Korea Research Institute of Bioscience and Biotechnology
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Isoliquiritigenin reduces LPS-induced inflammation by preventing mitochondrial fission in BV-2 microglial cells
No full textExcessive microglial cell activation in the brain can lead to the production of various neurotoxic factors (e.g., pro-inflammatory cytokines, nitric oxide) which can, in turn, initiate neurodegenerative processes. Recent research has been reported that mitochondrial dynamics regulate the inflammatory response of lipopolysaccharide (LPS). Isoliquiritigenin (ISL) is a compound found in Glycyrrhizae radix with anti-inflammatory and antioxidant properties. In this study, we investigated the function of ISL on the LPS-induced pro-inflammatory response in BV-2 microglial cells. We showed that ISL reduced the LPS-induced increase in pro-inflammatory mediators (e.g., nitric oxide and pro-inflammatory cytokines) via the inhibition of ERK/p38/NF-κB activation and the generation of reactive oxygen species (ROS). Furthermore, ISL inhibited the excessive mitochondrial fission induced by LPS, regulating mitochondrial ROS generation and pro-inflammatory response by suppressing the calcium/calcineurin pathway to dephosphorylate Drp1 at the serine 637 residue. Interestingly, the ISL pretreatment reduced the number of apoptotic cells and levels of cleaved caspase3/PARP, compared to LPS-treated cells. Our findings suggested that ISL ameliorated the pro-inflammatory response of microglia by inhibiting dephosphorylation of Drp1 (Ser637)-dependent mitochondrial fission. This study provides the first evidence for the effects of ISL against LPS-induced inflammatory response related and its link to mitochondrial fission and the calcium/calcineurin pathway. Consequently, we also identified the protective effects of ISL against LPS-induced microglial apoptosis, highlighting the pharmacological role of ISL in microglial inflammation-mediated neurodegeneration.
Crystal structure of human LC8 bound to a peptide from Ebola virus VP35
No full textZaire ebolavirus, commonly called Ebola virus (EBOV), is an RNA virus that causes severe hemorrhagic fever with high mortality. Viral protein 35 (VP35) is a virulence factor encoded in the EBOV genome. VP35 inhibits host innate immune responses and functions as a critical cofactor for viral RNA replication. EBOV VP35 contains a short conserved motif that interacts with dynein light chain 8 (LC8), which serves as a regulatory hub protein by associating with various LC8-binding proteins. Herein, we present the crystal structure of human LC8 bound to the peptide comprising residues 67-76 of EBOV VP35. Two VP35 peptides were found to interact with homodimeric LC8 by extending the central β-sheets, constituting a 2:2 complex. Structural analysis demonstrated that the intermolecular binding between LC8 and VP35 is mainly sustained by a network of hydrogen bonds and supported by hydrophobic interactions in which Thr73 and Thr75 of VP35 are involved. These findings were verified by binding measurements using isothermal titration calorimetry. Biochemical analyses also verified that residues 67-76 of EBOV VP35 constitute a core region for interaction with LC8. In addition, corresponding motifs from other members of the genus Ebolavirus commonly bound to LC8 but with different binding affinities. Particularly, VP35 peptides originating from pathogenic species interacted with LC8 with higher affinity than those from noninfectious species, suggesting that the binding of VP35 to LC8 is associated with the pathogenicity of the Ebolavirus species.
