Rigosertib has demonstrated therapeutic activity for patients with high-risk myelodysplastic syndrome (MDS) in clinical trials. However, the role of rigosertib in MDS has not been thoroughly characterized. In this study, we found out that rigosertib induced apoptosis, blocked the cell cycle at the G2/M phase and subsequently inhibited the proliferation of CD34+ cells from MDS, while it minimally affected the normal CD34+ cells. Further studies showed that rigosertib acted via the activation of the P53 signaling pathway. Bioinformatics analysis based on gene expression profile and flow cytometry analysis revealed the abnormal activation of the Akt-PI3K, Jak-STAT and Wnt pathways in high-grade MDS, while the p38 MAPK, SAPK/JNK and P53 pathways were abnormally activated in low-grade MDS. Rigosertib could markedly inhibit the activation of the Akt-PI3K and Wnt pathways, whereas it activated the SAPK/JNK and P53 pathways in high-grade MDS. A receptor tyrosine kinase phosphorylation array demonstrated that rigosertib could increase the activation of RET and PDGFR-β while reducing the activation of Tie2 and VEGFR2 in MDS cells. Taken together, these data indicate that rigosertib is a selective and promising anti-tumor agent that could ameliorate multiple dysregulated signaling transduction pathways in high-grade MDS.

The dry root and rhizome of Salvia miltiorrhiza Bunge, or Danshen, is a well-known, traditional Chinese medicine. Tanshinones are active compounds that accumulate in the periderm, resulting in red-colored roots. However, lines with orange roots have been observed in cultivated fields. Here, we performed metabolome and transcriptome analyses to investigate the changes of orange-rooted Danshen.

Andrographis paniculata (Burm.f.) Wall. ex Nees is widely used as medicinal herb in Southern and Southeastern Asia and andrographolide is its main medicinal constituent. Based on the structure of andrographolide, it has been proposed that cytochrome P450 enzymes play vital roles on its biosynthesis. NADPH:cytochrome P450 reductase (CPR) is the most important redox partner of multiple P450s. In this study, three CPRs were identified in the genomic data of A. paniculata (namely ApCPR1, ApCPR2, and ApCPR3), and their coding regions were cloned. They varied from 62% to 70% identities to each other at the amino acid sequence level. ApCPR1 belongs to Class I of dicotyledonous CPR while both ApCPR2 and ApCPR3 are grouped to Class II. The recombinant enzymes ApCPR1 and ApCPR2 reduced cytochrome c and ferricyanide in an NADPH-dependent manner. In yeast, they supported the activity of CYP76AH1, a ferruginol-forming enzyme. However, ApCPR3 did not show any enzymatic activities either in vitro or in vivo. Quantitative real-time PCR analysis showed that both ApCPR1 and ApCPR2 expressed in all tissues examined, but ApCPR2 showed higher expression in leaves. Expression of ApCPR2 was inducible by MeJA and its pattern matched with andrographolide accumulation. Present investigation suggested ApCPR2 involves in the biosynthesis of secondary metabolites including andrographolide.

Despite the improvements in prognostication of the revised International Prognostic Scoring System (IPSS-R) in myelodysplastic syndrome (MDS), there remain a portion of patients with lower risk (low/intermediate risk, LR) but poor prognostics. This study aimed to evaluate the relative contribution of mutational status when added to the IPSS-R, for estimating overall survival (OS) and progression-free survival (PFS) in patients with LR-MDS. We retrospectively analyzed clinical and laboratory variables of 328 patients diagnosed with MDS according to the FAB criteria. Twenty-nine-gene NGS assay was applied to bone marrow samples obtained at diagnosis. 233 (71.04%) patients were classified as LR-MDS. Univariate analysis showed association between inferior outcome (OS and PFS) and presence of JAK2 (p = 0.0177, p = 0.0002), RUNX1 (p = 0.0250, p = 0.0387), and U2AF1 (p = 0.0227, p = 0.7995) mutations. Multivariable survival analysis revealed JAK2 (p < 0.0001) and RUNX1 (p = 0.0215) mutations were independently prognostic for PFS in LR-MDS. Interestingly, bone marrow blast >1.5% could further predict disease progression of patients with LR-MDS (HR 8.06, 95%CI 2.95-22.04, p < 0.0001). Incorporation of JAK2, RUNX1 mutation and bone marrow blast in the IPSS-R can improve risk stratification in patients with LR-MDS. In summary, our result provided new risk factors for LR-MDS prognostics to identify candidates for early therapeutic intervention.

