Regulated virulence controls the ability of a pathogen to compete with the gut microbiota

The virulence mechanisms that allow pathogens to colonize the intestine remain unclear. Here, we show that germ-free animals are unable to eradicate Citrobacter rodentium, a model for human infections with attaching and effacing bacteria. Early in infection, virulence genes were expressed and required for pathogen growth in conventionally raised mice but not germ-free mice. Virulence gene expression was down-regulated during the late phase of infection, which led to relocation of the pathogen to the intestinal lumen where it was outcompeted by commensals. The ability of commensals to outcompete C. rodentium was determined, at least in part, by the capacity of the pathogen and commensals to grow on structurally similar carbohydrates. Thus, pathogen colonization is controlled by bacterial virulence and through competition with metabolically related commensals.     

Involvement of TNF-α and MAPK pathway in the intramammary MMP-9 release via degranulation of cow neutrophils during acute mammary gland involution

Neutrophil-derived MMP-9 activity is regulated more promptly and efficiently at the level of degranulation than at other levels of regulation. In human neutrophils, degranulation is one of the earliest responses to TNF-α stimulation, which involves protein kinase C and mitogen-activating protein kinase (MAPK) pathways. The level of MMP-9 in mammary secretion of cows increases drastically following milk-stasis, which is partially explained by increases of both neutrophil infiltration and neutrophil degranulation per se. Since MMP-9 represents one of the major remodeling capacities in the mammary gland of cows during early dry period, the current study attempted to explore the involved intracellular mechanisms in the up-regulated MMP-9 secretion. We repeatedly measured on the somatic cells of mammary secretion along the early dry period of cows the expression of TNF-α protein and the phosphorylation of p38 MAPK, ERK, and JNK. Also, cultures of bovine peripheral neutrophils were conducted to examine the mode of short-term MMP-9 secretion in response to TNF-α stimulation and the blocking effects of TNF-α antibody and inhibitors of MAPK pathways. Ex vivo measurements show that conventional cow milk has fully transformed into a neutrophil-abundant, lactoferrin-rich, and high-MMP-9 mammary secretion by d 7 in milk-stasis. No significant (P>0.05) change, however, was found in the expression of TNF-α or the phosphorylation extent of MAPK pathway intermediates on the somatic cells of mammary secretion during the first 3 weeks in milk-stasis. In vitro studies indicate linear increase of short-term MMP-9 release in response to TNF-α stimulation in dosages between 0.1 and 10 ng/ml. In the presence of preparations of d 7-dry secretion of cows, the short-term release of MMP-9 from bovine peripheral neutrophils was significantly (P<0.05) blocked by inhibitor of p38 MAPK but was significantly (P<0.05) promoted by ERK inhibitor while TNF-α antibody or JNK inhibitor exerted no effect. In conclusion, the current ex vivo measurements suggest no apparent association of TNF-α and MAPK pathway with long term intramammary accumulation of MMP-9 during the early dry period of cows, whereas cultures of bovine peripheral neutrophils under a simulated acute involution intramammary environment of cows suggest a role played by TNF-α and MAPK pathways in the short-term MMP-9 release via degranulation.     

Porcine MHC classical class I genes are coordinately expressed in superantigen-activated mononuclear cells

