Analysis of clinical and genomic profiles of therapy-related myeloid neoplasm in Korea.

BACKGROUND: Therapy-related myeloid neoplasm (T-MN) rarely occurs among cancer survivors, and was characterized by poor prognosis. T-MN has germline predisposition in a considerable proportion. Here, clinical characteristics and germline/somatic variant profiles in T-MN patients were investigated, and the findings were compared with those of previous studies. METHODS: A review of medical records, cytogenetic study, targeted sequencing by next-generation sequencing, and survival analysis were performed on 53 patients with T-MN at a single institution in Korea. RESULTS: The patients were relatively younger compared to T-MN patients in other studies. Our T-MN patients showed a high frequency of complex karyotypes, -5/del(5q), and -7/del(7q), which was similar to the Japanese study group but higher than the Australian study group. The most common primary disease was non-Hodgkin lymphoma, followed by breast cancer. The detailed distributions of primary diseases were different across study groups. Seven patients (13.2%) harbored deleterious presumed/potential germline variants in cancer predisposition genes (CPG) such as BRIP1, CEBPA, DDX41, FANCM, NBN, NF1, and RUNX1. In the somatic variant profile, TP53 was the most frequently mutated gene, which was consistent with the previous studies about T-MN. However, the somatic variant frequency in our study group was lower than in other studies. Adverse factors for overall survival were male sex, older age, history of previous radiotherapy, previous longer cytotoxic therapy, and -5/del(5q). CONCLUSION: The findings of our study corroborate important information about T-MN patients. As well as a considerable predisposition to CPG, the clinical characteristics and somatic variant profile showed distinctive patterns. Germline variant testing should be recommended for T-MN patients. If the T-MN patients harbor pathogenic germline variants, the family members for stem cell donation should be screened for carrier status through germline variant testing to avoid donor-derived myeloid neoplasm. For the prediction of the prognosis in T-MN patients, sex, age, past treatment history, and cytogenetic findings can be considered.

Identifying Terpenoid Biosynthesis Genes in Euphorbia maculata via Full-Length cDNA Sequencing.

The annual herb Euphorbia maculata L. produces anti-inflammatory and biologically active substances such as triterpenoids, tannins, and polyphenols, and it is used in traditional Chinese medicine. Of these bioactive compounds, terpenoids, also called isoprenoids, are major secondary metabolites in E. maculata. Full-length cDNA sequencing was carried out to characterize the transcripts of terpenoid biosynthesis reference genes and determine the copy numbers of their isoforms using PacBio SMRT sequencing technology. The Illumina short-read sequencing platform was also employed to identify differentially expressed genes (DEGs) in the secondary metabolite pathways from leaves, roots, and stems. PacBio generated 62 million polymerase reads, resulting in 81,433 high-quality reads. From these high-quality reads, we reconstructed a genome of 20,722 genes, in which 20,246 genes (97.8%) did not have paralogs. About 33% of the identified genes had two or more isoforms. DEG analysis revealed that the expression level differed among gene paralogs in the leaf, stem, and root. Whole sets of paralogs and isoforms were identified in the mevalonic acid (MVA), methylerythritol phosphate (MEP), and terpenoid biosynthesis pathways in the E. maculata L. The nucleotide information will be useful for identifying orthologous genes in other terpenoid-producing medicinal plants.

Clinical and Genomic Profiles of Korean Patients with MECOM Rearrangement and the t(3;21)(q26.2;q22.1) Translocation.

The translocation (3;21)(q26.2;q22.1) is a unique cytogenetic aberration that characterizes acute myeloid leukemia with myelodysplasia-related changes (AML-MRC) in patients with AML and myelodysplastic syndrome (MDS) or a therapy-related myeloid neoplasm. Using multigene target sequencing and FISH, we investigated the clinical and genomic profiles of patients with t(3;21) over the past 10 years. The frequency of t(3;21) among myeloid malignancies was very low (0.2%). Half of the patients had a history of cancer treatment and the remaining patients had de novo MDS. Twenty-one somatic variants were detected in patients with t(3;21), including in CBL, GATA2, and SF3B1. Recurrent variants in RUNX1 (c.1184A>C, p.Glu395Ala) at the same site were detected in two patients. None of the patients with t(3;21) harbored germline predisposition mutations for myeloid neoplasms. MECOM rearrangement was detected at a higher rate using FISH than using G-banding, suggesting that FISH is preferable for monitoring. Although survival of patients with t(3;21) is reportedly poor, the survival of patients with t(3;21) in this study was not poor when compared with that of other AML patients in Korea.

