Pig slurry acidification,separation technology and thermal conversion affect phosphorus availability in soil amended with the derived solid fractions,chars or ashes

Aims

The aim was to study effects of slurry acidification, separation technology and thermal processing on the availability of P in soil amended with the solid fraction of pig slurry.

Methods

Acidified and non-acidified slurry were separated using different technologies: screw press (SCR), decanting centrifuge (DEC) and drainage after chemical pretreatment (CHE). Solids and pyrolysed (400 °C or 600 °C, char) or combusted (625 °C, ash) solids were applied to two soils, with triple superphosphate (TSP) as a reference. Soil P availability was determined over 12 weeks using diffusive gradients in thin films (DGT).

Results

The initial availability of P from DEC and CHE solids was similar to TSP in both soils. After 6 weeks no significant difference was observed between DEC solids and DEC chars. Acidification did not significantly affect P availability in the solids-amended soils over time, but it did affect P availability with the thermally processed materials.

Conclusions

Application of separated pig slurry solids generally increased soil P availability initially, but declining with time, indicating soil P fixing reactions. Chars and ashes on the other hand showed lower initial P availability, but remained constant or increased slightly with chars yielding P availability similar to solids after 12 weeks.

     

Temporal and spatial mapping of red grouper Epinephelus morio sound production

The goals of this project were to determine the daily, seasonal and spatial patterns of red grouper Epinephelus morio sound production on the West Florida Shelf (WFS) using passive acoustics. An 11 month time series of acoustic data from fixed recorders deployed at a known E. morio aggregation site showed that E. morio produce sounds throughout the day and during all months of the year. Increased calling (number of files containing E. morio sound) was correlated to sunrise and sunset, and peaked in late summer (July and August) and early winter (November and December). Due to the ubiquitous production of sound, large‐scale spatial mapping across the WFS of E. morio sound production was feasible using recordings from shorter duration‐fixed location recorders and autonomous underwater vehicles (AUVs). Epinephelus morio were primarily recorded in waters 15–93 m deep, with increased sound production detected in hard bottom areas and within the Steamboat Lumps Marine Protected Area (Steamboat Lumps). AUV tracks through Steamboat Lumps, an offshore marine reserve where E. morio hole excavations have been previously mapped, showed that hydrophone‐integrated AUVs could accurately map the location of soniferous fish over spatial scales of <1 km. The results show that passive acoustics is an effective, non‐invasive tool to map the distribution of this species over large spatial scales.     

Vaccination with an Adenoviral Vector That Encodes and Displays a Retroviral Antigen Induces Improved Neutralizing Antibody and CD4+ T-Cell Responses and Confers Enhanced Protection

