Faecalicatena faecalis sp. nov., a moderately alkaliphilic bacterial strain isolated from swine faeces

An obligately anaerobic, Gram-stain-positive, non-motile, non-spore-forming and rod-shaped strain AGMB00832^T was isolated from swine faeces. Phylogenetic analysis based on the 16S rRNA gene, together with the housekeeping genes, gyrB and rpoD, revealed that strain AGMB00832^T belonged to the genus Faecalicatena and was most closely related to Faecalicatena orotica KCTC 15331^T. In biochemical analysis, strain AGMB00832^T was shown to be negative for catalase, oxidase and urease. Furthermore, the isolate was positive for β-glucosidase, β-glucuronidase, glutamic acid decarboxylase, proline arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase. The major cellular fatty acids (>?10%) of the isolate were C_14:0, C_16:0 and C_18:1ω11t DMA. Based on the whole genome sequence analysis, the DNA G?+?C content of strain AGMB00832^T was 44.2 mol%, and the genome size and numbers of rRNA and tRNA genes were 5,175,159 bp, 11 and 53, respectively. The average nucleotide identity and digital DNA–DNA hybridization values between strain AGMB00832^T and related strains were ≤?77.4 and 22.5%, respectively. Furthermore, the genome analysis revealed the presence of genes for alkaline shock protein 23 and cation/proton antiporters, which may facilitate growth of strain AGMB00832^T in alkaline culture condition. On the basis of polyphasic taxonomic approach, strain AGMB00832^T represents a novel species within the genus Faecalicatena, for which the name Faecalicatena faecalis sp. nov. is proposed. The type strain is AGMB00832^T (=?KCTC 15946^T?=?NBRC 114613^T).

     

Application of Biofilm-Forming Bacteria on the Enhancement of Organophosphorus Fungicide Degradation

This study aimed to develop technology enhancing the biodegradation efficacy against organophosphorus fungicide with biofilm-forming bacteria in situ. Using the crystal violet staining method, two bacterial strains having biofilm formation capability were isolated and identified as Pseudomonas sp. C7 and Bacillus sp. E5. Compared with the culture of tolclofos-methyl degrader Sphingomonas sp. 224, biofilm formation was improved by co-inoculation with biofilm-forming bacterium Bacillus sp. E5. Evaluated in liquid culture conditions, this two-species mixed consortium was observed to degrade tolclofos-methyl more effectively than Sphingomonas sp. 224 alone, with an approximately 90% degradation efficiency within 48 h of dosing. The improved effectiveness of the consortium biofilm was reflected using soil in situ with an approximately 7% increased degradation ratio over Sphingomonas sp. 224 alone. This is the first report demonstrating improved bioremediation degradation efficacy against tolclofos-methyl exhibited by a consortium biofilm. This work presents a possible effective bioremediation strategy using a specific biofilm composition against pollutants containing organophosphorus compounds in situ.     

Combination of 1,4-naphthoquinone with benzothiazoles had selective algicidal effects against harmful algae

A series of naphthoquinone-benzothiazole conjugates were synthesized as algicides, and their efficacies against harmful algal blooming species, such as Chattonella marina, Heterosigma akashiwo and Cochlodinium polykrikoides, were examined. The introduction of substituted benzothiazole at the C2 position of 1,4-naphthoquinone (compounds 1–9) resulted in higher algicidal activity against C. polykrikoides than the C6 conjugates (compounds 10–20). On the other hand, of the C6 conjugates, compounds 11 and 12 exhibited better algicidal activity against H. akashiwo, C. marina, and C. polykrikoides than the C2 conjugates. Further structure-activity analysis indicated that a replacement of the methoxy groups with hydroxyl groups (compounds 21–26) decreased the algicidal activity significantly. Among the various synthetic naphthoquinonebezothiazole conjugates tested, compound 12 was found to affect the most significant decrease in the level of C. polykrikoides growth, with an IC₅₀ of 0.19 μM. Compound 11 was found to be the most potent inhibitor against H. akashiwo and C. polykrikoides, with IC₅₀ values of 0.32 and 0.12 μM, respectively. Overall, these results highlight a possible method for controlling and inhibiting red tide forming algae using NQ derivatives.     

In vitro antiviral activity of phlorotannins isolated from Ecklonia cava against porcine epidemic diarrhea coronavirus infection and hemagglutination

Despite the prepdominat agent causing severe entero-pathogenic diarrhea in swine, there are no effective therapeutical treatment of porcine epidemic diarrhea virus (PEDV). In this study, we evaluated the antiviral activity of five phlorotannins isolated from Ecklonia cava (E. cava) against PEDV. In vitro antiviral activity was tested using two different assay strategies: (1) blockage of the binding of virus to cells (simultaneous-treatment assay) and (2) inhibition of viral replication (post-treatment assay). In simultaneous-treatment assay, compounds 2–5 except compound 1 exhibited antiviral activities of a 50% inhibitory concentration (IC₅₀) with the ranging from 10.8 ± 1.4 to 22.5 ± 2.2 μM against PEDV. Compounds 1–5 were completely blocked binding of viral spike protein to sialic acids at less than 36.6 μM concentrations by hemagglutination inhibition. Moreover, compounds 4 and 5 of five phlorotannins inhibited viral replication with IC₅₀ values of 12.2 ± 2.8 and 14.6 ± 1.3 μM in the post-treatment assay, respectively. During virus replication steps, compounds 4 and 5 exhibited stronger inhibition of viral RNA and viral protein synthesis in late stages (18 and 24 h) than in early stages (6 and 12 h). Interestingly, compounds 4 and 5 inhibited both viral entry by hemagglutination inhibition and viral replication by inhibition of viral RNA and viral protein synthesis, but not viral protease. These results suggest that compounds isolated from E. cava have strong antiviral activity against PEDV, inhibiting viral entry and/or viral replication, and may be developed into natural therapeutic drugs against coronavirus infection.     

