Particulate matter-mediated oxidative stress induces airway inflammation and pulmonary dysfunction through TXNIP/NF-κB and modulation of the SIRT1-mediated p53 and TGF-ß/Smad3 pathways in mice.

Exposure to particulate matter is currently recognized as a serious aggravating factor of respiratory diseases. In this study, we investigated the effects of particulate matter (PM) on the respiratory system in BALB/c mice and NCI-H292 cells. PM (0, 2.5, 5 and 20 mg/kg) was administered to mice by intra-tracheal instillation for 7 days. After a 7 day-repeated treatment of PM, we evaluated inflammatory cytokines/cell counts in bronchoalveolar lavage fluid (BALF) and conducted pulmonary histology and functional test. We also investigated the role of TXNIP/NF-κB and SIRT1-mediated p53 and TGF-ß/Smad3 pathways in PM-induced airway inflammation and pulmonary dysfunction. PM caused a significant increase in pro-inflammatory cytokines, inflammatory cell counts in bronchoalveolar lavage fluid. PM-mediated oxidative stress down-regulated thioredoxin-1 and up-regulated thioredoxin-interacting protein and activation of nuclear factor-kappa B in the lung tissue and PM-treated NCI-H292 cells. PM suppressed sirtuin1 protein levels and increased p53 acetylation in PM-exposed mice and PM-treated NCI-H292 cells. In addition, PM caused inflammatory cell infiltration and the thickening of alveolar walls by exacerbating the inflammatory response in the lung tissue. PM increased levels of transforming growth factor-ß, phosphorylation of Smad3 and activation of α-smooth muscle actin, and collagen type1A2 in PM-exposed mice and PM-treated NCI-H292 cells. In pulmonary function tests, PM exposure impaired pulmonary function resembling pulmonary fibrosis, characterized by increased resistance and elastance of the respiratory system, and resistance, elastance, and damping of lung tissues, whereas decreased compliance of the respiratory system, forced expired volume and forced vital capacity. Overall, PM-mediated oxidative stress caused airway inflammation and pulmonary dysfunction with pulmonary fibrosis via TXNIP pathway/NF-κB activation and modulation of the SIRT1-mediated TGF-ß/Smad3 pathways. The results of this study can provide fundamental data on the potential adverse effects and underlying mechanism of pulmonary fibrosis caused by PM exposure as a public health concern. Due to the potential toxicity of PM, people with respiratory disease must be careful with PM exposure.

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) terpolymer production from volatile fatty acids using engineered Ralstonia eutropha.

One of the advantages of microbial synthesis of polyhydroxyalkanoates (PHAs) is the production of diverse polymers with different properties by the addition of different monomers, such as 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx). Considering the number of possible variables, terpolymers can have more variations than copolymers. In this study, we aimed to synthesize the terpolymer P(3HB-co-3HV-co-3HHx) from volatile fatty acids such as propionate and butyrate using the recombinant Ralstonia eutropha strain (Re2133/pCB81), containing deletions in the phaB1, phaB2, and phaB3 genes, and overexpression of synthetic PHA operon (phaC2, phaA, phaJ). This strain produced terpolymers depending on the ratio of two different carbon sources, namely, propionic and butyric acids; however, wild type R. eutropha could not produce the same type of polymer. The incorporation of 3-hydroxyvalerate and 3-hydroxyhexanoate monomers was confirmed by gas chromatography and H-nuclear magnetic resonance spectroscopy, and the parameters affecting the terpolymer composition were obtained based on regression. In addition, the thermal analysis showed that this terpolymer has properties different from those of the copolymer, obtained from the same composition of volatile acids. Depending on the ratio of two volatile acids, the composition of the terpolymer can be regulated resulting in different properties.

Isolation and Characterization of Bifidobacterium longum subsp. longum BCBL-583 for Probiotic Applications in Fermented Foods

Recent human gut microbiome studies have supported that the genus Bifidobacterium is one of the most beneficial bacteria for human intestinal health. To develop a new probiotic strain for functional food applications, fourteen fecal samples were collected from healthy Koreans and the strain BCBL-583 was newly selected and isolated from a 25-year-old Korean woman's fecal sample using the selective medium for Bifidobacterium. Subsequent fructose-6-phosphate phosphoketolase (F6PPK) test and 16S rRNA gene sequencing analysis of the strain BCBL-583 confirmed that it belongs to B. longum subsp. longum. The stress resistance tests showed that it has oxygen and heat tolerance activities (5- and 3.9-fold increase for 24 h at 60 and 120 rpm, respectively; 78.61 ± 6.67% survival rate at 45°C for 24 h). In addition, gut environment adaptation tests revealed that this strain may be well-adapted in the gut habitat, with gastric acid/bile salt resistance (85.79 ± 1.53%, survival rate under 6 h treatments of gastric acid and bile salt) and mucin adhesion (73.72 ± 7.36%). Furthermore, additional tests including cholesterol lowering assay showed that it can reduce 86.31 ± 1.85% of cholesterol. Based on these results, B. longum BCBL-583 has various stress resistance for survival during food processing and environmental adaptation activities for dominant survival in the gut, suggesting that it could be a good candidate for fermented food applications as a new probiotic strain.

