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Absicoccus porci gen. nov., sp. nov., a member of the family Erysipelotrichaceae isolated from pig faece
No full textAn obligately anaerobic, Gram-stain-positive and coccus-shaped bacterium, designated strain YH-panp20T, was isolated from pig faeces. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the isolate belongs to the family Erysipelotrichaceae, and is most closely related to Catenisphaera adipataccumulans KCTC 15517T (93.5？% sequence similarity), followed by Faecalitalea cylindroides KCTC 5815T (92.2？%), Faecalicoccus acidiformans KCTC 15521T (90.2？%) and Holdemanella biformis KCTC 5969T (89.6？%). Average nucleotide identity values between YH-panp20T and its closest relatives were lower than 71？%. The G+C content of the isolate was 38.4？mol%, and its cell-wall peptidoglycan was found to be of A1γ type and contained meso-diaminopimelic acid. The predominant fatty acids were C18？:？1 cis 9, C18？:？0 DMA and C16？:？0. The major end-products of glucose fermentation were lactate, acetate and formate. Therefore, based on the phenotypic, phylogenetic and chemotaxonomic properties, a novel genus and species, Absicoccus porci gen. nov., sp. nov., is proposed for isolate YH-panp20T (=KCTC 15747T=JCM 32769T).
Kanakugiol, a compound isolated from Lindera erythrocarpa, promotes cell death by inducing mitotic catastrophe after cell cycle arrest
No full textA novel compound named 'kanakugiol' was recently isolated from Lindera erythrocarpa and showed free radical-scavenging and antifungal activities. However, the details of the anticancer effect of kanakugiol on breast cancer cells remain unclear. We investigated the effect of kanakugiol on the growth of MCF-7 human breast cancer cells. Kanakugiol affected cell cycle progression, and decreased cell viability in MCF-7 cells in a dose-dependent manner. It also enhanced PARP cleavage (50 kDa), whereas DNA laddering was not induced. FACS analysis with annexin V-FITC/PI staining showed necrosis induction in kanakugiol-treated cells. Caspase-9 cleavage was also induced. Expression of death receptors was not altered. However, Bcl-2 expression was suppressed, and mitochondrial membrane potential collapsed, indicating limited apoptosis induction by kanakugiol. Immunofluorescence analysis using α-tubulin staining revealed mitotic exit without cytokinesis (4N cells with two nuclei) due to kanakugiol treatment, suggesting that mitotic catastrophe may have been induced via microtubule destabilization. Furthermore, cell cycle analysis results also indicated mitotic catastrophe after cell cycle arrest in MCF-7 cells due to kanakugiol treatment. These findings suggest that kanakugiol inhibits cell proliferation and promotes cell death by inducing mitotic catastrophe after cell cycle arrest. Thus, kanakugiol shows potential for use as a drug in the treatment of human breast cancer.
Comprehensive DNA methylation profiling identifies novel diagnostic biomarkers for thyroid cancer
No full textBackground: There are no reliable biomarkers to accurately differentiate indolent thyroid tumors from more aggressive thyroid cancers. This study aimed to develop new DNA methylation markers for diagnosis and recurrence risk stratification of papillary thyroid carcinoma (PTC). Methods: Thyroid tumor-specific DNA methylation profiling was investigated in 34 fresh frozen tissues, which included nontumor (n？=？7), noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTP, n？=？6) and PTC (n？=？21), using the Illumina HumanMethylation EPIC array. We performed a genome-wide assessment of thyroid tumor-specific differentially methylated CpG sites in the discovery set, then validated the top candidate markers in an independent set of 293 paraffin tissue samples comprised of follicular adenoma (FA, n？=？61), Hurthle cell adenoma (HA, n？=？24), NIFTP (n？=？56), PTC (n？=？120), follicular thyroid carcinoma (n？=？27), and Hurthle cell carcinoma (n？=？5), by pyrosequencing. Results: Three selected markers (cg10705422, cg17707274, and cg26849382) differentiated nonmalignant (FA, HA, and NIFTP) tumors from differentiated thyroid cancers with area under the receiver operating characteristic curve of 0.83, 0.83, and 0.80, respectively. Low DNA methylation levels for three markers were significantly associated with recurrent or persistent disease (odds ratio (OR)？=？3.860 [95% confidence interval (CI) 1.194-12.475]) and distant metastasis (OR？=？4.009 [CI 1.098-14.632]) in patients with differentiated thyroid cancer. A subgroup analysis for the validation set showed that PTC patients with low DNA methylation levels more frequently had aggressive histology, extrathyroidal extension, lymph node metastasis, BRAFV600E mutations, and recurrent or persistent disease than those with high levels of methylation markers. All PTC patients who developed disease recurrence had low DNA methylation levels for three markers. Conclusions: DNA methylation levels of three markers can be useful for differentiating differentiated thyroid cancer from nonmalignant follicular thyroid lesions, and may serve as prognostic biomarkers for predicting recurrent or persistent disease after surgery for differentiated thyroid cancer.
