Microbiological Identification and Analysis of Swine Lungs Collected from Carcasses in Swine Farms, China

The primary objective of this 3 years study was to determine the prevalence of porcine pathogens of the lungs of swine in swine farms in southern China. A total of 5,420 samples were collected from 200 swine farms. The bacterium that was most commonly isolated was Streptococcus suis, with 10.24 % of the samples being positive, 114 lungs (2.1 %) were positive for pseudorabies virus and 263 (4.85 %) were positive for classical swine fever virus; much lower than positive for PRRSV (15.1 %, p = 0.023) and PCV2 (13.8 %, p = 0.038). lungs that were positive for PRRSV and/or PCV-2 have significantly increased odds of being positive for any of the S. suis (9.79 vs. 0.44 %, p = 0.003).     

Membrane Damage Induced by Supercritical Carbon Dioxide in Rhodotorula mucilaginosa

To clarify the mechanism of microbial inactivation by supercritical carbon dioxide (SCCO₂), membrane damage of Rhodotorula mucilaginosa was investigated within specific pressure (10 Mpa), temperature (37 °C), and treatment time (10–70 min) ranges, including cell morphological structure, membrane permeability and fluidity. SEM and TEM observations showed morphological changes in the cell envelope and intracellular organization after SCCO₂ treatment. Increase of membrane permeability was measured as increased uptake of the trypan blue dye with microscopy, and leakage of intracellular substances such as UV-absorbing materials and ions by determining the change of protein and electrical conductivity. The SCCO₂ mediated reduction in CFU ml⁻¹ was 0.5–1 log higher at 37 °C and 10 MPa for 60 min in Rose Bengal Medium containing 4 % sodium than a similar treatment in Rose Bengal Medium. Membrane fluidity analyzed by fluorescence polarization method using 1,6-diphenyl-1,3,5-hexatriene showed that the florescence polarization and florescence anisotropy of the SCCO₂-treated cells were increased slightly and gently compared with the untreated cells. The correlation between membrane damage and death of cells under SCCO₂ was clear, and the membrane damage was a key factor induced the inactivation of cells.     

Aggregate cell suspension cultures of Tripterygium wilfordii Hook. f. for triptolide, wilforgine, and wilforine production

The relationships between aggregate cell types, cell growth, and the triptolide, wilforgine, and wilforine content in aggregate cell suspension cultures of Tripterygium wilfordii Hook. f. were examined. Aggregate cells larger than 2?mm grew quickly and constituted the majority of the white aggregates. The accumulation of triptolide was strongly correlated with the size of the aggregates and the length of the culture period. The aggregates 0.5?C2?mm in diameter accumulated higher triptolide content than those with other sizes throughout the culture. However, the size of the aggregate cells did not significantly affect on the wilforgine and wilforine content. Two other kinds of aggregate cells, the brown and green aggregate cells, also formed in the suspension cultures. The smallest aggregates (0.1?C0.5?mm) had a lower biomass and growth rate and had more chloroplasts and higher alkaloid content. The results of this study can be used to improve the selection process for the mass production of triptolide, wilforgine, and wilforine from cell suspension cultures.     

Interactions between soybean ABA receptors and type 2C protein phosphatases

The plant hormone abscisic acid (ABA) plays important roles in regulating plant growth, development, and responses to environmental stresses. Proteins in the PYR/PYL/RCAR family (hereafter referred to as PYLs) are known as ABA receptors. Since most studies thus far have focused on Arabidopsis PYLs, little is known about PYL homologs in crop plants. We report here the characterization of 21 PYL homologs (GmPYLs) in soybean. Twenty-three putative GmPYLs can be found from soybean genome sequence and categorized into three subgroups. GmPYLs interact with AtABI1 and two GmPP2Cs in diverse manners. A lot of the subgroup I GmPYLs interact with PP2Cs in an ABA-dependent manner, whereas most of the subgroup II and III GmPYLs bind to PP2Cs in an ABA-independent manner. The subgroup III GmPYL23, which cannot interact with any of the tested PP2Cs, differs from other GmPYLs. The CL2/gate domain is crucial for GmPYLs-PP2Cs interaction, and a mutation in the conserved proline (P109S) abolishes the interaction between GmPYL1 and AtABI1. Furthermore, the ABA dependence of GmPYLs-PP2Cs interactions are partially correlated with two amino acid residues preceding the CL2/gate domain of GmPYLs. We also show that GmPYL1 interacts with AtABI1 in an ABA-dependent manner in plant cells. Three GmPYLs differentially inhibit AtABI1 and GmPP2C1 in an ABA-dependent or -enhanced manner in vitro. In addition, ectopically expressing GmPYL1 partially restores ABA sensitivity of the Arabidopsis triple mutant pyr1/pyl1/pyl4. Taken together, our results suggest that soybean GmPYLs are ABA receptors that function by interacting and inhibiting PP2Cs.     