Sensitive change of iso-branched fatty acid (iso-15:0) in Bacillus pumilus PAMC 23174 in response to environmental changes.

In this study, the environmental adaptive metabolic processes were investigated using a psychrotrophic polar bacterium Bacillus pumilus PAMC 23174 in response to various temperatures and nutrients, especially in regard to the synthesis of fatty acids. Fatty acid methyl ester analysis was performed using gas chromatography-mass spectrometry and we found that a sensitive changes in iso-branched fatty acid (iso-15:0) synthesis occurred when adjusting the nutritional ratio of branched chain fatty acids (anteiso/iso) with different temperatures, resulting in a change in the balance of anteiso- and iso-form fatty acids. We also observed that this Arctic bacterium preferred amino acid leucine for the synthesis of fatty acids. The increased and decreased synthesis of iso-form fatty acids in response to different temperatures and leucine preference, changes the fatty acid ratio in bacteria, which further affects the membrane fluidity and it is also directly correlated with survival of bacteria in an extreme environment. Hence, this study suggests that B. pumilus PAMC 23174 is a potential model organism for the analysis of the unique ecological adaptations of polar bacteria in changing and the extreme environments.

Increased vulnerability to physical stress by inactivation of NdgR in Streptomyces coelicolor.

The antibiotic production and spore formation process in Streptomyces coelicolor need complex decision making processes by several regulatory units. These regulatory units are involved in both primary and secondary metabolism. As a result, most regulators have several functions, and those are worthwhile themes to study about different functions of a known regulator. In this study, a deletion mutant of ndgR, which encodes the nitrogen-dependent growth regulator, was examined by the cell viability test, TEM, and growth in N-acetylglucosamine/asparagine (GlcNAc/Asn) liquid medium. The results of the study show that NdgR is also involved in the structure of the cell membrane affecting survival under physical shocks. Deletion of ndgR leads to abnormal cell membrane resulting in the vulnerable cells to physical stress caused by shaking with beads in liquid culture condition. This empirical observation is the first meaningful explanation to why ndgR mutant could not grow well in a liquid minimal medium due to the defect of N-acetylglucosamine (GlcNAc) utilization and phospholipid synthesis.

Starch based polyhydroxybutyrate production in engineered Escherichia coli.

Every year, the amount of chemosynthetic plastic accumulating in the environment is increasing, and significant time is required for decomposition. Bio-based, biodegradable plastic is a promising alternative, but its production is not yet a cost effective process. Decreasing the production cost of polyhydroxyalkanoate by utilizing renewable carbon sources for biosynthesis is an important aspect of commercializing this biodegradable polymer. An Escherichia coli strain that expresses a functional amylase and accumulate polyhydroxybutyrate (PHB), was constructed using different plasmids containing the amylase gene of Panibacillus sp. and PHB synthesis genes from Ralstonia eutropha. This engineered strain can utilize starch as the sole carbon source. The maximum PHB production (1.24 g/L) was obtained with 2% (w/v) starch in M9 media containing 0.15% (w/v) yeast extract and 10 mM glycine betaine. The engineered E. coli SKB99 strain can accumulate intracellular PHB up to 57.4% of cell dry mass.

Lipase-Catalyzed Production of 6-O-cinnamoyl-sorbitol from D-sorbitol and Cinnamic Acid Esters.

To overcome the poor properties of solubility and stability of cinnamic acid, cinnamate derivatives with sugar alcohols were produced using the immobilized Candida antarctica lipase with vinyl cinnamate and D-sorbitol as substrate at 45 °C. Immobilized C. antarctica lipase was found to synthesize 6-O-cinnamoyl-sorbitol and confirmed by HPLC and (1)H-NMR and had a preference for vinyl cinnamate over other esters such as allyl-, ethyl-, and isobutyl cinnamate as co-substrate with D-sorbitol. Contrary to D-sorbitol, vinyl cinnamate, and cinnamic acid, the final product 6-O-cinnamoyl-sorbitol was found to have radical scavenging activity. This would be the first report on the biosynthesis of 6-O-cinnamoyl-sorbitol with immobilized enzyme from C. antarctica.

Production of rapamycin in Streptomyces hygroscopicus from glycerol-based media optimized by systemic methodology.