Unique microbial module regulates the harmful algal bloom (Cochlodinium polykrikoides) and shifts the microbial community along the Southern Coast of Korea
No full textHarmful algal blooms (HABs) of Cochlodinium (aka Margalefidinium) polykrikoides cause huge economic and ecological damages and thus are considered environmental problems. Previous studies uncovered that the formation and collapse of phytoplankton blooms could be closely related to their associated microbes although their roles in C. polykrikoides bloom have not been elucidated yet. To explore the potential interactions between C. polykrikoides and other microbes (archaea, bacteria, and phytoplankton), we collected water samples in the free-living (FL) (0.22 to 3 μm), nanoparticle-associated (NP) (3 to 20 μm), and microparticle-associated (MP) (>20 μm) fractions when C. polykrikoides blooms occurred from July to August in 2016, 2017, and 2018 in the South Sea of Korea. The microbial composition of the C. polykrikoides-associated microbial cluster (Module I) significantly differed from those of other modules associated with Alexandrium, Chaetoceros or Chattonella. Over half of the interspecies interactions in Module I occurred within the module. That is, specific microbial clusters were associated with the C. polykrikoides bloom. Structural equation modeling (SEM) further confirmed the stronger effects of Module I on C. polykrikoides blooms compared to environmental factors. Among the operational taxonomic units (OTUs) directly correlated with C. polykrikoides, Marine Group I was presumed to supply vitamin B12, the essential element for C. polykrikoides growth, while the potential fish pathogens (Micrococcaceae and Piscirickettsiaceae) could contribute to the massive fish death together with C. polykrikoides itself. In addition, the zoospores of Syndiniales, a parasitoid to dinoflagellates, might be related to the sudden collapse of C. polykrikoides blooms. These microbial groups also contributed to significant alterations of the local microbial community structures. Collectively, network analysis and SEM revealed that the C. polykrikoides bloom is concomitant with distinct microbial communities, contributing to the rise and fall of the bloom, and finally determining the local microbial community structures.
The microtubule-associated histone methyltransferase SET8, facilitated by transcription factor LSF, methylates alpha-tubulin
No full textMicrotubules are cytoskeletal structures critical for mitosis, cell motility, and protein and organelle transport and are a validated target for anticancer drugs. However, how tubulins are regulated and recruited to support these distinct cellular processes is incompletely understood. Posttranslational modifications of tubulins are proposed to regulate microtubule function and dynamics. Although many of these modifications have been investigated, only one prior study reports tubulin methylation and an enzyme responsible for this methylation. Here we used in vitro radiolabeling, MS, and immunoblotting approaches to monitor protein methylation and immunoprecipitation, immunofluorescence, and pulldown approaches to measure protein-protein interactions. We demonstrate that N-lysine methyltransferase 5A (KMT5A or SET8/PR-Set7), which methylates lysine 20 in histone H4, bound α-tubulin and methylated it at a specific lysine residue, Lys311 Furthermore, late SV40 factor (LSF)/CP2, a known transcription factor, bound both α-tubulin and SET8 and enhanced SET8-mediated α-tubulin methylation in vitro In addition, we found that the ability of LSF to facilitate this methylation is countered by factor quinolinone inhibitor 1 (FQI1), a specific small-molecule inhibitor of LSF. These findings suggest the general model that microtubule-associated proteins, including transcription factors, recruit or stimulate protein-modifying enzymes to target tubulins. Moreover, our results point to dual functions for SET8 and LSF not only in chromatin regulation but also in cytoskeletal modification.
Morpho-molecular characterization of diatom Skeletonema pseudocostatum (Thalassiosirales, Bacillariophyta) from the Korean coast
No full textMorphology of a strain of unspecified Skeletonema species established from Korean coast was examined by light, fluorescence and scanning electron microscopy, and SSU (small subunit) and LSU (large subunit) rDNA of the strain were also sequenced. The specimen was characterized by solitary or short chains, and each cell contained 1-2 chloroplasts. The valve face was slightly convex, and the terminal fultoportula processes (TFPPs) were open and showed narrow distal ends that could be truncated or spiny with claw-like protrusions. The basal part of the TFPPs was tubular and oblique to the cell axis. The intercalary fultoportula processes (IFPPs) were also narrow, completely open, and joined in a 1 : 1 junction. Occasionally, one IFPP was connected with two opposing IFPPs. The morphological features of the specimen were identical to those of Skeletonema pseudocostatum. Molecular phylogeny based on SSU rDNA revealed that the Korean strain is nested within a clade comprising S. pseudocostatum and S. tropicum. However, based on D1-D2 LSU rDNA sequences, a clade including S. pseudocostatum and a Korean strain was separated from the S. tropicum clade. This indicates that the Korean strain can be identified as S. pseudocostatum. This species represents the first record from Korean coastal waters.