We present a new type of adenoviral vector that both encodes and displays a vaccine antigen on the capsid, thus combining in itself gene-based and protein vaccination; this vector resulted in an improved vaccination outcome in the Friend virus (FV) model. For presentation of the envelope protein gp70 of Friend murine leukemia virus on the adenoviral capsid, gp70 was fused to the adenovirus capsid protein IX. When compared to vaccination with conventional FV Env- and Gag-encoding adenoviral vectors, vaccination with the adenoviral vector that encodes and displays pIX-gp70 combined with an FV Gag-encoding vector resulted in significantly improved protection against systemic FV challenge infection, with highly controlled viral loads in plasma and spleen. This improved protection correlated with improved neutralizing antibody titers and stronger CD4⁺ T-cell responses. Using a vector that displays gp70 without encoding it, we found that while the antigen display on the capsid alone was sufficient to induce high levels of binding antibodies, in vivo expression was necessary for the induction of neutralizing antibodies. This new type of adenovirus-based vaccine could be a valuable tool for vaccination.Adenoviruses have been a focus of interest as vaccine vectors for more than a decade and have been tested in various preclinical and clinical studies for vaccination against viral and bacterial infections (reviewed in reference 38). This interest is based on the ability of adenoviral vectors to induce high antibody titers and robust cytotoxic T-lymphocyte (CTL) responses and on the high immunogenicity of the vector, which might have an adjuvant effect on vaccination (17). Adenoviral vectors have also been extensively evaluated for immunization against HIV (reviewed in reference 1), where they were used either alone or in combination with plasmid DNA or protein in prime-boost immunizations. However, vaccination with adenoviral vectors against HIV showed no effectiveness in a large phase IIb study (4), but it is conceivable that the observed lack of effectiveness was due to the choice of vaccine antigen rather than the vector itself, as the vaccine relied exclusively on the induction of CTL responses, and the outcome was unexpected given previous results from studies in nonhuman primates (33, 42). The findings of the phase IIb study brought about a shift of focus from the CTL response to a more balanced immune response, including neutralizing antibodies, that is now expected to be necessary for protection from HIV infection.Apart from adenoviral vectors that encode vaccine antigens, there have also been approaches to modify adenoviral capsid proteins to include antigenic epitopes. These were mostly inserted into external loops of the hexon protein (5, 22, 25, 26, 43), which is the main component of the adenovirus capsid, but also other components of the capsid, such as fiber, protein IX, and penton base, have been evaluated (22). These studies showed that incorporation of single epitopes into capsid proteins of adenovirus leads to induction of antibody and CD4⁺ T-cell responses, suggesting that incorporation of epitopes into the adenovirus capsid is a useful tool for epitope-based vaccination.Fusion of a polylysine sequence or an arginine-glycine-aspartic acid motif to adenovirus pIX has been shown to be a tool for redirection of adenovirus tropism to heparan sulfate and α_vβ integrins, respectively (9, 41). By fusing green fluorescent protein and luciferase to the C terminus of pIX, it was shown that relatively large proteins can be displayed on the adenovirus capsid while maintaining the protein''s conformation and function as well as virion integrity (24, 28).Here we describe a novel vaccination approach that combines genetic and protein vaccination by using adenoviral vectors not only as gene expression vectors but also as nanoparticle carriers for a vaccine antigen to improve the vaccination efficiency through enhanced induction of antibodies. Display of the vaccine antigen on the adenovirus capsid was achieved by fusion of the antigen to the C terminus of the adenovirus capsid protein pIX. It was shown before that the presentation of antigens in ordered arrays leads to improved antibody responses by cross-linking of B-cell receptors (13). As the adenoviral capsid is highly structured, we hypothesized that fusion to pIX would result in an ordered display of the antigen, presumably facilitating antibody induction.We evaluated this vaccine approach using the Friend virus (FV) infection model. FV is an immunosuppressive retroviral complex that consists of Friend murine leukemia virus (F-MuLV) and the replication-deficient, F-MuLV-dependent spleen focus-forming virus. FV infection of susceptible mice induces rapid polyclonal erythroblast proliferation, which leads to splenic enlargement and erythroleukemia and takes a lethal course also in adult mice (14). Protection from FV infection has been shown to require complex immune responses involving antibodies as well as CD4⁺ and CD8⁺ T cells (7). FV is regarded as a useful retrovirus infection model because basic requirements for vaccine protection seem to be similar for FV and HIV infection (8). We demonstrated previously that the FV model is suitable to evaluate and improve adenoviral vectors for antiretroviral vaccination (2), as we showed that a heterologous prime-boost vaccination with adenovirus type 5 (Ad5) and fiber chimeric Ad5F35 vectors led to better protection from FV infection than homologous vaccination, which correlated with improved induction of neutralizing antibodies.For vaccination with expression/display vectors against FV we constructed a fusion protein of the adenoviral capsid protein pIX and the F-MuLV envelope protein gp70 and produced adenoviral vectors expressing the pIX-gp70 fusion protein, which was incorporated into the viral capsid. We vaccinated FV-susceptible CB6F1 hybrid mice with antigen expression/display vectors or with conventional antigen-expressing adenoviral vectors and analyzed the protection conferred by these two vaccines. Having demonstrated that the expression/display vector leads to better protection of mice from FV challenge, we constructed a panel of expression/display vectors displaying different fusion proteins containing F-MuLV Env or Gag in order to elucidate the underlying immunological mechanisms of the improved protection conferred by the adenoviral expression/display vectors.     