Rapamycin, produced by the soil bacterium Streptomyces hygroscopicus, has the ability to suppress the immune system and is used as an antifungal, anti-inflammatory, antitumor, and immunosuppressive agent. In an attempt to increase the productivity of rapamycin, mutagenesis of wild-type Streptomyces hygroscopicus was performed using ultraviolet radiation, and the medium composition was optimized using glycerol (which is one of the cheapest starting substrates) by applying Plackett-Burman design and response surface methodology. Plackett-Burman design was used to analyze 14 medium constituents: M100 (maltodextrin), glycerol, soybean meal, soytone, yeast extract, (NH4)2SO4, L-lysine, KH2PO4, K2HPO4, NaCl, FeSO4·7H2O, CaCO3, 2-(N-morpholino) ethanesulfonic acid, and the initial pH level. Glycerol, soytone, yeast extract, and CaCO3 were analyzed to evaluate their effect on rapamycin production. The individual and interaction effects of the four selected variables were determined by Box-Behnken design, suggesting CaCO3, soytone, and yeast extract have negative effects, but glycerol was a positive factor to determine rapamycin productivity. Medium optimization using statistical design resulted in a 45% (220.7 ± 5.7 mg/l) increase in rapamycin production for the Streptomyces hygroscopicus mutant, compared with the unoptimized production medium (151.9 ± 22.6 mg/l), and nearly 588% compared with wildtype Streptomyces hygroscopicus (37.5 ± 2.8 mg/l). The change in pH showed that CaCO3 is a critical and negative factor for rapamycin production.

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)) from butyrate using engineered Ralstonia eutropha.

Polyhydroxyalkanoates (PHAs), a promising family of bio-based polymers, are considered to be alternatives to traditional petroleum-based plastics. Copolymers like poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (P(HB-co-HHx)) have been shown to exhibit favorable physical and mechanical properties, due to decreased crystallinity resulting from the presence of medium-chain-length 3-hydroxyhexanoate (3HHx) monomers. In this study, we produced P(HB-co-HHx) using engineered Ralstonia eutropha strains containing deletions of the acetoacetyl-CoA reductase (phaB) genes and replacing the native PHA synthase with phaC2 from Rhodococcus aetherivorans I24 and by using butyrate, a short-chain organic acid, as the carbon source. Although the wild-type R. eutropha did not produce P(HB-co-HHx) when grown on mixed acids or on butyrate as the sole carbon source, we are able to produce polymer containing up to 40 wt% 3HHx monomer with the aforementioned engineered R. eutropha strains using various concentrations of just butyrate as the sole carbon source. This is the first report for the production of P(HB-co-HHx) copolymer in R. eutropha using butyrate.

Putative role of a Streptomyces coelicolor-derived α-mannosidase in deglycosylation and antibiotic production.

SCO0948 was found to be the single open reading frame annotated to encode an α-mannosidase (AM1) in Streptomyces coelicolor M145. To characterize the protein, we overexpressed SCO0948 in Escherichia coli BL21(DE3). Recombinant AM1, with a molecular weight of 110 kDa, exhibited α-mannosidase activity toward 4-nitrophenyl-α-D-mannopyranoside with a K m of 4.61 mM, a V(max) of 101.6 mM/min, and a specific activity of 47.96 U/mg. Treatment of ovalbumin, a glycoprotein, with AM1 resulted in partial deglycosylation, as assessed by glycostaining and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The S. coelicolor deletion mutant for SCO0948 failed to produce α-mannosidase activity, confirming AM1 as the only α-mannosidase in S. coelicolor M145. Interestingly, the deletion mutant and a complementation strain produced lower levels of the antibiotics actinorhodin and undecylprodigiosin in glucose minimal media. The results indicate that AM1 as an α-mannosidase influences deglycosylation and antibiotic production in S. coelicolor M145.

Functional analysis of the gene SCO1782 encoding Streptomyces hemolysin (S-hemolysin) in Streptomyces coelicolor M145.

In the process of evaluating the growth of Streptomyces coelicolor on rich media such as blood agar, we found that S. coelicolor a non-pathogenic, well-known antibiotic producer had the ability to grow and produce a prominent hemolytic zone. By comparing the growth with an agarase gene mutant of S. coelicolor, a similar prominent hemolytic zone was found to develop due to the organism's hemolytic activity. After the confirmation of hemolytic activity from S. coelicolor, the genome was searched for hemolysin-coding genes; consequently, SCO1782, SCO2534, and SCO3882 were identified, whose products were annotated as a putative, membrane, and hypothetical proteins, respectively. Functional characterization of all the recombinant proteins expressed in Escherichia coli BL21(DE3) revealed that only SCO1782 exhibited hemolytic activity. This S. coelicolor protein, designated as S-hemolysin, showed sequence similarity toward hemolysins from Brachyspira hyodysenteriae (35%) and Mycobacterium tuberculosis (62%). Recombinant hemolysin exhibited activity against sheep blood erythrocytes and cytolytic activity against human fibroblast cells. Deletion of SCO1782 resulted in complete loss of hemolysin activity in S. coelicolor.