Evaluation of a fosmid-clone-based microarray for comparative analysis of swine fecal metagenomes

Glass slide arrayed with fosmid clone DNAs generated from swine feces as probes were fabricated and used as a metagenome microarray (MGA). MGA appeared to be specific to their corresponding target genomic fragments. The detection limit was 10 ng of genomic DNA (ca. 10(6) bacterial cells) in the presence of 1000 ng of background DNA. Linear relationships between the signal intensity and the target DNA (20-100 ng) were observed (r ( 2 )=0.98). Application of MGA to the comparison of swine fecal metagenomes suggested that the microbial community composition of swine intestine could be dependent on the health state of swine.     

Genetic co-transfer of CCR7 ligands enhances immunity and prolongs survival against virulent challenge of pseudorabies virus

The CC chemokine receptor 7 (CCR7) and cognate CCR7 ligands, CCL19 and CCL21, help establish microenvironments in lymphoid tissue that can facilitate encounters between naive T cells and mature dendritic cells (DCs). This study was conducted to determine if CCR7 ligands can augment the immunogenicity of a DNA vaccine that expresses glycoprotein B (gB) of the pseudorabies virus (PrV). The genetic co-transfer of CCR7 ligands along with a PrV DNA vaccine increased the levels of serum PrV-specific immunoglobulin (Ig) G by 2- to 2.5-fold. In addition, the level of PrV-specific IgG2a isotype was significantly enhanced by co-injection of CCR7 ligand DNA, which indicates that CCR7 ligand biases the humoral immunity toward the Th1-type pattern. The co-injection of CCR7 ligand DNA consistently enhanced the level of Th1-type cytokines (IL-2 and IFN-gamma) produced by stimulated immune cells when compared with a group that was vaccinated with the PrV DNA vaccine. Also, the genetic co-transfer of CCR7 ligand DNAs with PrV DNA vaccine provided prolonged survival against a virulent challenge by PrV. Moreover, the co-administration of CCR7 ligand DNA increased the number of mature DCs into the secondary lymphoid tissues, which appeared to enhance the proliferation of PrV-immune CD4(+) T cells. Taken together, these findings indicate that CCR7 ligands are an attractive adjuvant for a PrV DNA vaccine that can offer protective immunity against the PrV.     

Ecology of deepwater rice-fields in Bangladesh 4. Nitrogen fixation by blue-green algal communities

1. Aquatic macrophytes formed dense beds in fallow areas during the four and a half months of the flood season in all but one deepwater rice-growing location in Bangladesh; these included several types of life-form, but the fine-leaved species, Myriophyllum sp., Najas indica, Utricularia stellaris were often especially abundant. The same species grew inside deepwater rice fields, but at much lower densities. A similar contrast occurred for the algae, although deepwater rice often developed dense masses of epiphytes on aquatic roots, stems and leaf sheaths, when plants were growing in isolated, well-illuminated situations.
2. Two widespread algae, Aulosira fertilissima and Scytonema mirabile, were equally successful on soil in the period prior to the arrival of floodwaters and floating on the surface of the water during the flood season. Other species common during the flood season differed from those common on soil.
3. Most blue-green algae inside deepwater rice fields were heterocystous; the only species not so, but forming distinct colonies, was Aphanothece stagnina. However only non-heterocystous forms were found at one location in south Bangladesh (Phaltita) and a change from heterocystous to non-heterocystous forms was noted at the main research site (near Sonargaon) during late September in at least one year. The water column at the former was almost entirely anoxic, while the change at the latter occurred at a time when the water sometimes became anoxic during the night. It is suggested that differences in ability to tolerate anoxic periods may be a key factor in determining the success of the algal and vascular plant species in the different micro-habitats within these DWR-growing areas.
4. Although diatoms were quantitatively only a minor component of the algal biomass, they became more frequent later in the season when the water became microaerobic or anoxic for part of the day. Navicula confervacea was overall the most abundant species at the two main research locations.

     

Structural and biochemical characterization of the broad substrate specificity of Bacteroides thetaiotaomicron commensal sialidase

Sialidases release the terminal sialic acid residue from a wide range of sialic acid-containing polysaccharides. Bacteroides thetaiotaomicron, a symbiotic commensal microbe, resides in and dominates the human intestinal tract. We characterized the recombinant sialidase from B. thetaiotaomicron (BTSA) and demonstrated that it has broad substrate specificity with a relative activity of 97, 100 and 64 for 2,3-, 2,6- and 2,8-linked sialic substrates, respectively. The hydrolysis activity of BTSA was inhibited by a transition state analogue, 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid, by competitive inhibition with a K_i value of 35 μM. The structure of BSTA was determined at a resolution of 2.3 Å. This structure exhibited a unique carbohydrate-binding domain (CBM) at its N-terminus (a.a. 23–190) that is adjacent to the catalytic domain (a.a. 191–535). The catalytic domain has a conserved arginine triad with a wide-open entrance for the substrate that exposes the catalytic residue to the surface. Unlike other pathogenic sialidases, the polysaccharide-binding site in the CBM is near the active site and possibly holds and positions the polysaccharide substrate directly at the active site. The structural feature of a wide substrate-binding groove and closer proximity of the polysaccharide-binding site to the active site could be a unique signature of the commensal sialidase BTSA and provide a molecular basis for its pharmaceutical application.     