Production and Activity of Cristazarin in the Lichen-Forming Fungus Cladonia metacorallifera.

Lichens are a natural source of bioactive compounds. Cladonia metacorallifera var. reagens KoLRI002260 is a rare lichen known to produce phenolic compounds, such as rhodocladonic, thamnolic, and didymic acids. However, these metabolites have not been detected in isolated mycobionts. We investigated the effects of six carbon sources on metabolite biosynthesis in the C. metacorallifera mycobiont. Red pigments appeared only in Lilly and Barnett's media with fructose at 15 °C after 3 weeks of culture and decreased after 6 weeks. We purified these red pigments using preparative-scale high performance liquid chromatography and analyzed them via nuclear magnetic resonance. Results indicated that 1% fructose-induced cristazarin and 6-methylcristazarin production under light conditions. In total, 27 out of 30 putative polyketide synthase genes were differentially expressed after 3 weeks of culture, implying that these genes may be required for cristazarin production in C. metacorallifera. Moreover, the white collar genes Cmwc-1 and Cmwc-2 were highly upregulated at all times under light conditions, indicating a possible correlation between cristazarin production and gene expression. The cancer cell lines AGS, CT26, and B16F1 were sensitive to cristazarin, with IC50 values of 18.2, 26.1, and 30.9 µg/mL, respectively, which highlights the value of cristazarin. Overall, our results suggest that 1% fructose under light conditions is required for cristazarin production by C. metacorallifera mycobionts, and cristazarin could be a good bioactive compound.

Colletotrichalactones A-Ca, unusual 5/6/10-fused tricyclic polyketides produced by an endophytic fungus, Colletotrichum sp. JS-0361.

Three unusual polyketides with a 5/6/10-fused ring system, named colletotrichalactones A-Ca (1-3a), were isolated from cultures of the endophytic fungus, Colletotrichum sp. JS-0361, which was isolated from a leaf of Morus alba. Their structures, including their absolute stereochemistries, were completely established using extensive spectroscopic methods together with a chemical reaction utilizing competing enantioselective acylation coupled with LC/MS. Compounds possessing this ring skeleton were previously reported in three studies. Our rigorous chemical investigation revealed the complete configuration of this skeleton, which agreed with the results for glabramycin B with this ring skeleton established by computational chemistry and enantioselective synthesis in previous reports. 1 and 2 had unstable aldehyde groups that were easily converted to acetal groups in the presence of solvents. Meanwhile, compound 3a, with terminal acetal functionality, was deduced to be an artefact originating from compound 3 with a terminal aldehyde group. Compounds 1 and 3a displayed moderate-to-potent cytotoxic activities against MCF7 cells with IC50s of 35.06 and 25.20 µM, respectively.

RNA-Seq De Novo Assembly and Differential Transcriptome Analysis of Korean Medicinal Herb Cirsium japonicum var. spinossimum.

Cirsium japonicum belongs to the Asteraceae or Compositae family and is a medicinal plant in Asia that has a variety of effects, including tumour inhibition, improved immunity with flavones, and antidiabetic and hepatoprotective effects. Silymarin is synthesized by 4-coumaroyl-CoA via both the flavonoid and phenylpropanoid pathways to produce the immediate precursors taxifolin and coniferyl alcohol. Then, the oxidative radicalization of taxifolin and coniferyl alcohol produces silymarin. We identified the expression of genes related to the synthesis of silymarin in C. japonicum in three different tissues, namely, flowers, leaves and roots, through RNA sequencing. We obtained 51,133 unigenes from transcriptome sequencing by de novo assembly using Trinity v2.1.1, TransDecoder v2.0.1, and CD-HIT v4.6 software. The differentially expressed gene analysis revealed that the expression of genes related to the flavonoid pathway was higher in the flowers, whereas the phenylpropanoid pathway was more highly expressed in the roots. In this study, we established a global transcriptome dataset for C. japonicum. The data shall not only be useful to focus more deeply on the genes related to product medicinal metabolite including flavolignan but also to study the functional genomics for genetic engineering of C. japonicum.

