K/Akt pathway and its implication in axonal growth and neuronal morphogenesis. Our data show that when oestrogen receptors are blocked the axonal and dendritic lengths are reduced in mouse primary neurons. We found that Akt/Rheb/mTORC1 responds to ER activation in neurons and that some elements of this pathway are able to restore a normal neuronal morphology even in the presence of oestrogen receptor antagonist. All these data demonstrate a new mechanism regulated by oestradiol, at least in neuronal morphogenesis.

WIP is a negative regulator of neuronal maturation and synaptic activity.

Wiskott-Aldrich syndrome protein (WASP) -interacting protein (WIP) is an actin-binding protein involved in the regulation of actin polymerization in cells, such as fibroblasts and lymphocytes. Despite its recognized function in non-neuronal cells, the role of WIP in the central nervous system has not been examined previously. We used WIP-deficient mice to examine WIP function both in vivo and in vitro. We report here that WIP(-)(/-) hippocampal neurons exhibit enlargement of somas as well as overgrowth of neuritic and dendritic branches that are more evident in early developmental stages. Dendritic arborization and synaptogenesis, which includes generation of postsynaptic dendritic spines, are actin-dependent processes that occur in parallel at later stages. WIP deficiency also increases the amplitude and frequency of miniature excitatory postsynaptic currents, suggesting that WIP(-)(/-) neurons have more mature synapses than wild-type neurons. These findings reveal WIP as a previously unreported regulator of neuronal maturation and synaptic activity.

Innate immune activation of swine intestinal epithelial cell lines (IPEC-J2 and IPI-2I) in response to LPS from Salmonella typhimurium.

The innate immune system has the basic function of identifying and eradicating microbial invaders and alerting the adaptative immune system to their presence. In this study, the porcine intestinal innate immune response was evaluated by analysing the expression of TLRs, cytokines and chemokines in two porcine epithelial cell lines from different regions: IPEC-J2 (jejunum) and IPI-2I (ileum). Both cells lines were stimulated with 1microg of LPS from Salmonella typhimurium. RNA was collected at 30min, 1, 2, 3 and 4h after treatment. Expression of TLR-1, -2, -3, -4, -6, -8, -9, -10, TNF-alpha, IL-1beta, -8 and MCP-1 was quantified relative to the quantity of Cyclophilin-A mRNA using real-time quantitative PCR (RTQ-PCR). The results obtained show up differences in the gene expression between both cell lines IPEC-J2 and IPI-2I as response to LPS from S. typhimurium during the activation time, which may suggest an in vivo variability in the innate immune response against pathogens in different regions of the host's gut.

Pharmacological inhibition of GSK-3 is not strictly correlated with a decrease in tyrosine phosphorylation of residues 216/279.

Recent evidence suggests that intramolecular autophosphorylation is responsible for the tyrosine phosphorylation (pY) of residues 279 or 216 of glycogen synthase kinase-3 (GSK-3alpha or beta), an event that appears to play an important role in regulating this kinase. This provocative hypothesis was based on the capacity of certain nonselective GSK-3 inhibitors to alter both the activity of GSK-3 and its pY. Inhibitors of GSK-3 are not always capable of preventing this tyrosine phosphorylation, which may require an extended period of time. For example, although lithium chloride inhibits GSK-3 activity, this inhibition does not alter its pY content. Furthermore, even when GSK-3 activity is impaired, GSK-3 pY can still be modified by physiological or pharmacological agents. Taken together, these data indicate that GSK-3 kinase activity is not necessarily correlated with the extent of GSK-3 pY. We hypothesized that some as-yet-unidentified tyrosine kinases and phosphatases may also regulate this kinase.

Retention of cobalt on a humin derived from brown coal.