Description of Paenibacillus dokdonensis s p. nov., a new bacterium isolated from soil
No full textTwo strains isolated from soil samples were designated as YH-JAE5T and YH-JAE2. The isolates were facultative anaerobic, Gram-stain-variable, motile, rod-shaped bacteria. Phylogenetic analysis indicated that the isolates belonged to the genus Paenibacillus , but the 16S rRNA gene sequence similarities were <98？%？when compared with other species within the genus. Analysis of rpoB gene revealed the isolates formed a sub-cluster with P. chibensis . The only menaquinone identified was MK-7. The two isolates contained meso-diaminopimelic acid within their cell wall peptidoglycan. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phospholipid, aminophospholipids, and lipids. The major fatty acids were C15？:？0 anteiso and C15？:？0 iso. The average nucleotide identity, average amino acid identity, and digital DNA？DNA hybridization values between isolate YH-JAE5T and the most closely related reference strain ( Paenibacillus chibensis KCTC 3758T) were 81.7, 84.8 and 23.4？%, respectively. The G+C content of the genomic DNA was 47.4？mol%. Thus, the polyphasic data revealed that YH-JAE2 (=KCTC 43239=JCM 34435) and YH-JAE5T (=KCTC 43059=JCM 33533) represent a new species. The name Paenibacillus dokdonensis sp. nov. is proposed.
Neobacillus endophyticus sp. nov., an endophytic bacterium isolated from Selaginella involvens roots
No full textA Gram-stain-positive, facultatively anaerobic, rod-shaped, endospore-forming, oxidase-positive, and catalase-negative strain designated as BRMEA1T was isolated from the surface-sterilized Selaginella involvens roots. Growth of strain BRMEA1T was found to occur at pH 6.0？8.0 (optimum, pH 7.0), 15？50？°C (optimum, 25？30？°C) and in the absence of NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain BRMEA1T formed a lineage within the genus Neobacillus (family Bacillaceae ) and showed the highest sequence similarity to Neobacillus drentensis DSM 15600T (98.3？%) and Neobacillus fumarioli KCTC 13885T (98.2？%), and less than 98.2？% 16S rRNA gene sequence similarity to the other members of the genus Neobacillus . Whole-genome analysis of strain BRMEA1T comprised a circular chromosome (5？632？809？bp in size) with 38.5？mol% G+C content. Digital DNA？DNA hybridization analyses revealed that strain BRMEA1T showed 20.5 and 22.0% genomic DNA relatedness with the closest species, N. drentensis DSM 15600T and N. fumarioli KCTC 13885T, respectively. The whole-genome sequence of strain BRMEA1T showed the presence of 11 specific conserved signature indels for the genus Neobacillus . The major cellular fatty acids (>10？%) of strain BRMEA1T were found to be iso-C15？:？0 and anteiso-C15？:？0, while the major polar lipids were found to be diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. Polyphasic analysis results revealed that BRMEA1T represents a novel species of the genus Neobacillus , with the proposed name Neobacillus endophyticus sp. nov. The type strain is BRMEA1T (=KCTC 43208T=CCTCC AB 2020071T).
Evaluation of the uninjured anterior talofibular ligament by ultrasound for assessing generalized joint hypermobility
No full textBackground: Most clinicians use the Beighton score to assess generalized joint hypermobility (GJH) when deciding on the treatment of chronic lateral ankle instability (CLAI). The purpose of the study was to evaluate anterior talofibular ligament (ATFL) status by ultrasound and correlate these values with Beighton scores and the manual anterior drawer test (ADT).
Methods: The participants were divided into two groups, those without GJH (24 ankles) and with GJH (20 ankles). For the investigation of ATFL, resting and stress ultrasonography was performed to assess the length, height (degree of loosening) and thickness. Beighton scores, manual ADT grades and ultrasound parameters of participants with and without GJH were compared. The correlation coefficients among those values were analyzed.
Results: The mean ATFL length, resting height, stress height and mean difference in height between resting and stress ATFL were all significantly different between the two groups (P < .05). The resting and stress ATFL length, height, and difference in height between resting and stress ATFL showed a positive linear relationship with Beighton scores and manual ADT grades (P < .05).
Conclusions: The ATFL stress ultrasound parameters showed significant differences between participants with high and low Beighton scores and were correlated with Beighton scores and manual ADT grades.