Quantitative phosphoproteomic analysis of prion-infected neuronal cells

Following cultivation of distinct mesenchymal stem cell (MSC) populations derived from human umbilical cord under hypoxic conditions (between 1.5% to 5% oxygen (O₂)) revealed a 2- to 3-fold reduced oxygen consumption rate as compared to the same cultures at normoxic oxygen levels (21% O₂). A simultaneous measurement of dissolved oxygen within the culture media from 4 different MSC donors ranged from 15 μmol/L at 1.5% O₂ to 196 μmol/L at normoxic 21% O₂. The proliferative capacity of the different hypoxic MSC populations was elevated as compared to the normoxic culture. This effect was paralleled by a significantly reduced cell damage or cell death under hypoxic conditions as evaluated by the cellular release of LDH whereby the measurement of caspase3/7 activity revealed little if any differences in apoptotic cell death between the various cultures. The MSC culture under hypoxic conditions was associated with the induction of hypoxia-inducing factor-alpha (HIF-1α) and an elevated expression of energy metabolism-associated genes including GLUT-1, LDH and PDK1. Concomitantly, a significantly enhanced glucose consumption and a corresponding lactate production could be observed in the hypoxic MSC cultures suggesting an altered metabolism of these human stem cells within the hypoxic environment.     

Cross-talk between Type Three Secretion System and Metabolism in Yersinia

Pathogenic yersiniae utilize a type three secretion system (T3SS) to inject Yop proteins into host cells in order to undermine their immune response. YscM1 and YscM2 proteins have been reported to be functionally equivalent regulators of the T3SS in Yersinia enterocolitica. Here, we show by affinity purification, native gel electrophoresis and small angle x-ray scattering that both YscM1 and YscM2 bind to phosphoenolpyruvate carboxylase (PEPC) of Y. enterocolitica. Under in vitro conditions, YscM1, but not YscM2, was found to inhibit PEPC with an apparent IC₅₀ of 4 μm (K_i = 1 μm). To analyze the functional roles of PEPC, YscM1, and YscM2 in Yop-producing bacteria, cultures of Y. enterocolitica wild type and mutants defective in the formation of PEPC, YscM1, or YscM2, respectively, were grown under low calcium conditions in the presence of [U-¹³C₆]glucose. The isotope compositions of secreted Yop proteins and nine amino acids from cellular proteins were analyzed by mass spectrometry. The data indicate that a considerable fraction of oxaloacetate used as precursor for amino acids was derived from [¹³C₃]phosphoenolpyruvate by the catalytic action of PEPC in the wild-type strain but not in the PEPC^- mutant. The data imply that PEPC is critically involved in replenishing the oxaloacetate pool in the citrate cycle under virulence conditions. In the YscM1^- and YscM2^- mutants, increased rates of pyruvate formation via glycolysis or the Entner-Doudoroff pathway, of oxaloacetate formation via the citrate cycle, and of amino acid biosynthesis suggest that both regulators trigger the central metabolism of Y. enterocolitica. We propose a “load-and-shoot cycle” model to account for the cross-talk between T3SS and metabolism in pathogenic yersiniae.Type three secretion systems (T3SSs)³ are used by several Gram-negative bacteria as microinjection devices to deliver effector proteins into host cells (1). The translocated effector proteins reprogram the host cell in favor of the microbial invader or symbiont. Pathogenic yersiniae (the enteropathogenic Yersinia enterocolitica and Yersinia pseudotuberculosis and the plague bacillus Yersinia pestis) utilize a plasmid-encoded T3SS to undermine the host primary immune response (2). This is mediated by the injection of a set of effector proteins called Yops (Yersinia outer proteins) into host cells, in particular into cells with innate immune functions, such as macrophages, dendritic cells, and neutrophils (3). The concerted action of Yops, targeting multiple signaling pathways, results in actin cytoskeleton disruption, suppression of proinflammatory signaling, and induction of apoptosis. This strategy enables yersiniae to multiply extracellularly in host