Korea Research Institute of Bioscience and Biotechnology
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Enhanced production of 1-deoxynojirimycin in Bacillus subtilis subsp. inaquosorum by random mutagenesis and culture optimization
No full text1-Deoxynojirimycin (DNJ) is a potent inhibitor of α-glucosidase having antidiabetic and antiviral activities. In the present study, DNJ production by Bacillus subtilis subsp. inaquosorum KCTC 13429 (B. subtilis IWT) was confirmed and a mutant B. subtilis I.247 strain showing 50% increased DNJ production than wild-type strain after cultivation in 5% defatted soybean meal (DFS) for five days was isolated by UV random mutagenesis. The optimum culture conditions to maximize DNJ production by B. subtilis I.247 was predicted using response surface methodology to cultivate in medium containing 3.4% sorbitol and 2.4% yeast extract as carbon and nitrogen sources, respectively, at a temperature of 32°C. Under these conditions B. subtilis I.247 was able to produce 359 mg/L after five days of cultivation. Furthermore, when the B. subtilis I.247 transformant harboring a vector expressing a gabT1-yktc1-gutB1 DNJ biosynthetic gene cluster was cultured under the optimized condition, DNJ production was increased to 773 mg/L, representing a level 6.2-fold higher than that of the wild-type strain cultured in 5% DFS for five days.
Callicarpa japonica Thunb. ameliorates allergic airway inflammation by suppressing NF-κB activation and upregulating HO-1 expression
No full textEthnopharmacological relevance: Callicarpa japonica Thunb., as an herbal medicine has been used for the treatment of inflammatory diseases in China and Korea.
Materials and methods: Ultra performance liquid chromatography？photodiode array？quadrupole time？of？flight mass spectrometer (UPLC？PDA？QTof MS) was used to detect the major phenylethanoid glycosides in the C. japonica extract. BALB/c mice were intraperitoneally sensitized by ovalbumin (OVA) (on days 0 and 7) and challenged by OVA aerosol (on days 11？13) to induce airway inflammatory response. The mice were also administered with C. japonica Thunb. (CJT) (20 and 40 mg/kg Per oral) on days 9？13. CJT pretreatment was conducted in lipopolysaccharide (LPS)-stimulated RAW264.7 or phorbol 12-myristate 13-acetate (PMA)-stimulated A549 cells.
Results: CJT administration significantly reduced the secretion of Th2 cytokines, TNF-α, IL-6, immunoglobulin E (IgE) and histamine, and the recruitment of eosinophils in an OVA-exposed mice. In histological analyses, the amelioration of inflammatory cell influx and mucus secretion were observed with CJT. The OVA-induced airway hyperresponsiveness (AHR), iNOS expression and NF-κB activation were effectively suppressed by CJT administration. In addition, CJT led to the upregulation of HO-1 expression. In an in vitro study, CJT pretreatment suppressed the LPS-induced TNF-α secretion in RAW264.7 cells and attenuated the PMA-induced IL-6, IL-8 and MCP-1 secretion in A549 cells. These effects were accompanied by downregulated NF-κB phosphorylation and by upregulated HO-1 expression.
Conclusion: These results suggested that CJT has protective activity against OVA-induced airway inflammation via downregulation of NF-κB activation and upregulation of HO-1, suggesting that CJT has preventive potential for the development of allergic asthma.
Native high-density lipoproteins (HDL) with higher paraoxonase exerts a potent antiviral effect against SARS-CoV-2 (COVID-19), while glycated HDL lost the antiviral activity
No full textHuman high-density lipoproteins (HDL) show a broad spectrum of antiviral activity in terms of anti-infection. Although many reports have pointed out a correlation between a lower serum HDL-C and a higher risk of COVID-19 infection and progression, the in vitro antiviral activity of HDL against SARS-CoV-2 has not been reported. HDL functionality, such as antioxidant and anti-infection, can be impaired by oxidation and glycation and a change to pro-inflammatory properties. This study compared the antiviral activity of native HDL with glycated HDL via fructosylation and native low-density lipoproteins (LDL). After 72 h of fructosylation, glycated HDL showed a typical multimerized protein pattern with an elevation of yellowish fluorescence. Glycated HDL showed a smaller particle size with an ambiguous shape and a loss of paraoxonase activity up to 51% compared to native HDL. The phagocytosis of acetylated LDL was accelerated 1.3-fold by glycated HDL than native HDL. Native HDL showed 1.7 times higher cell viability and 3.6 times higher cytopathic effect (CPE) inhibition activity against SARS-CoV-2 than that of glycated HDL under 60 μg/mL (approximately final 2.2 μM) in a Vero E6 cell. Native HDL showed EC50 = 52.1 ± 1.1 μg/mL (approximately final 1.8 μM) for the CPE and CC50 = 79.4 ± 1.5 μg/mL (around 2.8 μM). The selective index (SI) of native HDL was calculated to be 1.52. In conclusion, native HDL shows potent antiviral activity against SARS-CoV-2 without cytotoxicity, while the glycation of HDL impairs its antiviral activity. These results may explain why patients with diabetes mellitus or hypertension are more sensitive to a COVID-19 infection and have a higher risk of mortality.
