Salmonella fecal shedding and immune responses are dose- and serotype- dependent in pigs

Despite the public health importance of Salmonella infection in pigs, little is known about the associated dynamics of fecal shedding and immunity. In this study, we investigated the transitions of pigs through the states of Salmonella fecal shedding and immune response post-Salmonella inoculation as affected by the challenge dose and serotype. Continuous-time multistate Markov models were developed using published experimental data. The model for shedding had four transient states, of which two were shedding (continuous and intermittent shedding) and two non-shedding (latency and intermittent non-shedding), and one absorbing state representing permanent cessation of shedding. The immune response model had two transient states representing responses below and above the seroconversion level. The effects of two doses [low (0.65×10(6) CFU/pig) and high (0.65×10(9) CFU/pig)] and four serotypes (Salmonella Yoruba, Salmonella Cubana, Salmonella Typhimurium, and Salmonella Derby) on the models' transition intensities were evaluated using a proportional intensities model. Results indicated statistically significant effects of the challenge dose and serotype on the dynamics of shedding and immune response. The time spent in the specific states was also estimated. Continuous shedding was on average 10-26 days longer, while intermittent non-shedding was 2-4 days shorter, in pigs challenged with the high compared to low dose. Interestingly, among pigs challenged with the high dose, the continuous and intermittent shedding states were on average up to 10-17 and 3-4 days longer, respectively, in pigs infected with S. Cubana compared to the other three serotypes. Pigs challenged with the high dose of S. Typhimurium or S. Derby seroconverted on average up to 8-11 days faster compared to the low dose. These findings highlight that Salmonella fecal shedding and immune response following Salmonella challenge are dose- and serotype-dependent and that the detection of specific Salmonella strains and immune responses in pigs are time-sensitive.     

Recognition and cleavage of the bacteriophage P1 packaging site (pac). II. Functional limits of pac and location of pac cleavage termini

Bacteriophage P1 initiates the processive packaging of its DNA at a unique site called pac. We show that a functional pac site is contained within a 161 base-pair segment of P1 EcoRI fragment 20. It extends from a position 71 base-pairs to a position 232 base-pairs from the EcoRI-22 proximal side of that fragment. The 3' and 5' pac termini are located centrally within that 161 base-pair region and are distributed over about a turn of the DNA helix. The DNA sequence of the terminus region is shown below, with the large arrows indicating the positions of termini that are frequently represented in the PI population and the small arrows indicating the positions of termini that are rarely represented in the P1 population. (Sequence: in text). Digestion of P1 virus DNA with EcoRI generates two major EcoRI-pac fragments, which differ in size by about five or six base-pairs. While the structure and position of the double-stranded pac ends of these fragments have not been determined precisely, the 5' termini at those ends probably correspond to the two major pac cleavage sites in the upper strand of the sequences shown above. The 161 base-pair pac site contains the hexanucleotide sequence 5'-TGATCAG-3' repeated four times at one end and three times at the other. Removal of just one of those elements from either the right or left ends of pac reduces pac cleavage by about tenfold. Moreover, the elements appear to be additive in their effect on pac cleavage, as removal of one and a half elements or all three elements from the right side of pac reduces pac cleavage 100-fold, and greater than 1000-fold, respectively.     

Ubiquitin function studied by disulfide engineering

Disulfide engineering was used to probe the role of conformational mobility in ubiquitin-mediated proteolysis. Six genes that encode cysteine-containing mutants of ubiquitin were constructed, expressed in Escherichia coli and the proteins purified. Single cysteine-containing mutants and a 4/14 disulfide were active in degradation of a substrate protein in vitro, while the 4/66 disulfide, which cross-links the NH2- and COOH-terminal strands of the protein, was only 20-30% active. The solution structure of the 4/66 mutant was solved: the disulfide is left-handed with no perturbations in the backbone from that of wild type ubiquitin. The results suggest that conformational mobility is required for the activity of ubiquitin in signaling proteolysis.     

Complexation of metal ions by pseudotripeptides with different functionalized N-alkyl residues

Summary Seven pseudotripeptides with the common structure Bz-His-ψ[CO−N(CH₂)n-X]Gly-His-NH₂ were synthesized on the solid phase using the Fmoc-strategy, trityl protection for both His residues and Boc-or-OBu^t-protection for N-aminoalkyl-and N-carboxyalkyl residues, respectively. Functionalized N-alkyl glycyl peptides were formed on the solid phase by amination of a bromoacetyl dipeptide. All seven pseudotripeptides are able to form chelate complexes with the metal ions Zn²⁺, Ni²⁺, Cu²⁺ and Co²⁺. The existence of monomeric 1∶1 complexes for these pseudopeptides was calculated from the MW estimated by MALDI-MS and from the isotope distribution pattern estimated by ESI.     