tissue.Expression of the Yersinia T3SS is up-regulated at 37 °C, and translocation of Yops across the host cell membrane is triggered by cell contact (4, 5). Pathogenic yersiniae cultivated under low calcium conditions at 37 °C express a phenotype referred to as “low calcium response” (LCR). The LCR is characterized by growth restriction as well as massive expression and secretion of Yops into the culture medium (6–9). The allocation of energy and metabolites for the massive synthesis and transport of Yops is demanding, and this burden is believed to be responsible for the observed growth inhibition (10). To give an idea of the metabolic requirements, Yops are secreted to the culture supernatant in 10-mg amounts per liter of culture within 2 h after calcium depletion of the medium. Furthermore, post-translationally secreted substrates need to be unfolded by a T3SS-specific ATPase prior to secretion (11–14). In addition, T3SS-dependent transport of Yops requires the proton motive force (15). However, there is evidence that growth cessation and Yop expression can be uncoupled (16, 17), suggesting a coordinated regulation of metabolism and protein transport rather than the LCR reflecting an inevitable physiological consequence.What are the candidate proteins that could be involved in such a coordination? YscM1 and YscM2 (57% identical to YscM1) are key candidates, since they act at a major nodal point of the T3SS regulatory network in Y. enterocolitica. In Y. pestis and Y. pseudotuberculosis, only the YscM1 homologue LcrQ exists (99% identical to YscM1). YscM1/LcrQ and YscM2 are secretion substrates of the T3SS that are involved in up-regulation of Yop expression after host cell contact. Upon cell contact, the decrease of intracellular levels of YscM1/LcrQ and YscM2 due to their translocation into host cells results in a derepression of Yop synthesis (18–21). The two yscM copies of Y. enterocolitica were presumed to be functionally equivalent, since deletion of either gene was found to be phenotypically silent (19, 22). Only deletion of both yscM genes could establish the lcrQ phenotype (19, 22), distinguished by temperature sensitivity for growth, derepressed Yop expression, and secretion of LcrV and YopD in the presence of calcium ions.YscM1/LcrQ as well as YscM2 exhibit homology to the N terminus of the effector YopH (19, 23, 24), a fact that may explain their shared assistance by SycH (specific Yop chaperone) (20, 25). It was shown that YscM1/LcrQ and YscM2 exert their influence on Yop expression in concert with the T3SS components SycH, SycD (LcrH in Y. pestis and Y. pseudotuberculosis), and YopD (21, 26–28). It is further described that YscM1 and/or YscM2 interact with several of the T3SS-specific chaperones, in particular with SycH, SycE, SycD, and SycO (20, 29–31). This has led to the model that YscM/LcrQ proteins might function as an interface that senses whether chaperones are loaded with Yops and transduces these signals into control of Yop expression (14).These features of YscM1/LcrQ and YscM2 prompted us to speculate about a key role of these proteins in coordination of metabolism and expression of T3SS components. Using recombinant GST-YscM1 and GST-YscM2 as bait for Y. enterocolitica cytosolic proteins, we identified phosphoenolpyruvate carboxylase (PEPC) as interaction partner of both YscM1 and YscM2. Under in vitro conditions, YscM1 down-regulated PEPC activity and bacterial growth/replication. Isotopologue profiling of Yop proteins and derived amino acids from Y. enterocolitica grown in the presence of [U-¹³C₆]glucose showed the functionality of the PEPC reaction under virulence conditions (isotopologues are molecular entities that differ only in isotopic composition (number of isotopic substitutions); e.g. CH₄, CH₃D, and CH₂D₂). Moreover, biosynthetic rates of amino acids were increased in mutants defective in YscM1 or YscM2, suggesting a general role of these regulators in the metabolism of Y. enterocolitica. Recently, evidence has been accumulating that the metabolic state contributes to the regulation of T3SSs of diverse pathogens, also including the flagellar T3SS in Pseudomonas and Salmonella (32–36).     