Bone marrow stromal cells from patients with myelodysplastic syndrome (MDS) display a senescence phenotype, but the underlying mechanism has not been elucidated. Pro-inflammatory signaling within the malignant clone and the bone marrow microenvironment has been identified as a key pathogenetic driver of MDS. Our study revealed that S100A9 is highly-expressed in lower-risk MDS. Moreover, normal primary mesenchymal stromal cells (MSCs) and the human stromal cell line HS-27a co-cultured with lower-risk MDS bone marrow mononuclear cells acquired a senescence phenotype. Exogenous supplemented S100A9 also induced cellular senescence in MSCs and HS-27a cells. Importantly, Toll-like receptor 4 (TLR4) inhibition or knockdown attenuated the cellular senescence induced by S100A9. Furthermore, we showed that S100A9 induces NLRP3 inflammasome formation, and IL-1β secretion; findings in samples from MDS patients further confirmed these thoughts. Moreover, ROS and IL-1β inhibition suppressed the cellular senescence induced by S100A9, whereas NLRP3 overexpression and exogenous IL-1β supplementation induces cellular senescence. Our study demonstrated that S100A9 promotes cellular senescence of bone marrow stromal cells via TLR4, NLRP3 inflammasome formation, and IL-1β secretion for its effects. Our findings deepen the understanding of the molecular mechanisms involved in MDS reprogramming of MSCs and indicated the essential role of S100A9 in tumor-environment interactions in bone marrow.

Manganese oxidizing bacteria can produce biogenic manganese oxides (BMO) on their cell surface and have been applied in the fields of agriculture, bioremediation, and drinking water treatment to remove toxic contaminants based on their remarkable chemical reactivity. Herein, we report for the first time the synthetic application of the manganese oxidizing bacteria, Pseudomonas putida MnB1 as a whole-cell biocatalyst for the effective oxidation of β-keto ester with excellent yield. Differing from known chemical protocols toward this transformation that generally necessitate the use of organic solvents, stoichiometric oxygenating agents and complex chemical catalysts, our strategy can accomplish it simply under aqueous and mild conditions with higher efficiency than that provided by chemical manganese oxides. Moreover, the live MnB1 bacteria are capable of continuous catalysis for this C-O bond forming reaction for several cycles and remain proliferating, highlighting the favorable merits of this novel protocol for sustainable chemistry and green synthesis.

MDS development is a dynamic process during which the accumulation of somatic mutations leads to specific malignant evolution. To elucidate the differential roles of gene mutations in typical MDS, we used targeted sequencing to investigate clonal patterns from 563 patients and focused on cases (199/563 cases) with initial mutations (ASXL1, DNMT3A and TET2) at MDS diagnosis. The consistency of frequency and distribution in patients with or without aberrant chromosomes suggested early events of these initial mutations. Some additional driver mutations (SF3B1, U2AF1 or RUNX1) played roles to keep the basic disease features, or give rise to different phenotypes (BCOR, EZH2 or TP53) in individual patients. Notably, analysis in paired samples before and after MDS progression showed that the mutations identified as last events (involving active signaling, myeloid transcription or tumor suppressor) seemed necessary for MDS development to be AML. Last mutations can exist at MDS diagnosis, or emerge at AML transformation, and involve a small group of genes. Single-allele CEBPA mutations and diverse TP53 mutations were checked as the most common last event mutations. Considering the necessity of last event mutations and limited gene involvement in AML transformations, it is possible to validate a small group of last events involved mutations to develop some new strategies to block MDS progression.