The importance of denitrification for N2O emissions from an N-saturated forest in SW China: results from in situ 15N labeling experiments

Long-term elevated atmogenic deposition (~5 g m^?2 year^?1) of reactive nitrogen (N) causes N saturation in forests of subtropical China which may lead to high nitrous oxide (N₂O) emissions. Recently, we found high N₂O emission rates (up to 1,730 μg N₂O–N m^?2 h^?1) during summer on well-drained acidic acrisols (pH = 4.0) along a hill slope in the forested Tieshanping catchment, Chongqing, southwest China. Here, we present results from an in situ ¹⁵N–NO₃ ^? labeling experiment to assess the contribution of nitrification and denitrification to N₂O emissions in these soils. Two loads of 99 at.% K¹⁵NO₃ (equivalent to 0.2 and 1.0 g N m^?2) were applied as a single dose to replicated plots at two positions along the hill slope (at top and bottom, respectively) during monsoonal summer. During a 6-day period after label application, we found that 71–100 % of the emitted N₂O was derived from the labeled NO₃ ^? pool irrespective of slope position. Based on this, we assume that denitrification is the dominant process of N₂O formation in these forest soils. Within 6 days after label addition, the fraction of the added ¹⁵N–NO₃ ^? emitted as ¹⁵N–N₂O was highest at the low-N addition plots (0.2 g N m^?2), amounting to 1.3 % at the top position of the hill slope and to 3.2 % at the bottom position, respectively. Our data illustrate the large potential of acid forest soils in subtropical China to form N₂O from excess NO₃ ^? most likely through denitrification.     

Poly-γ-glutamate from Bacillus subtilis inhibits tyrosinase activity and melanogenesis

Poly-γ-glutamate (γ-PGA) has been considered as one of the most promising biomaterials with a wide range of applications, but there has been no report that directly shows the anti-tyrosinase and anti-melanogenesis properties of γ-PGA. In the present study, we investigated the inhibitory effects of γ-PGA with low molecular weight (Mw; lγ-PGA) and high Mw (hγ-PGA) on mushroom tyrosinase and murine tyrosinase activities and on melanogenesis in B16 melanoma cells. First, we showed that both lγ-PGA and hγ-PGA could effectively inhibit mushroom tyrosinase activities including monophenolase and diphenolase activities in a dose-dependent manner. Second, both lγ-PGA and hγ-PGA showed strong anti-tyrosinase activity and anti-melanogenesis in B16 melanoma cells. Third, both lγ-PGA and hγ-PGA inhibited forskolin-induced tyrosinase activity and melanogenesis by decreasing the levels of intracellular reactive oxygen species and nitric oxide while increasing the catalase activity in B16 cells. This is the first report on the anti-melanogenesis effect of γ-PGA, which suggests that γ-PGA could have a potential in the cosmetic skin whitening business, therapeutic applications and the food industry.     

Purification of clinical-grade disulfide stabilized antibody fragment variable—Pseudomonas exotoxin conjugate (dsFv-PE38) expressed in Escherichia coli

Immunotoxins are rationally designed cancer targeting and killing agents. Disulfide stabilized antibody Fv portion—toxin conjugates (dsFv-toxin) are third generation immunotoxins containing only the antibody fragment variable portions and a toxin fused to the V_H or V_L. Pseudomonas exotoxin fragment (PE-38) is a commonly used toxin in immunotoxin clinical trials. dsFv-toxin purification was previously published, but the recovery was not satisfactory. This report describes the development of a cGMP production process of the dsFv-toxin that incorporated a novel purification method. The method has been successfully applied to the clinical manufacturing of two dsFv-PE38 immunotoxins, MR1-1 targeting EGFRvIII and HA22 targeting CD22. The two subunits, V_L and V_H PE-38 were expressed separately in Escherichia coli using recombinant technology. Following cell lysis, inclusion bodies were isolated from the biomass harvested from fermentation in animal source component-free media. The dsFv-toxin was formed after denaturation and refolding, and subsequently purified to homogeneity through ammonium sulfate precipitation, hydrophobic interaction and ion-exchange chromatography steps. It was shown, in a direct comparison experiment using MR1-1 as model protein, that the recovery from the new purification method was improved three times over that from previously published method. The improved recovery was also demonstrated during the clinical production of two dsFv-PE38 immunotoxins—MR1-1 and HA22.     

Degradation of paracetamol by pure bacterial cultures and their microbial consortium

Three bacterial strains utilizing paracetamol as the sole carbon, nitrogen, and energy source were isolated from a paracetamol-degrading aerobic aggregate, and assigned to species of the genera Stenotrophomonas and Pseudomonas. The Stenotrophomonas species have not included any known paracetamol degraders until now. In batch cultures, the organisms f1, f2, and fg-2 could perform complete degradation of paracetamol at concentrations of 400, 2,500, and 2,000 mg/L or below, respectively. A combination of three microbial strains resulted in significantly improved degradation and mineralization of paracetamol. The co-culture was able to use paracetamol up to concentrations of 4,000 mg/L, and mineralized 87.1 % of the added paracetamol at the initial of 2,000 mg/L. Two key metabolites of the biodegradation pathway of paracetamol, 4-aminophenol, and hydroquinone were detected. Paracetamol was degraded predominantly via 4-aminophenol to hydroquinone with subsequent ring fission, suggesting new pathways for paracetamol-degrading bacteria. The degradation of paracetamol could thus be performed by the single isolates, but is stimulated by a synergistic interaction of the three-member consortium, suggesting a possible complementary interaction among the various isolates. The exact roles of each of the strains in the consortium need to be further elucidated.