Genetic association study identifies HSPB7 as a risk gene for idiopathic dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a structural heart disease with strong genetic background. Monogenic forms of DCM are observed in families with mutations located mostly in genes encoding structural and sarcomeric proteins. However, strong evidence suggests that genetic factors also affect the susceptibility to idiopathic DCM. To identify risk alleles for non-familial forms of DCM, we carried out a case-control association study, genotyping 664 DCM cases and 1,874 population-based healthy controls from Germany using a 50K human cardiovascular disease bead chip covering more than 2,000 genes pre-selected for cardiovascular relevance. After quality control, 30,920 single nucleotide polymorphisms (SNP) were tested for association with the disease by logistic regression adjusted for gender, and results were genomic-control corrected. The analysis revealed a significant association between a SNP in HSPB7 gene (rs1739843, minor allele frequency 39%) and idiopathic DCM (p = 1.06×10⁻⁶, OR = 0.67 [95% CI 0.57–0.79] for the minor allele T). Three more SNPs showed p < 2.21×10⁻⁵. De novo genotyping of these four SNPs was done in three independent case-control studies of idiopathic DCM. Association between SNP rs1739843 and DCM was significant in all replication samples: Germany (n = 564, n = 981 controls, p = 2.07×10⁻³, OR = 0.79 [95% CI 0.67–0.92]), France 1 (n = 433 cases, n = 395 controls, p = 3.73×10⁻³, OR = 0.74 [95% CI 0.60–0.91]), and France 2 (n = 249 cases, n = 380 controls, p = 2.26×10⁻⁴, OR = 0.63 [95% CI 0.50–0.81]). The combined analysis of all four studies including a total of n = 1,910 cases and n = 3,630 controls showed highly significant evidence for association between rs1739843 and idiopathic DCM (p = 5.28×10⁻¹³, OR = 0.72 [95% CI 0.65–0.78]). None of the other three SNPs showed significant results in the replication stage.This finding of the HSPB7 gene from a genetic search for idiopathic DCM using a large SNP panel underscores the influence of common polymorphisms on DCM susceptibility.     

Overlapping Binding Sites of Structurally Different Antiarrhythmics Flecainide and Propafenone in the Subunit Interface of Potassium Channel Kv2.1

Kv2.1 channels, which are expressed in brain, heart, pancreas, and other organs and tissues, are important targets for drug design. Flecainide and propafenone are known to block Kv2.1 channels more potently than other Kv channels. Here, we sought to explore structural determinants of this selectivity. We demonstrated that flecainide reduced the K⁺ currents through Kv2.1 channels expressed in Xenopus laevis oocytes in a voltage- and time-dependent manner. By systematically exchanging various segments of Kv2.1 with those from Kv1.2, we determined flecainide-sensing residues in the P-helix and inner helix S6. These residues are not exposed to the inner pore, a conventional binding region of open channel blockers. The flecainide-sensing residues also contribute to propafenone binding, suggesting overlapping receptors for the drugs. Indeed, propafenone and flecainide compete for binding in Kv2.1. We further used Monte Carlo-energy minimizations to map the receptors of the drugs. Flecainide docking in the Kv1.2-based homology model of Kv2.1 predicts the ligand ammonium group in the central cavity and the benzamide moiety in a niche between S6 and the P-helix. Propafenone also binds in the niche. Its carbonyl group accepts an H-bond from the P-helix, the amino group donates an H-bond to the P-loop turn, whereas the propyl group protrudes in the pore and blocks the access to the selectivity filter. Thus, besides the binding region in the central cavity, certain K⁺ channel ligands can expand in the subunit interface whose residues are less conserved between K⁺ channels and hence may be targets for design of highly desirable subtype-specific K⁺ channel drugs.     