Au@ZIF-8 SERS paper for food spoilage detection
No full textPutrescine and cadaverine are important volatile indicators for the evaluation of food spoilage. In this study, a metal-organic framework (MOF)-coated surface-enhanced Raman scattering (SERS) paper platform for the detection of putrescine and cadaverine is developed. Au@ zeolite imidazolate framework-8 (ZIF-8) SERS paper is fabricated by the coating of ZIF-8 layer on a Au nanoparticle-impregnated paper that is prepared by dry plasma reduction. The Au@ZIF-8 SERS paper is characterized by scanning electron microscope, energy-dispersive X-ray spectroscopy, X-ray diffraction, and N2 sorption isotherm. The ZIF-8 layer enables the accumulation of gaseous molecules and also provides enhancement of SERS signals. The fluorescence, SERS, and simulation results prove the improved detection ability of the Au@ZIF-8 platform for the volatile molecules. For the selective detection of putrescine and cadaverine, the Au@ZIF-8 SERS paper is functionalized with 4-mercatobenzaldehyde (4-MBA). The 4-MBA molecule acts as a Raman reporter and also a specific receptor for the volatile amine molecules. Using the intensity ratiometric detection of 4-MBA-functionalized Au@ZIF-8 SERS paper, putrescine and cadaverine are quantitatively detected with detection limits of 76.99 and 115.88 parts per billion, respectively. Furthermore, the detection of volatile amine molecules released from spoiled salmon, chicken, beef, and pork samples is demonstrated. It is anticipated that the MOF-coated SERS paper platforms will be applicable not only in food safety but other applications including disease diagnosis and environmental monitoring.
Spirosoma taeanense sp. nov., a radiation resistant bacterium isolated from a coastal sand dune
No full textAn aerobic, Gram-negative, non-motile, non-spore-forming, rod-shaped, and pale yellow-colored bacterial strain, designated TS118T, was isolated from a sand sample obtained from a coastal sand dune after exposure to 3 kGy of gamma radiation. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolate was a member of the genus Spirosoma and most closely related to Spirosoma metallicum PR1014kT (95.1% similarity). The genome of strain TS118T is constituted by one chromosome (5,691,492 bp) and one plasmid (28,440 bp) and has a G？+？C content of 52.7%. The genome contains 4641 protein coding sequences (CDSs), 38 tRNAs, and 11 rRNAs. The predominant fatty acids of strain TS118T were C16:1 ω5c, iso-C15:0, C16:0, summed feature 3 (C16:1 ω6c and/or C16:1 ω7c), and iso-C17:0 3-OH. The major polar lipids were phosphatidylethanolamine, an unidentified amino lipid and an unidentified aminophospholipid. The main respiratory quinone was menaquinone-7 (MK-7). The novel strain showed resistance to gamma radiation with a D10 value (i.e., the dose required to reduce the bacterial population by tenfold) of 4.3 kGy. Based on the phylogenetic, physiological, and chemotaxonomic characteristics, strain TS118T represents a novel species, for which the name Spirosoma taeanense sp. nov. is proposed. The type strain is TS118T (=KCTC 72898T =JCM 34024T).
Orphan nuclear receptor ERRγ is a transcriptional regulator of CB1 receptor-mediated TFR2 gene expression in hepatocytes
No full textOrphan nuclear receptor estrogen-related receptor γ (ERRγ) is an important transcription factor modulating gene transcription involved in endocrine control of liver metabolism. Transferrin receptor 2 (TFR2), a carrier protein for transferrin, is involved in hepatic iron overload in alcoholic liver disease (ALD). However, TFR2 gene transcriptional regulation in hepatocytes remains largely unknown. In this study, we described a detailed molecular mechanism of hepatic TFR2 gene expression involving ERRγ in response to an endocannabinoid 2-arachidonoylglycerol (2-AG). Treatment with 2-AG and arachidonyl-2′-chloroethylamide, a selective cannabinoid receptor type 1 (CB1) receptor agonist, increased ERRγ and TFR2 expression in hepatocytes. Overexpression of ERRγ was sufficient to induce TFR2 expression in both human and mouse hepatocytes. In addition, ERRγ knockdown significantly decreased 2-AG or alcohol-mediated TFR2 gene expression in cultured hepatocytes and mouse livers. Finally, deletion and mutation analysis of the TFR2 gene promoter demonstrated that ERRγ directly modulated TFR2 gene transcription via binding to an ERR-response element. This was further confirmed by chromatin immunoprecipitation assay. Taken together, these results reveal a previously unrecognized role of ERRγ in the transcriptional regulation of TFR2 gene expression in response to alcohol.