Development of microsatellite markers for Viscum coloratum (Santalaceae) and their application to wild populations.

PREMISE OF THE STUDY: Microsatellite primers were developed for Viscum coloratum (Santalaceae), a semiparasitic medicinal plant that is known for its anticancer properties. Due to excessive human harvesting and loss of suitable habitat of its populations, it has become a potentially threatened species requiring immediate conservation efforts. METHODS AND RESULTS: Based on transcriptome data for V. coloratum, 124 primer pairs were randomly selected for initial validation, of which 19 yielded polymorphic microsatellite loci, with two to six alleles per locus. The usefulness of these markers was assessed for 60 individuals representing three populations of V. coloratum. Observed and expected heterozygosity values ranged from 0.033 to 0.833 and 0.032 to 0.672, respectively. Cross-species amplification for 19 loci in the related species V. album was conducted. CONCLUSIONS: The 19 newly developed loci are expected to be useful for studying the population genetics and ecological conservation of V. coloratum.

Annulohpoxylotol A and B, new sesquiterpenoids from the endophytic fungus Annulohypoxylon truncatum, are natural NF-κB inhibitors.

Two sesquiterpenoids, annulohpoxylotol A and B, were isolated along with five sterols from an ethyl acetate extract of cultures of the endophytic fungus Annulohypoxylon truncatum growing on leaves of Zizania caduciflora. The structures of the isolated compounds were established using one-dimensional (1D) and two-dimensional (2D)-NMR and mass spectrometry. The nuclear factor-kappa B (NF-κB) inhibitory activities of the isolated compounds stimulated with tumor necrosis factor-alpha (TNF-α) were measured using a luciferase reporter system. Annulohpoxylotol A (1) significantly inhibited NF-κB activation in a dose-dependent manner, with an IC50 of 7.11 µM, whereas annulohpoxylotol B (2) and ergone (7) moderately inhibited NF-κB transcriptional activity, with IC50 values of 19.24 and 17.51 µM, respectively.

Isochromans and Related Constituents from the Endophytic Fungus Annulohypoxylon truncatum of Zizania caduciflora and Their Anti-Inflammatory Effects.

Six new isochroman derivatives (annulohypoxylomans A-C, 1-3; annulohypoxylomanols A and B, 6 and 7; and annulohypoxyloside, 8), an isocoumarin (annulohypoxylomarin A, 4), and an azaphilone derivative (xylariphilone, 5) were isolated from an ethyl acetate extract derived from cultures of the endophytic fungus JS540 found in the leaves of Zizania caduciflora. The JS540 strain was identified as Annulohypoxylon truncatum. The structures of the isolated compounds were elucidated by one- and two-dimensional nuclear magnetic resonance and mass spectrometry and by comparison with related compounds from the literature. The anti-inflammatory activities of the isolated compounds were evaluated in lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells. Xylariphilone (5) exhibited significant inhibitory effects on LPS-induced interleukin (IL)-6, IL-12 p40, and tumor necrosis factor (TNF)-α production with IC50 values of 5.3, 19.4, and 37.6 µM, respectively.

MP-V1 from the Venom of Social Wasp Vespula vulgaris Is a de Novo Type of Mastoparan that Displays Superior Antimicrobial Activities.