Simultaneous pro？ling of Arabidopsis thaliana and Vibrio vulni？cus MO6-24/O transcriptomes by dual RNA-seq analysis
No full textWe previously demonstrated that a marine bacterial pathogen Vibrio vulnificus isolated from sea foods modulated gene expression levels and defense responses of a land plant Arabidopsis thaliana. Although the interaction between V. vulnificus and A. thaliana was verified under artificial and greenhouse conditions, the simultaneous changes in host and pathogen transcriptomes remained obscure. In this study, we simultaneously analyzed the transcriptome of V. vulnificus MO6-24/O and A. thaliana by dual RNA-sequencing analysis. Disease symptoms appeared at 5 and 7 days post-inoculation in vitro and post-infiltration in planta, respectively. A total of 31, 128, 303, 219, and 130 differentially expressed genes (DEGs) were identified in V. vulnificus MO6-24/O at 3, 6, 12, 24, and 48 h post-infiltration. Out of these, 14 genes involved in the virulence and pathogenicity of V. vulnificus MO6 were characterized. These genes were clustered into six categories, including adherence, antiphagocytosis, chemotaxis and motility, iron uptake, toxin and secretion system. In plant side, the bacterium DEGs potentially played a pivotal role in activating pattern recognition receptors (PRRs)-mediated defense responses. A. thaliana genes related to PRRs, reactive oxygen species burst, mitogen-activated protein kinase cascade induction, salicylic acid, jasmonic acid, ethylene, abscisic acid, auxin, gibberellin, and cytokinin were highly induced by V. vulnificus MO6-24/O challenge. Taken together, our results indicate that the sophisticated communication between a marine bacterial pathogen V. vulnificus and A. thaliana occurs. It is the first report demonstration that V. vulnificus actively modulates its virulence factors and potential host immune regulator in a land plant species.
Engineering Yarrowia lipolytica for de novo production of tetraacetyl phytosphingosine
No full textAims: To genetically engineer the oleaginous yeast Yarrowia lipolytica for de novo production of tetraacetylphytosphingosine (TAPS), a precursor of phytosphingosine, and optimization of fermentation conditions for high yield.
Methods and results: We successfully constructed a TAPS-producing Y. lipolytica CE3 strain by co-expression of Wickerhamomyces ciferrii-derived acetyl transferases, Sli1p and Atf2p. Next, we optimized several environmental factors including temperature, initial pH and C/N ratio for TAPS production in a shake culture. Deletion of LCB4 in CE3 strain increased the volumetric TAPS titre and cell-specific yield to 142·1 ± 10·7 mgTAPS l-1 and 3·08 ± 0·11 mgTAPS gDCW -1 , respectively, in a shake flask culture incubated for 120 h at 28°C with glycerol as the carbon source. Finally, we developed a 5-l fed-batch process with NaOH-mediated pH control and olive oil as a carbon source, exhibiting 650 ± 24 mgTAPS l-1 of TAPS production within 56 h of the fermentation.
Conclusions: The introduction of codon-optimized Sli1p and Atf2p, deletion of LCB4 gene and sexual hybridization, accompanied by specific fermentation conditions, enhanced TAPS yield in Y. lipolytica.
Significance and impact of the study: Our results highlight Y. lipolytica as a promising candidate for the industrial production of TAPS, an important component of cosmetic formulations.
Expression and purification of soluble and active human enterokinase light chain in Escherichia coli
No full textHuman enterokinase light chain (hEKL) specifically cleaves the sequence (Asp)4-Lys↓X (D4K), making this a frequently used enzyme for site-specific cleavage of recombinant fusion proteins. However, hEKL production from Escherichia coli is limited due to intramolecular disulphide bonds. Here, we present strategies to obtain soluble and active hEKL from E. coli by expressing the hEKL variant C112S fused with maltose-binding protein (MBP) through D4K and molecular chaperons including GroEL/ES. The fusion protein self-cleaved in vivo, thereby removing the MBP in the E. coli cells. Thus, the self-cleaved hEKL variant was released into the culture medium. One-step purification using HisTrap™ chromatography purified the hEKL variant exhibiting an enzymatic activity of 3.1 × 103 U/mL (9.934 × 105 U/mg). The approaches presented here greatly simplify the purification of hEKL from E. coli without requiring refolding processes.