Difference in microchip electrophoretic mobility between partially and fully PEGylated poly(amidoamine) dendrimers

The objective of this study was to investigate the difference in electrophoretic mobility between partially and fully poly(ethylene glycol)-conjugated poly(amidoamine) dendrimers (part-PEG-PAMAM and full-PEG-PAMAM, respectively) using a microchip capillary gel electrophoresis (MCGE). While MCGE allowed size-based separation of PEG-PAMAMs prepared with monomethoxy PEG-nitrophenyl carbonate, full-PEG-PAMAMs migrated slower than part-PEG-PAMAMs that were similar in size or larger. When the measured molecular weights obtained from MCGE analysis and the calculated molecular weights were plotted, each part-PEG-PAMAM and full-PEG-PAMAM showed correlation coefficients greater than 0.98. This study indicates that MCGE would be useful for characterizing PEG-PAMAMs with different PEGylation degrees.     

Selenite negatively regulates caspase-3 through a redox mechanism

Selenium, an essential biological trace element, exerts its modulatory effects in a variety of cellular events including cell survival and death. In our study we observed that selenite protects HEK293 cells from cell death induced by ultraviolet B radiation (UVB). Exposure of HEK293 cells to UVB radiation resulted in the activation of caspase-3-like protease activity, and pretreatment of the cells with z-DEVD-fmk (N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone), a caspase-3 inhibitor, prevented UVB-induced cell death. Interestingly, enzymatic activity of caspase-3-like protease in cell lysates of UVB-exposed cells was repressed in vitro by the presence of selenite. Selenite also inhibited the in vitro activity of purified recombinant caspase-3 in cleaving Ac-DEVD-pNA (N-acetyl-Asp-Glu-Asp-p-nitroanilide) or ICAD(L) (inhibitor of a caspase-activated deoxyribonuclease) and in the induction of DNA fragmentation. The inhibitory action of selenite on a recombinant active caspase-3 could be reversed by sulfhydryl reducing agents, such as dithiothreitol and beta-mercaptoethanol. Furthermore, pretreatment of cells with selenite suppressed the stimulation of the caspase-3-like protease activity in UVB-exposed cells, whereas dithiothreitol and beta-mercaptoethanol reversed this suppression of the enzymatic activity. Taken together, our data suggest that selenite inhibits caspase-3-like protease activity through a redox mechanism and that inhibition of caspase-3-like protease activity may be the mechanism by which selenite exerts its protective effect against UVB-induced cell death.     

incR: a new R package to analyse incubation behaviour

Incubation represents a life stage of crucial importance for the optimal development of avian embryos. For most birds, incubation poses a trade‐off between investing in self‐maintenance and offspring care. Furthermore, incubation is affected by environmental temperatures and, therefore, will be likely impacted by climate change. Despite its relevance and readily available temperature logging methods, avian incubation research is hindered by recognised limitations in available software. In this paper, a new quantitative approach to analyse incubation behaviour is presented. This new approach is embedded in a free R package, incR. The flexibility of the R environment eases the analysis, validation and visualisation of incubation temperature data. The core algorithm in incR is validated here and it is shown that the method extracts accurate metrics of incubation behaviour (e.g. number and duration of incubation bouts). This paper also presents a suggested workflow along with detailed R code to aid the practical implementation of incR.     

Assessing the short‐term effects of capture,handling and tagging of sandgrouse

Capturing and marking free‐living birds permits the study of important aspects of their biology but may have undesirable effects. Bird welfare should be a primary concern, so it is necessary to evaluate and minimize any adverse effects of procedures used. We assess short‐term effects associated with the capture, handling and tagging with backpack‐mounted transmitters of Pin‐tailed Pterocles alchata and Black‐bellied Pterocles orientalis Sandgrouse, steppe birds of conservation concern. There was a significantly higher mortality (15%) during the first week after capture than during the following weeks (< 2.5%) in Pin‐tailed Sandgrouse, but no significant temporal mortality pattern in Black‐bellied Sandgrouse. In Pin‐tailed Sandgrouse, mortality rate during the first week increased with increasing relative transmitter and harness weight regardless of season, and with increasing handling time during the breeding season. There were no significant differences in mortality rate between study areas, type of tag, sex or age or an effect of restraint time. These results suggest the use of lighter transmitters (< 3% of the bird's weight) and a reduction of handling time (< 20 min), particularly during the breeding season, as essential improvements in procedure to reduce the mortality risk associated with the capture, handling and tagging of these vulnerable species.