In this work, the retention of cobalt on a humin (HU) derived from a brown coal is studied. Through a systematic and coordinated investigation of the behavior of the metal ions in solution (speciation diagrams as a function of pH) and their adsorption and precipitation processes with reactive functional groups of the solid (sorption isotherms), the interactions of different Co(II) species with HU are probed. To further confirm the nature of these interactions, the complementary spectroscopic techniques of FTIR, Raman microspectroscopy, UV-visible absorption and XRD are employed. Molecular modeling techniques are used to gain information about the stability of different Co(II) species as a function of pH, as well as the stability of Co(II) species complexed with benzoic acid, a common surface component of humic substances. It is found that the selectivity that humin has for different Co(II) species, as well as the amount of Co(II) that it can retain, are both highly dependent on pH. Through Raman microspectroscopy measurements, the presence and location of Co(OH)(2) precipitates on the surface of HU is confirmed.

Theoretical study on fulvic acid structure, conformation and aggregation. A molecular modelling approach.

The ubiquitous presence of humic substances (HS), combined with their ability to provide multiple sites for chemical reaction, makes them relevant to numerous biogeochemical processes such as mineral weathering, nutrient bioavailability, and contaminant transport. The reactivity of HS depends on their functional group chemistry and microstructure, which are in turn influenced by the composition of the surrounding media. In order to help towards an understanding of structure conformations and aggregation process of HS in soils and waters and to get a better knowledge of these kinds of materials, a fulvic acid (FA) has been modelled as a function of its ionic state under different conditions. Our proposed theoretical model based on the Temple-Northeastern-Birmingham (TNB) monomer fits well with experimental observations on the solubility (dipolar moment) and electronic and vibrational spectra of FAs. The presence of water molecules has a great stabilization effect on the electrostatic energy; this effect is greater as ionized rate increases. In vacuum, the non-ionized aggregated species are more stable than monomers because of the increase in their interaction due to H-bonding and non-bonding forces. When the molecules are ionized, no aggregation process takes place. In solution, the FA concentration is a critical factor for the aggregation. The system containing two FA molecules probably did not form aggregates because its equivalent concentration was too low. When the concentration was increased, the system gave rise to the formation of aggregates. The ionic state is another critical factor in the aggregation process. The ionized FA has a higher electric negative charge, which increases the energetic barriers and inhibits the approximation of FA caused by the Brownian movement.

Effect of pH on the aggregation of a gray humic acid in colloidal and solid states.

Gray humic acids have a marked colloidal character, a large number of surface functional groups, and are subject to aggregation phenomena. They are able to complex soluble pollutants, and initiate flocculation processes as a function of environmental conditions. The aim of this work is to study the aggregation of a gray humic acid, which is stable in colloidal dispersion, by means of photon correlation spectroscopy, and molecular modeling. The effect of this aggregation in the solid state is also studied by means of N2 (to 77 K) and CO2 (to 273 K) adsorption isotherms, as well as FT-IR absorption. The variation of the colloid's zeta potential and size, with pH, reflects the ionization of the carboxylic and phenolic acidic groups, and a linear dependence of size on zeta potential. The decrease in the size of the colloids seems to be more affected by the ionization of the phenolic acid groups, than by that of the carboxylic acid groups, which is likely because in the case of the ionized carboxylic groups the humic colloids are still capable of generating H-bonds. In the solid state, aggregation effects are illustrated by a decrease in surface area, and a disappearance of certain micropores, with increasing pH. These features are likely due to an inhibition of aggregation in the colloidal state as a consequence of the increase in charge that results from ionization of the acidic groups, and also to an increased hindrance to H-bond formation, due to the loss of protons during the above-mentioned ionization process.

Modeling the adsorption and precipitation processes of Cu(II) on humin.

Humins (HU) are the most insoluble fraction of humic substances. Chemically, they can be considered as humic macromolecules bonded to the mineral matter of soil. The HU have a marked colloidal character and they are extremely important in retention of pollutants in soils. The aim of this work is to combine adsorption data with spectroscopic techniques in order to study the adsorption and precipitation processes of Cu(II) on HU. Analysis of sorption isotherms by means of several single-adsorption-process-based models makes it possible to obtain the speciation diagrams of Cu(II) species on HU surfaces. Further, FTIR (which provides information about the changes in the surface groups of the HU) and DRX (which shows the formation of possible crystalline phases on the HU surface) were used to determine the specific interactions of Cu(II) cations with the surface reactive groups of HU. The shape of the isotherms at constant pH varies with pH from L1-type (pH 2-4) to L3-type (pH 5-6) and S-type (pH 8), which indicates a change in the retention mechanism. When pH is 2 the retention of Cu(II), as [Cu(H(2)O)(6)](2+), is the preferred retention mechanism. The retained quantity of Cu(II) as [Cu(OH)(H(2)O)(5)](+) increases with pH. Starting from pH 4 the Cu(II) begins its precipitation, which is the preferred retention mechanism at pH 8. The presence of HU has a great influence on the precipitation process of Cu(II), giving rise to botalackite, which reveals epitaxial growth of crystals.