Identification of Human Embryonic Progenitor Cell Targeting Peptides Using Phage Display

Human pluripotent stem (hPS) cells are capable of differentiation into derivatives of all three primary embryonic germ layers and can self-renew indefinitely. They therefore offer a potentially scalable source of replacement cells to treat a variety of degenerative diseases. The ability to reprogram adult cells to induced pluripotent stem (iPS) cells has now enabled the possibility of patient-specific hPS cells as a source of cells for disease modeling, drug discovery, and potentially, cell replacement therapies. While reprogramming technology has dramatically increased the availability of normal and diseased hPS cell lines for basic research, a major bottleneck is the critical unmet need for more efficient methods of deriving well-defined cell populations from hPS cells. Phage display is a powerful method for selecting affinity ligands that could be used for identifying and potentially purifying a variety of cell types derived from hPS cells. However, identification of specific progenitor cell-binding peptides using phage display may be hindered by the large cellular heterogeneity present in differentiating hPS cell populations. We therefore tested the hypothesis that peptides selected for their ability to bind a clonal cell line derived from hPS cells would bind early progenitor cell types emerging from differentiating hPS cells. The human embryonic stem (hES) cell-derived embryonic progenitor cell line, W10, was used and cell-targeting peptides were identified. Competition studies demonstrated specificity of peptide binding to the target cell surface. Efficient peptide targeted cell labeling was accomplished using multivalent peptide-quantum dot complexes as detected by fluorescence microscopy and flow cytometry. The cell-binding peptides were selective for differentiated hPS cells, had little or no binding on pluripotent cells, but preferential binding to certain embryonic progenitor cell lines and early endodermal hPS cell derivatives. Taken together these data suggest that selection of phage display libraries against a clonal progenitor stem cell population can be used to identify progenitor stem cell targeting peptides. The peptides may be useful for monitoring hPS cell differentiation and for the development of cell enrichment procedures to improve the efficiency of directed differentiation toward clinically relevant human cell types.     

IgM-antibody responses of chickens to salivary antigens of Triatoma infestans as early biomarkers for low-level infestation of triatomines

The recombinant form of a highly immunogenic 14.6 kDa protein in Triatoma infestans saliva (rTiSP14.6) is a potential epidemiological marker for the detection of triatomine bug populations using IgG responses in peridomestic chickens. However, the persistence of the IgG response prevents it being of value for several months in areas where triatomine control programmes have been implemented. In this investigation, IgM-antibody reactions to crude salivary antigens or rTiSP14.6 decayed rapidly after exposure of chickens and were measurable for only 18 days after a single challenge with T. infestans. In serial exposure experiments, chickens from low and high exposure groups showed no significant differences in anti-saliva and anti-rTiSP14.6 IgM-antibody titres. Highly immunogenic salivary antigens of 12 and 14 kDa were recognised by all chicken sera. Sera from peridomestic chickens from sites of known T. infestans infestation in Bolivia also recognised these two antigens and no differences in the IgM responses of sera from chickens from low and high infestation households were detected. IgM responses were specific to infested households and could not be detected in sera from non-infested households. Cross-reactivity studies showed that at least four other triatomine species share the 14.6 kDa salivary antigen. No IgM responses were detected against salivary proteins of mosquitoes and sandflies. Thus, we believe that rTiSP14.6 represents a promising epidemiological marker for the detection of low numbers of triatomines in peridomestic habitats, and the comparison of IgM and IgG responses can be used to detect re-infestation soon after insecticide-based control programmes.     

Not all forests are expanding over central Brazilian savannas

Recently we reported on the expansion of riparian forests into savannas in central Brazil. To enlarge the scope of the earlier study we investigated whether upland deciduous and xeromorphic forests behaved similarly. We investigated past vegetation changes that occurred in forest/savanna transitions using carbon isotope ratios (δ¹³C) measured in the soil organic matter as a tracer. We analyzed the ¹⁴C activity where δ¹³C showed major shifts in vegetation. The role of soil chemical and physical attributes in defining vegetation distribution is discussed. Structural changes in vegetation were found to be associated with shifts in the isotope composition (δ¹³C) of soil organic matter. This was attributed to intrinsic differences in the biomass of trees and grasses and allowed for the determination of past shifts in vegetation by evaluating δ¹³C at different depths. The deciduous forest decreased in area approximately 980 years ago. Tree cover increased in the xeromorphic forest, but the border stayed stable through time. The deciduous forest and adjacent savanna have eutrophic soils while the xeromorphic forest and adjacent savanna have dystrophic soils. However, greater organic carbon, nitrogen and phosphorus concentrations are observed in the forests. We provide concrete evidence of deciduous forest retreat unlike the stability observed in the xeromorphic forest/savanna boundary. These results contrast with the expansion of riparian forests recently reported in the same region.