Sulfated cholecystokinin-8 activates phospho-mTOR immunoreactive neurons of the paraventricular nucleus in rats

The serin/threonin-kinase, mammalian target of rapamycin (mTOR) was detected in the arcuate nucleus (ARC) and paraventricular nucleus of the hypothalamus (PVN) and suggested to play a role in the integration of satiety signals. Since cholecystokinin (CCK) plays a role in the short-term inhibition of food intake and induces c-Fos in PVN neurons, the aim was to determine whether intraperitoneally injected CCK-8S affects the neuronal activity in cells immunoreactive for phospho-mTOR in the PVN. Ad libitum fed male Sprague-Dawley rats received 6 or 10 μg/kg CCK-8S or 0.15 M NaCl ip (n = 4/group). The number of c-Fos-immunoreactive (ir) neurons was assessed in the PVN, ARC and in the nucleus of the solitary tract (NTS). CCK-8S increased the number of c-Fos-ir neurons in the PVN (6 μg: 103 ± 13 vs. 10 μg: 165 ± 14 neurons/section; p < 0.05) compared to vehicle treated rats (4 ± 1, p < 0.05), but not in the ARC. CCK-8S also dose-dependently increased the number of c-Fos neurons in the NTS. Staining for phospho-mTOR and c-Fos in the PVN showed a dose-dependent increase of activated phospho-mTOR neurons (17 ± 3 vs. 38 ± 2 neurons/section; p < 0.05), while no activated phospho-mTOR neurons were observed in the vehicle group. Triple staining in the PVN showed activation of phospho-mTOR neurons co-localized with oxytocin, corresponding to 9.8 ± 3.6% and 19.5 ± 3.3% of oxytocin neurons respectively. Our observations indicate that peripheral CCK-8S activates phospho-mTOR neurons in the PVN and suggest that phospho-mTOR plays a role in the mediation of CCK-8S's anorexigenic effects.     

Spatial analysis of land cover determinants of malaria incidence in the Ashanti Region, Ghana

Malaria belongs to the infectious diseases with the highest morbidity and mortality worldwide. As a vector-borne disease malaria distribution is strongly influenced by environmental factors. The aim of this study was to investigate the association between malaria risk and different land cover classes by using high-resolution multispectral Ikonos images and Poisson regression analyses. The association of malaria incidence with land cover around 12 villages in the Ashanti Region, Ghana, was assessed in 1,988 children <15 years of age. The median malaria incidence was 85.7 per 1,000 inhabitants and year (range 28.4–272.7). Swampy areas and banana/plantain production in the proximity of villages were strong predictors of a high malaria incidence. An increase of 10% of swampy area coverage in the 2 km radius around a village led to a 43% higher incidence (relative risk [RR] = 1.43, p<0.001). Each 10% increase of area with banana/plantain production around a village tripled the risk for malaria (RR = 3.25, p<0.001). An increase in forested area of 10% was associated with a 47% decrease of malaria incidence (RR = 0.53, p = 0.029).Distinct cultivation in the proximity of homesteads was associated with childhood malaria in a rural area in Ghana. The analyses demonstrate the usefulness of satellite images for the prediction of malaria endemicity. Thus, planning and monitoring of malaria control measures should be assisted by models based on geographic information systems.     

Codelivery of the chemokine CCL3 by an adenovirus-based vaccine improves protection from retrovirus infection

Processing and presentation of vaccine antigens by professional antigen-presenting cells (APCs) is of great importance for the efficient induction of protective immunity. We analyzed whether the efficacy of an adenovirus-based retroviral vaccine can be enhanced by coadministration of adenovirus-encoded chemokines that attract and stimulate APCs. In the Friend retrovirus (FV) mouse model we coexpressed CCL3, CCL20, CCL21, or CXCL14 from adenoviral vectors, together with FV Gag and Env antigens, and then analyzed immune responses and protection from pathogenic FV infection. Although most tested chemokines did not improve protection against FV challenge, mice that received adenoviral vectors encoding CCL3 together with FV antigens showed significantly better control over viral loads and FV-induced disease than mice immunized with the viral antigens only. Improved protection correlated with enhanced virus-specific CD4+ T cell responses and higher neutralizing antibody titers. To apply these results to an HIV vaccine, mice were immunized with adenoviral vectors encoding the HIV antigens Env and Gag-Pol and coadministered vectors encoding CCL3. Again, this combination vaccine induced higher virus-specific antibody titers and CD4+ T cell responses than did the HIV antigens alone. These results indicate that coexpression of the chemokine CCL3 by adenovirus-based vectors may be a promising tool to improve antiretroviral vaccination strategies.