Genetic manipulation of a lipolytic yeast Candida aaseri SH14 using CRISPR-Cas9 system
No full textA lipolytic yeast Candida aaseri SH14 that can utilise long-chain fatty acids as the sole carbon source was isolated from oil palm compost. To develop this strain as a platform yeast for the production of bio-based chemicals from renewable plant oils, a genetic manipulation system using CRISPR-Cas9 was developed. Episomal vectors for expression of Cas9 and sgRNA were constructed using an autonomously replicating sequence isolated from C. aaseri SH14. This system guaranteed temporal expression of Cas9 for genetic manipulation and rapid curing of the vector from transformed strains. A β-oxidation mutant was directly constructed by simultaneous disruption of six copies of acyl-CoA oxidases genes (AOX2, AOX4 and AOX5) in diploid cells using a single sgRNA with 70% efficiency and the Cas9 vector was efficiently removed. Blocking of β-oxidation in the triple AOX mutant was confirmed by the accumulation of dodecanedioic acid from dodecane. Targeted integration of the expression cassette for C. aaseri lipase2 was demonstrated with 60% efficiency using this CRISPR-Cas9 system. This genome engineering tool could accelerate industrial application of C. aaseri SH14 for production of bio-based chemicals from renewable oils.
Simple, rapid, and accurate malaria diagnostic platform using microfluidic-based immunoassay of Plasmodium falciparum lactate dehydrogenase
No full textThis work reports on a rapid diagnostic platform for the detection of Plasmodium falciparum lactate dehydrogenase (PfLDH), a representative malaria biomarker, using a microfluidic microplate-based immunoassay. In this study, the microfluidic microplate made it possible to diagnose PfLDH with a small volume of sample (only 5 μL) and short time (<？90 min) compared to conventional immunoassays such as enzyme-linked immunosorbent assay (ELISA). Moreover, the diagnostic performance of PfLDH showed high sensitivity, specificity, and selectivity (i.e., 0.025 pg/μL in phosphate-buffered saline and 1 pg/μL in human serum). The microfluidic-based microplate sensing platform has the potential to adapt simple, rapid, and accurate diagnoses to the practical detection of malaria.
Ethylene glycol and glycolic acid production from xylonic acid by Enterobacter cloacae
No full textBACKGROUND:
Biological routes for ethylene glycol production have been developed in recent years by constructing the synthesis pathways in different microorganisms. However, no microorganisms have been reported yet to produce ethylene glycol naturally.
RESULTS:
Xylonic acid utilizing microorganisms were screened from natural environments, and an Enterobacter cloacae strain was isolated. The major metabolites of this strain were ethylene glycol and glycolic acid. However, the metabolites were switched to 2,3-butanediol, acetoin or acetic acid when this strain was cultured with other carbon sources. The metabolic pathway of ethylene glycol synthesis from xylonic acid in this bacterium was identified. Xylonic acid was converted to 2-dehydro-3-deoxy-D-pentonate catalyzed by D-xylonic acid dehydratase. 2-Dehydro-3-deoxy-D-pentonate was converted to form pyruvate and glycolaldehyde, and this reaction was catalyzed by an aldolase. D-Xylonic acid dehydratase and 2-dehydro-3-deoxy-D-pentonate aldolase were encoded by yjhG and yjhH, respectively. The two genes are part of the same operon and are located adjacent on the chromosome. Besides yjhG and yjhH, this operon contains four other genes. However, individually inactivation of these four genes had no effect on either ethylene glycol or glycolic acid production; both formed from glycolaldehyde. YqhD exhibits ethylene glycol dehydrogenase activity in vitro. However, a low level of ethylene glycol was still synthesized by E. cloacae ΔyqhD. Fermentation parameters for ethylene glycol and glycolic acid production by the E. cloacae strain were optimized, and aerobic cultivation at neutral pH were found to be optimal. In fed batch culture, 34 g/L of ethylene glycol and 13 g/L of glycolic acid were produced in 46 h, with a total conversion ratio of 0.99 mol/mol xylonic acid.
CONCLUSIONS:
A novel route of xylose biorefinery via xylonic acid as an intermediate has been established.
Enhancer function of microRNA-3681 derived from long terminal repeats represses the activity of variable number tandem repeats in the 3’ UTR of SHISA7
No full textMicroRNAs (miRNAs) are non-coding RNA molecules involved in the regulation of gene expression. MiRNAs inhibit gene expression by binding to the 3' untranslated region (UTR) of their target gene. MiRNAs can originate from transposable elements (TEs), which comprise approximately half of the eukaryotic genome and one type of TE, called the long terminal repeat (LTR) is found in class of retrotransposons. Amongst the miRNAs derived from LTR, hsa-miR-3681 was chosen and analyzed using bioinformatics tools and experimental analysis. Studies on hsa-miR-3681 have been scarce and this study provides the relative expression analysis of hsa-miR-3681-5p from humans, chimpanzees, crab-eating monkeys, and mice. Luciferase assay for hsa-miR-3681-5p and its target gene SHISA7 supports our hypothesis that the number of miRNA binding sites affects target gene expression. Especially, the variable number tandem repeat (VNTR) and hsa-miR-3681-5p shares the binding sites in the 3' UTR of SHISA7, which leads the enhancer function of hsa-miR-3681-5p inhibits the activity of VNTR. To conclude, hsa-miR-3681-5p acts as a super-enhancer and the enhancer function of hsa-miR-3681-5p act as a repressor of VNTR activity in the 3' UTR of SHISA7.