The expression of the major histocompatibility complex (MHC) classical class I genes is important for the adaptive immune response to target virus-infected cells and cancer cells. The up-regulation of the MHC is achieved by hormonal/cytokine signals including IFN-γ-inducible elements. The swine leukocyte antigen (SLA), the MHC class I region of pigs, consists of the duplicated classical class I genes, SLA-1, SLA-2 and SLA-3, but the molecular mechanisms involved in their up-regulation after T cell stimulation have not been fully elucidated. In order to better understand some of the putative regulatory mechanisms of SLA class I gene expression in activated T cells, we examined the coordinated expression of the SLA classical class I, IFN-γ and interferon regulatory factor-1 (IRF-1) genes in the peripheral blood mononuclear cells (PBMCs) of SLA homozygous Clawn miniature swine stimulated for 72h with either IFN-γ or an enterotoxin produced by Staphylococcus aureus. This enterotoxin, toxic shock syndrome-1 (TSST-1), is known to act as a superantigen (sAG) to activate the T cells in various vertebrate species. We showed by using mAbs and flow cytometry that the CD4(+)CD25(+) cell number of swine PBMCs was also increased by TSST-1 and to a lesser degree by IFN-γ. Time course analyses of the expression of the IFN-γ, IRF-1 and the three classical class I genes, SLA-1, SLA-2, and SLA-3, in PBMCs by quantitative real-time PCR revealed a transitory response to TSST-1 or IFN-γ stimulation. The IFN-γ mRNA levels in the PBMCs were continuously up-regulated over the first 48h by TSST-1 or IFN-γ. In contrast, SLA class I expression moderately increased at 24h and then decreased to a baseline level or less at 72h of IFN-γ or TSST-1 stimulation. The three classical SLA class I genes showed similar expression kinetics, although SLA-3 mRNA level was consistently lower than those of SLA-1 and -2. The expression of IRF-1, a modulator of SLA expression, showed similar kinetics to those of the three classical SLA class I genes. The expression profiles detected by flow cytometry of the SLA molecules on the cell surface of PBMCs were maintained at a consistently high level during cell stimulation with either TSST-1 or IFN-γ, which was distinct from the kinetics of mRNA expression. These results showed that miniature swine SLA class I mRNA expression was effectively and equally up-regulated among the three loci and coordinately with IRF-1 gene expression after stimulation of T cell activation by sAG or IFN-γ.     

Development and characterization of mouse monoclonal antibodies reactive with chicken CD83

This study was carried out to develop and characterize mouse monoclonal antibodies (mAbs) against chicken CD83 (chCD83), a membrane-bound glycoprotein belonging to the immunoglobulin superfamily that is primarily expressed on mature dendritic cells (DCs). A recombinant chCD83/IgG4 fusion protein containing the extracellular region of chCD83 was expressed in Chinese Hamster Ovary (CHO) cells and isolated from the spent cell culture medium by protein G affinity chromatography. The extracellular region of the chCD83 protein was purified and used to immunize mice. A cell fusion was performed, from which 342 hybridomas were screened for mAbs to chCD83. Two mAbs, chCD83-159 and chCD83-227, stained the greatest percentage of chCD83-transfected CHO cells and were selected for further characterization. By flow cytometry, both mAbs reacted with a chicken macrophage cell line, HD11. Both mAbs also recognized a single 53 kDa protein on Western blots of lysates from lipopolysaccharide-stimulated spleen mononuclear cells or unstimulated HD11 cells. Immunostaining of chicken secondary lymphoid organs identified chCD83(+) cells with morphologic and subtissue localization properties comparable to mammalian DCs. In vitro stimulation of spleen mononuclear cells with concanavalin A (Con A) decreased the percentage of chCD83(+) cells compared with cells treated with medium alone. Interestingly, spleen cells treated with Con A in the presence of chCD83-227 mAb exhibited decreased percentage of MHCII(+) cells compared with cells treated with an isotype-matched negative control mAb. These chCD83 mAbs may be useful for future investigations of chicken immune cell maturation and mechanisms of action.     

Participatory agroforestry development for restoring degraded sloping land in DPR Korea

Participatory approaches in agroforestry combine land, labor, and knowledge, by blending local experience with external expert support for sloping land restoration. We describe and analyze over a decade of bottom-up agroforestry development processes that today are influencing national policies. In the 1990s, after economic upheaval following the collapse of trade with the USSR (Soviet Union) rapid conversion of sloping lands to agriculture, in association with heavy rainfall events, caused widespread erosion and landslides. In response, pilot scale ??user groups?? obtained rights-to-use, rights-to-harvest and rights-to-plan or access to sloping lands for tree products and food. All three rights were novel in the DPR Korea and jointly contributed to success, together with active research support. Innovations in double-cropping annual food crops together with non-competitive contour strips of valuable fruits (aronia berry: Aronia melanocarpa) and/or high-value timber (larch: Larix leptolepis) emerged as preferred local agroforestry systems. Broad support for agroforestry practices has now emerged within the Ministry of Land and Environmental Protection as well as a number of universities and research centres. Further development will require increased engagement with agricultural and horticultural agencies, while the social dimensions of participatory agroforestry continue to provide rich learning.     