Flavonoids from the seeds of Psoralea corylifolia inhibit diacylglycerol acyltransferase
No full textThree new flavonoids, including a new prenylated aurone, 7,2',5'-trihydroxy-8-prenylaurone (1), and two new flavanones, 2(S)-4'-hydroxy-6-methoxy-7-(2”-hydroxy-3”-methybult-3”-enyl)flavanone (2) and 2(S)-4'-hydroxy-7-methoxy-6-(1”,2”-epoxy-3”-hydroxy-dimethyl)flavanone (3), together with eight known flavonoids were isolated from the seeds of Psoralea corylifolia L. The structures were elucidated by detailed spectroscopic analysis and comparison with reported data. Among them, compounds 1 ？ 3, 10 and 11 exhibited inhibitory effects on the enzyme activity of DGAT1 in an in vitro assay with IC50 values ranging from 52.2 ± 1.2？89.2 ± 1.4 μM.
One-pot, solid-phase immunosensing platform consisting of a nanometer-thick Au/TiO2 photocatalytic film and Cy5/capture antibody/gold nanorod conjugates
No full textThere is a demand for one-pot, portable (solid-phase), sensitive, and user-friendly immunosensors for future point-of-care (POC) self-testing. However, current immunoassays such as the enzyme-linked immunosorbent assay (ELISA) typically involve several complicated steps, and they are not readily adaptable by nonexpert users. Herein, we present a rapid (∼30 min) one-pot, solid-phase immunosensor, based on nanomaterials, by combining a nanometer-thick Au/TiO2 photocatalytic film and Cy5/capture antibody/gold nanorod (GNR) conjugates immobilized on a membrane (the fluorescence of Cy5 was enhanced by the GNR). The one-pot immunoassay is started by adding a drop of a mixture containing 4-chloro-1-naphthol (CN), a horseradish peroxidase (HRP)-labeled detection antibody, and an antigen onto the one-pot immunosensor and illuminating UV light. The UV illumination on the Au/TiO2 film results in the production of H2O2, which promotes a CN precipitation reaction. 4-Chloro-1-naphthol precipitates produced by the HRP, which was bound to the conjugates via the antibodies and antigens, could preliminarily quench Cy5 fluorescence via Forster resonance energy transfer, because of their proximity to Cy5. The sensitivity of the developed one-pot immunosensor was similar to that of a commercially available ELISA kit. Given the increasing interest in the early diagnosis of various diseases, including cancers, dementia, and coronavirus disease 2019, the application of nanomaterials such as a porous thin-film photocatalyst and GNR-based fluorescent probes could pave way for the development of next-generation POC biosensors.
Genetic and molecular determinants of polymicrobial interactions in Fusobacterium nucleatum
No full textA gram-negative colonizer of the oral cavity, Fusobacterium nucleatum not only interacts with many pathogens in the oral microbiome but also has the ability to spread to extraoral sites including placenta and amniotic fluid, promoting preterm birth. To date, however, the molecular mechanism of interspecies interactions？termed coaggregation？by F. nucleatum and how coaggregation affects bacterial virulence remain poorly defined. Here, we employed genome-wide transposon mutagenesis to uncover fusobacterial coaggregation factors, revealing the intertwined function of a two-component signal transduction system (TCS), named CarRS, and a lysine metabolic pathway in regulating the critical coaggregation factor RadD. Transcriptome analysis shows that CarR modulates a large regulon including radD and lysine metabolic genes, such as kamA and kamD, the expression of which are highly up-regulated in the ΔcarR mutant. Significantly, the native culture medium of ΔkamA or ΔkamD mutants builds up abundant amounts of free lysine, which blocks fusobacterial coaggregation with streptococci. Our demonstration that lysine-conjugated beads trap RadD from the membrane lysates suggests that lysine utilizes RadD as its receptor to act as a metabolic inhibitor of coaggregation. Lastly, using a mouse model of preterm birth, we show that fusobacterial virulence is significantly attenuated with the ΔkamA and ΔcarR mutants, in contrast to the enhanced virulence phenotype observed upon diminishing RadD (ΔradD or ΔcarS mutant). Evidently, F. nucleatum employs the TCS CarRS and environmental lysine to modulate RadD-mediated interspecies interaction, virulence, and nutrient acquisition to thrive in the adverse environment of oral biofilms and extraoral sites.
The groESL ISR sequence-based species-specific identification of GRAS and non-GRAS Lactiplantibacillus as an alternative to 16S rRNA sequencing
No full textThe untranscribed DNA sequences of the intergenic spacer regions (ISR) in the groESL were analyzed to resolve the ambiguity within phylogenetically close GRAS and non-GRAS species. The sequencing results of amplified polymerase chain reaction (PCR) products using groESL-ISR-specific primers accurately distinguished L. plantarum and L. pentosus, which were not distinguished by 16 S rRNA sequences. Furthermore, the 20 selected major probiotics species were divided into several groups according to the length (22？91 bp) and homology (65？99%) of their ISR sequences, and this discrimination was consistent with core-genome-based differentiation. Therefore, ISR sequence-based species identification showed more accurate results than 16 S rRNA sequence-based identification and represented genome-based speciation.