Induction of MiR-21 by Stereotactic Body Radiotherapy Contributes to the Pulmonary Fibrotic Response

Radiation-induced lung fibrosis, the most serious effect of lung cancer radiotherapy on normal tissue, remains a major technical obstacle to the broader application of radiotherapy to patients with lung cancer. This study describes the use of an image-guided irradiation system in mice mimicking stereotactic body radiotherapy (SBRT) to examine the molecular features of chronic fibrotic response after radiation injury. MicroRNA (miR) array analysis of injured pulmonary tissue identified a set of miRs whose expression was significantly increased in damaged lung tissue. In particular, miR-21 expression was increased at the radiation injury site, concurrent with collagen deposition. Although the inhibition of miR-21 by its specific inhibitor anti-miR-21 only marginally affected endothelial-mesenchymal transition (EndMT) in lung endothelial cells, this inhibition significantly reduced collagen synthesis in lung fibroblasts. Furthermore, ectopic expression of miR-21 was sufficient to promote a fibrotic response in lung fibroblasts, enhancing Smad2 phosphorylation concurrent with Smad7 downregulation. These findings indicate that the induction of miR-21 expression is responsible for fibrotic responses observed in mesenchymal cells at the injury site through the potentiation of TGF-β signaling. Local targeting of miR-21 at the injured area could have potential therapeutic utility in mitigating radiation-induced lung fibrosis.     

Production and characterization of keratinase from<Emphasis Type="Italic">Paracoccus</Emphasis> sp. WJ-98

A bacterial strain WJ-98 found to produce active extracellular keratinase was isolated from the soil of a poultry factory. It was identified asParacoccus sp. based on its 16S rRNA sequence analysis, morphological and physiological characteristics. The optimal culture conditions for the production of keratinase byParacoccus sp. WJ-98 were investigated. The optimal medium composition for keratinase production was determined to be 1.0% keratin, 0.05% urea and NaCl, 0.03% K₂HPO₄, 0.04% KH₂PO₄, and 0.01% MgCl₂·6H₂O. Optimal initial pH and temperature for the production of keratinase were 7.5 and 37°C, respectively. The maximum keratinase production of 90 U/mL was reached after 84 h of cultivation under the optimal culturing conditions. The keratinase fromParacoccus sp. WJ-98 was partially purified from a culture broth by using ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, followed by gel filtration chromatography on Sephadex G-75. Optimum pH and temperature for the enzyme reaction were pH 6.8 and 50°C, respectively and the enzymes were stable in the pH range from 6.0 to 8.0 and below 50°C. The enzyme activity was significantly inhibited by EDTA, Zn²⁺ and Hg²⁺. Inquiry into the characteristics of keratinase production from these bacteria may yield useful agricultural feed processing applications.     

Reactive oxygen species modulates the intracellular level of HBx viral oncoprotein

HBx (hepatitis B virus X) viral oncoprotein is a multifunctional protein of which the cellular level may be one of the important factors in determining HBV-mediated pathological progression of liver diseases, chronic hepatitis, and hepatocellular carcinoma. Our previous work revealed that adriamycin, a chemotherapeutic agent, caused a marked increase in the intracellular level of HBx by retarding its rapid degradation. In the present study, modulation of HBx expression was found to be confined to adriamycin but not to other chemotherapeutic agents, cisplatin and 5-fluorouracil. Interestingly, adriamycin caused a rapid increase of reactive oxygen species (ROS) and its accumulation continued until 24h. In contrast, two other agents had little effect on ROS generation, suggesting the possible involvement of ROS in the HBx regulation. In fact, direct addition of H(2)O(2) to the cells significantly increased the level of HBx protein in HBx-expressing ChangX-34 cells as well as in hepatitis B virus-related hepatoma cells, PLC/PRF/5 and HepG2.2.15 cells. Furthermore, antioxidants, N-acetyl-cysteine and pyrrolidinedithiocarbamate (PDTC), completely abolished the increase of HBx protein induced by adriamycin, indicating that adriamycin modulates the intracellular HBx level via ROS generation. Together, these findings provide a novel aspect of HBx regulation by cellular ROS level. Therefore, intracellular microenvironments generating ROS such as severe inflammation may aggravate the pathogenesis of liver disease by accumulating the HBx level.     

Production of tagatose by a recombinant thermostable L-arabinose isomerase from Thermus sp. IM6501

A gene (thaI) corresponding to l-arabinose isomerase from Thermus strain IM6501 was cloned by PCR. It comprised 1488 nucleotides and encoded a polypeptide of 496 residues with a predicted molecular weight of 56019 Da. The deduced amino acid sequence had 96.8% identity with the l-arabinose isomerase of Geobacillus stearothermophilus. Recombinant ThaI with N-terminal hexa-tistidine tags was over-expressed in Escherichia coli and purified by affinity chromatography using Ni-NTA resin. The purified ThaI was thermostable with maximal activity at 60°C at pH 8 for 30 min of reaction. Zn²⁺ and Ni²⁺ inactivated the catalytic activity of ThaI, 5 mM Mn²⁺ enhanced the bioconversion yield by 90%. The bioconversion yield of 54% from d-galactose to d-tagatose was obtained by recombinant ThaI at 60°C over 3 d.