Computed tomography and radiographic lymphography of the thoracic duct by subcutaneous or submucosal injection

A simple method of lymphography of the thoracic duct was investigated. Using three female beagles, contrast media were administered rectally, vaginally and into the perianal tissue. The administration sites were gently massaged, and imaging was carried out at constant intervals using computed tomography and radiograph. Moreover, Indian ink was administered into the rectum mucous membrane in dogs for proof of this method of lymphography, and the lymph drainage routes were observed. The investigation showed that clear computed tomography and radiographic contrast images of the thoracic duct were obtained by subcutaneous and submucosa injection of angiography contrast medium and 3D processing of these images revealed the three-dimensional positions and course of the thoracic duct and cisterna chyli.     

The effects of replacement of antibiotics with by‐products of oriental medicinal plants on growth performance and meat qualities in fattening pigs

The effect of by‐products of oriental medicinal plants (OMP; T1) containing 0.03% herb extracts (T2) or 0.1% aminolevulinic acid (T3) on the production performance of swine during the finishing period and on its meat quality were investigated. No significant differences were found in the weight gain, feed intake and feed conversion rate among the tested groups (P > 0.05). But the treated group showed higher (P < 0.05) moisture and ash and lower protein than the control group. The T3 group showed a lower meat cholesterol content (38.42 mg/100 g) compared to the other groups (P < 0.05). The vitamin E content of the muscle in the treated groups was higher compared to the control group. No antibiotic content was detected in all treated and control samples. The values of the volatile basic nitrogen (VBN) and thiobarbituric acid reactive substance (TBARS) of the treated groups were significantly lower (P < 0.01) than the control group. The treated groups had significantly better (P < 0.05) sensory‐test scores for color, flavor, off‐flavor and total acceptability compared to the control group.     

Changes in the anatomy of surface and liquid uptake of wood after laser incising

The effects of laser incising on the surface of heat-affected zones (HAZs) and liquid uptake of wood were examined. Deep pin holes were incised on the tangential section of the wood with a laser beam of 1 kW in power with varied pulse widths, and the HAZs of the holes were investigated using scanning electron microscopy. The length of liquid uptake passing through HAZs surfaces was then measured. It was observed that the HAZs were unable to maintain normal porous structure, and smooth surfaces such as that of wood charcoal were also not observed. This is because the composite of the middle lamella completely melted, which led to a decrease in the length of liquid uptake. In addition, the release of high gas-pressure and thermal energy during the laser-incising process did not damage the tylose and the aspirated pit in the longitudinal direction. Because of this, the liquid movement remained blocked. However, the incising holes provided additional liquid-intake points, resulting in greater intake capacity. Moreover, it was observed that improvement of liquid impregnation is related to hole diameter.     

Cellulose nanowhisker-incorporated poly(lactic acid) composites for high thermal stability

In this study, biodegradable composites were prepared using cellulose nanowhiskers and poly(lactic acid). For processing at high temperature over 200 °C, cellulose nanowhiskers were prepared by ultra-sound treatment, with the high thermal stability of natural cellulose. The nanowhiskers were confirmed using transmission electron microscopy, X-ray diffraction, and thermo-gravimetric analysis. Surface modification of the cellulose nanowhiskers was performed to increase the adhesion between hydrophilic nanofillers and hydrophobic polymer matrix. The dynamic mechanical thermal analysis of the composites showed better reinforcing effect of the modified cellulose nanocrystals. The effects of cellulose nanowhiskers on the biodegradability of poly(lactic acid) were studied using a microbial oxidative degradation analyzer.     

Colorimetric detection of transition metal ions with azopyridine-based probing molecule in aqueous solution and in PMMA film

We report on an azopyridine derivative for probing transition metal ions and fabrication of its films. The probe, AP, showed a sensitive absorption change toward transition metal ions, especially cobalt ion, even at the concentration of ppm range, accompanied by yellow-to-red color transition with noticeable isosbestic point. In addition, the AP-containing PMMA film with high transparency can be fabricated by spin-casting without any aggregation of AP. The film with PMMA matrix shows good sensitivity toward cobalt ion similar to the case in the solution with a feature of metallochromic transition.