Mastoparans from the venom of social wasps have attracted considerable attention as effective antibiotic candidates. In this study, mastoparan V1 (MP-V1) from Vespula vulgaris was first disclosed to have a peptide amino acid sequence distinct from typical mastoparans and its biochemical properties and antimicrobial effects were compared with those of typical mastoparans MP-L, -X(V) and -B. Circular dichroism (CD) spectroscopy revealed that MP-V1 and -X(V) form more stable α-helical conformations in lipid membrane-like environments than MP-L and -B. In parallel, these two also showed more effective antimicrobial activities against the pathogens than did MP-L and -B. Although MP-V1 had a less stable α-helical conformation than MP-X(V), it showed stronger antimicrobial effects against Streptococcus mutans and Salmonella enterica than MP-X(V). In the meantime, analysis of hemolytic activity revealed a range of doses (~50 µM) that exhibited little potent cytotoxicity on human erythrocytes. Finally, the atypical MP-V1 peptide amino acid sequence provided important clues to understanding its antimicrobial mechanism from a structural perspective. Therefore, it has been concluded that MP-V1 is a de novo type of mastoparan with superior antimicrobial activities against both pathogenic bacteria and fungi, which may be useful in developing multipurpose antimicrobial drugs against infectious diseases.

Draft Genome Sequence of Lichen-Forming Fungus Cladonia metacorallifera Strain KoLRI002260.

The lichen-forming fungus Cladonia metacorallifera strain KoLRI002260 is capable of producing a number of secondary metabolites, including usnic, didymic, and squamatic acids, which have antitumor, antioxidant, and antibiotic activities. The draft genome assembly has a size of 36,682,060 bp, with a G+C content of 44.91%, and consists of 30 scaffolds.

Cyclophilin 20-3 relays a 12-oxo-phytodienoic acid signal during stress responsive regulation of cellular redox homeostasis.

The jasmonate family of phytohormones plays central roles in plant development and stress acclimation. However, the architecture of their signaling circuits remains largely unknown. Here we describe a jasmonate family binding protein, cyclophilin 20-3 (CYP20-3), which regulates stress-responsive cellular redox homeostasis. (+)-12-Oxo-phytodienoic acid (OPDA) binding promotes CYP20-3 to form a complex with serine acetyltransferase 1, which triggers the formation of a hetero-oligomeric cysteine synthase complex with O-acetylserine(thiol)lyase B in chloroplasts. The cysteine synthase complex formation then activates sulfur assimilation that leads to increased levels of thiol metabolites and the buildup of cellular reduction potential. The enhanced redox capacity in turn coordinates the expression of a subset of OPDA-responsive genes. Thus, we conclude that CYP20-3 is a key effector protein that links OPDA signaling to amino acid biosynthesis and cellular redox homeostasis in stress responses.

The colchicine derivative CT20126 shows a novel microtubule-modulating activity with apoptosis.

New colchicine analogs have been synthesized with the aim of developing stronger potential anticancer activities. Among the analogs, CT20126 has been previously reported to show immunosuppressive activities. Here, we report that CT20126 also shows potential anticancer effects via an unusual mechanism: the modulation of microtubule integrity and cell cycle arrest at the G2/M phase before apoptosis. When we treated COS-7 cells with CT20126 (5 µM), the normal thread-like microtubules were disrupted into tubulin dimers within 10 min and thereafter repolymerized into short, thick filaments. In contrast, cells treated with the same concentration of colchicine exhibited microtubule depolymerization after 20 min and never underwent repolymerization. Furthermore, optical density (OD) analysis (350 nm) with purified tubulin showed that CT20126 had a higher repolymerizing activity than that of Taxol, a potent microtubule-polymerizing agent. These results suggest that the effects of CT20126 on microtubule integrity differ from those of colchicine: the analog first destabilizes microtubules and then stabilizes the disrupted tubulins into short, thick polymers. Furthermore, CT20126 induced a greater level of apoptotic activity in Jurkat T cells than colchicine (assessed by G2/M arrest, caspase-3 activation and cell sorting). At 20 nM, CT20126 induced 47% apoptosis among Jurkat T cells, whereas colchicine induced only 33% apoptosis. Our results suggest that the colchicine analog CT20126 can potently induce apoptosis by disrupting microtubule integrity in a manner that differs from that of colchicine or Taxol.

Design, synthesis and identification of novel colchicine-derived immunosuppressant.

Synthesis and biological evaluation of various colchicine analogues through the mixed-lymphocyte reaction (MLR), lymphoproliferation, and inhibitory effects on the inflammatory genes are described. In addition, a new series of immunosuppressive agents developed on the structural basis of colchicine, as well as their structure-activity relationships is reported. The most potent analogue 20a exhibited an excellent immunosuppressive activity on in vivo skin-allograft model, which is comparable to that of cyclosporin A.