¿Institucionalización o marginación?: análisis etnográfico de una residencia de Tercera Edad / Institucionalization or isolation?: etnographical analysis of an Elderly Residence

El presente trabajo muestra, mediante una actividad multidisciplinar entre enfermeros y antropólogos, cómo las Residencias de Tercera Edad, uno de los recursos actualmente imprescindibles dentro del sistema sociosanitario, pueden llegar a convertirse en un centro de marginación social para los ancianos allí ingresados. Igualmente, se plantean una serie de propuestas para evitar dicha marginación y contribuir tanto a la mejora de la salud de los ancianos residentes en dichos centros como para mantener su integración social (AU)

Cu(II) retention on a humic substance.

Humic substances (HS) are macromolecular products derived from a physical, chemical, and microbiological process called "humification." These substances play an important role in the mobility and bioavailability of nutrients and contaminants in the environment. Adsorption isotherms provide a macroscopic view of the retention phenomena. However, complementary techniques are needed in order to study the retention mechanism. The application of the classical models and some modern ones, based on humic substances chemistry, do not accurately describe these adsorption data. The aim of this paper is to model isotherms and combine adsorption data with spectroscopy and microscopy techniques to study the Cu(II) retention on a HS. The adsorption isotherms shape varies significantly with the solution pH from L-type (pH 2-6) to S-type (pH 8). FTIR shows that, when pH is 2 the retention of Cu(II), as [Cu(H(2)O)(6)](2+), is the preferred retention mechanism. The quantity of Cu(II) retained as [Cu(OH)(H(2)O)(6)](+) rises, as pH increases. At pH 4, Cu(II) begins to precipitate, which is the preferred mechanism at pH 8.02. The presence of HS has a great influence on the precipitation process of Cu(II), giving rise to amorphous precipitates. As it is shown by SEM-XRF, Cu(II) distributes heterogeneously on HS surface and accumulates on the humic phases. The presence of different anions (chloride and nitrate) slightly modifies the HS behavior as cation exchanger. When Cl(-) ions are present, part of the Cu(II) form [CuCl(4)](2-), which is stable in solution due to its negative charge; when the anion present is NO(3)(-) the formed complex, [CuNO(3)](+), is retained on the HS.

Analysis of swine beta1 integrin (CD29) epitopes through monoclonal antibodies developed using two immunization strategies.

This report describes the production and characterization of monoclonal antibodies (MAbs) to swine beta1 integrin subunit (CD29) using two different immunization strategies. MAb GP4B4 was developed from a mouse immunized with porcine peripheral blood mononuclear cells (PBMC), while MAbs GP1A5, GP1A6, and GP4A1 were produced by immunizing mice with a porcine CD29 recombinant protein (rpCD29) developed in our laboratory, which includes the ligand binding site. GP4B4 and UCP1D2 (specific to porcine CD29) immunoprecipitated two bands of approximately 115 and 150 kDa under reducing conditions. The molecule recognized by these two antibodies was studied using flow cytometry and was found in practically all cells studied, displaying a similar reaction pattern. Western blot assays performed with rpCD29 indicated that MAbs GP1A5, GP1A6, and GP4A1 recognized the 30-kDa band for this recombinant protein, confirming their specificity for the beta1, integrin subunit. Immunohistochemical analyses of these MAbs disclosed a morphological pattern associated with smooth muscle, epithelium, and myeloid cells, as expected in MAbs recognizing CD29. This MAb panel could be useful as a general anti-CD29 reagent and would allow further research into this important integrin in swine.