MoCRZ1, a gene encoding a calcineurin-responsive transcription factor, regulates fungal growth and pathogenicity of Magnaporthe oryzae.

Ca(2+)-dependent signaling plays important roles in cellular development and metabolism in fungi. Pharmacological and molecular evidence clearly indicates that Ca(2+)-dependent signaling is required for infection-related development and pathogenicity in the rice blast fungus Magnaporthe oryzae. However, little information is available on downstream regulators in the Ca(2+)-dependent signaling pathway. To understand the role of a calcineurin-dependent transcription factor in the rice blast fungus, an ortholog of Saccharomyces cerevisiae CRZ1 in M. oryzae, MoCRZ1, was identified and functionally characterized. The Deltamocrz1 mutant exhibited impaired growth in the presence of Ca(2+) ions or cell wall perturbing agents. The Deltamocrz1 mutant also showed reduced conidiation and reduced pathogenicity, which is mainly due to a defect in host penetration. MoCRZ1 fused to EGFP was trans-localized into the nucleus in a Ca(2+)/calcineurin-dependent manner. The MoCRZ1 gene is also required for the calcineurin-dependent transcriptional induction of FKS1, a gene encoding a beta-1,3 glucan synthase, CHS2 and CHS4, genes encoding two chitin synthases, and PMC and PMR gene families encoding P-type ATPases in response to Ca(2+). These results suggest that MoCRZ1 is a downstream regulator in Ca(2+)-dependent signaling for pathogenicity in M. oryzae, and its biochemical mechanisms are well conserved among fungal species.

CT20126, a novel immunosuppressant, prevents collagen-induced arthritis through the down-regulation of inflammatory gene expression by inhibiting NF-kappaB activation.

The colchicine-derived CT20126 compound has recently been shown to exert an immune regulatory effect and prolong the survival of allograft skins. In this study, we explored the anti-inflammatory and anti-arthritic effects of CT20126 in vivo and in vitro as well as investigated its underlying action mechanism. CT20126 suppressed the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2, tumor necrosis factor-alpha, and interleukin-1beta as well as the production of nitric oxide and prostaglandin E(2) in lipopolysaccharide (LPS)-treated macrophages as well as LPS-administered mice. This drug also inhibited the production of nitric oxide, prostaglandin E(2), and the chemokines, RANTES, GROalpha, and ENA-78, in cytokine-stimulated human synoviocytes. CT20126 suppressed NF-kappaB activation and iNOS promoter activity, which correlated with its inhibitory effect on phosphorylation-dependent IkappaB kinase activation, IkappaB phosphorylation and degradation, and NF-kappaB nuclear translocation, in LPS-stimulated macrophages. This compound also inhibited LPS-induced NF-kappaB-inducing kinase (NIK) and Akt phosphorylation, which are upstream of NF-kappaB activation. Furthermore, CT20126 significantly decreased the incidence and severity of arthritis as well as inhibited the expression of inflammatory cytokines, chemokines, iNOS, and cyclooxygenase-2 in the paws of collagen-induced arthritic mice. These findings indicate that CT20126 exerts an anti-inflammatory effect through NF-kappaB-responsive inflammatory gene expression by inhibiting the NIK- and Akt-dependent canonical NF-kappaB pathway and can be used as a therapeutic agent for rheumatoid arthritis related to chronic inflammation.

Colchicine-derived compound CT20126 promotes skin allograft survival by regulating the balance of Th1 and Th2 cytokine production.

Colchicine has been shown to regulate the expression of inflammatory gene, but this compound possesses much weaker anti-inflammatory activity. In this study, we synthesized a new colchicine derivative CT20126 and examined its immunomodulatory property. CT20126 was found to have immunosuppressive effects by inhibiting lymphocyte proliferation without cytotoxicity and effectively inhibit the transcriptional expression of the inflammatory genes, iNOS, TNF-alpha, and IL-1beta, in macrophages stimulated by LPS. This effect was nearly comparable to that of cyclosporine A. This compound also significantly suppressed the production of nitric oxide and Th1-related pro-inflammatory cytokines, IL-1beta, TNF-alpha, and IL-2, with minimal suppression of Th2-related anti-inflammatory cytokines IL-4 and IL-10 in the sponge matrix allograft model. Moreover, administration of CT20126 prolonged the survival of allograft skins from BALB/c mice (H-2(d)) to the dorsum of C57BL/6 (H-2(b)) mice. The in vivo immune suppressive effects of CT20126 were similar to that of cyclosporine A. These results indicate that this compound may have potential therapeutic value for transplantation rejection and other inflammatory diseases.

MHP1, a Magnaporthe grisea hydrophobin gene, is required for fungal development and plant colonization.

Fungal hydrophobins are implicated in cell morphogenesis and pathogenicity in several plant pathogenic fungi including the rice blast fungus Magnaporthe grisea. A cDNA clone encoding a hydrophobin (magnaporin, MHP1) was isolated from a cDNA library constructed from rice leaves infected by M. grisea. The MHP1 codes for a typical fungal hydrophobin of 102 amino acids containing eight cysteine residues spaced in a conserved pattern. Hydropathy analysis of amino acids revealed that MHP1 belongs to the class II group of hydrophobins. The amino acid sequence of MHP1 exhibited about 20% similarity to MPG1, an M. grisea class I hydrophobin. Expression of MHP1 was highly induced during plant colonization and conidiation, but could hardly be detected during mycelial growth. Transformants in which MHP1 was inactivated by targeted gene replacement showed a detergent wettable phenotype, but were not altered in wettability with water. mhp1 mutants also exhibited pleiotropic effects on fungal morphogenesis, including reduction in conidiation, conidial germination, appressorium development and infectious growth in host cells. Furthermore, conidia of mhp1 mutants were defective in their cellular organelles and rapidly lose viability. As a result, mhp1 mutants exhibited a reduced ability to infect and colonize a susceptible rice cultivar. These phenotypes were recovered by re-introduction of an intact copy of MHP1. Taken together, these results indicate that MHP1 has essential roles in surface hydrophobicity and infection-related fungal development, and is required for pathogenicity of M. grisea.

TRF2 is in neuroglial cytoplasm and induces neurite-like processes.

TRF2 is a ubiquitous protein that protects telomeres in the nucleus. We found that TRF2 was present at the peripheral nerve axons and the brain neuroglial cell processes extensively. It was in the cytoplasmic membrane as well as nuclear fractions, but not in the soluble cytoplasmic fraction of SH-SY5Y neuroblastoma cells. TRF2 was up-regulated in P19 embryonal carcinoma cells at the early stage of induced neural differentiation with retinoic acid treatment. Upon transfection, TRF2-expressing COS cells often produced neurite-like long cytoplasmic processes. TRF2 is a component of neuroglial cells and appears to be involved in the cytoplasmic process formation that is necessary for neural differentiation.

Expression profiling and subtype-specific expression of stomach cancer.

The expression profiling and molecular grouping of stomach cancers has been a challenging task because of their complexity and variation. We have analyzed gene expression profiles of 22 gastric cancer/nontumor mucosa couples using 14K cDNA microarray chips designed for gastric cancer analysis. Upon pairwise analysis of the individual couples at the false significance rate 0.91%, 79 and 398 genes were reported to be up-regulated and down-regulated in tumors, respectively. Tumors were clustered into two groups having high and low inflammatory infiltration, respectively. The latter consisted of three subgroups, including diffuse type carcinomas and intestinal types with distinct pathological characteristics of aggressive behavior. When the pooled tumor was hybridized against the pooled nontumor mucosa samples, more genes were detected to express differentially than those detected by the pairwise analysis at the same threshold level. However, they did not render satisfactory clustering of individual tumors. Our data showed that stomach cancers could be clustered effectively using stomach-specific microarrays and pairwise analysis of tumor/nontumor mucosa couples. It is suggested that the application of specific goal-oriented experimental designing would be advantageous for efficient analysis of expression profiles of such a complex disease as gastric cancer.