BCOR (BCL6 corepressor) is an epigenetic regulator gene involved in the specification of cell differentiation and body structure development. Recurrent somatic BCOR mutations have been identified in myelodysplastic syndrome (MDS). However, the clinical impact of BCOR mutations on MDS prognosis is controversial and the response of hypomethylating agents in MDS with BCOR mutations (BCORMUT) remains unknown.

Mutations in the U2 small nuclear RNA auxiliary factor 1 (U2AF1) gene are the common feature of a major subset in myelodysplastic syndromes (MDS). However, the genetic landscape and molecular pathogenesis of oncogenic U2AF1S34F mutation in MDS are not totally understood. We performed comprehensive analysis for prognostic significance of U2AF1 mutations in acute myeloid leukemia (AML) cohort based on The Cancer Genome Atlas (TCGA) database. Functional analysis of U2AF1S34F mutation was performed in vitro. Differentially expressed genes (DEGs) and significantly enriched pathways were identified by RNA sequencing. The forkhead box protein O3a (FOXO3a) was investigated to mediate the function of U2AF1S34F mutation in cell models using lentivirus. Chromatin immunoprecipitation, immunoblotting analyses, and immunofluorescence assays were also conducted. U2AF1 mutations were associated with poor prognosis in MDS and AML samples, which significantly inhibited cell proliferation and induced cellular apoptosis in cell models. Our data identified that U2AF1-mutant cell lines undergo FOXO3a-dependent apoptosis and NLRP3 inflammasome activation, which induces pyroptotic cell death. Particularly, an increase in the level of FOXO3a promoted the progression of MDS in association with restored autophagy program leading to NLRP3 inflammasome activation in response to U2AF1S34F mutation. Based on the result that U2AF1S34F mutation promoted the transcriptional activity of Bim through upregulating FOXO3a with transactivation of cell cycle regulators p21Cip1 and p27Kip1, FOXO3a, a potentially cancer-associated transcription factor, was identified as the key molecule on which these pathways converge. Overall, our studies provide new insights that U2AF1S34F mutation functions the crucial roles in mediating MDS disease progression via FOXO3a activation, and demonstrate novel targets of U2AF1 mutations to the pathogenesis of MDS.

CYP76AH1 is the key enzyme in the biosynthesis pathway of tanshinones in Salvia miltiorrhiza, which are famous natural products with activities against various heart diseases and others. CYP76AH1 is a membrane-associated typical plant class II cytochrome P450 enzyme and its catalytic mechanism has not to be clearly elucidated. Structural determination of eukaryotic P450 enzymes is extremely challenging. Recently, we solved the crystal structures of CYP76AH1 and CYP76AH1 in complex with its natural substrate miltiradiene. The structure of CYP76AH1 complexed with miltiradiene is the first plant cytochrome P450 structure in complex with natural substrate. The studies revealed a unique array pattern of amino acid residues, which may play an important role in orienting and stabilizing the substrate for catalysis. This work would provide structural insights into CYP76AH1 and related P450s and the basis to efficiently improve tanshinone production by synthetic biology techniques.

DHX9 is a member of the DEAH (Asp-Glu-Ala-His) helicase family and regulates DNA replication and RNA processing. DHX9 dysfunction promotes tumorigenesis in several solid cancers. However, the role of DHX9 in MDS is still unknown. Here, we analyzed the expression of DHX9 and its clinical significance in 120 MDS patients and 42 non-MDS controls. Lentivirus-mediated DHX9-knockdown experiments were performed to investigate its biological function. We also performed cell functional assays, gene microarray, and pharmacological intervention to investigate the mechanistic involvement of DHX9. We found that overexpression of DHX9 is frequent in MDS and associated with poor survival and high risk of acute myeloid leukemia (AML) transformation. DHX9 is essential for the maintenance of malignant proliferation of leukemia cells, and DHX9 suppression increases cell apoptosis and causes hypersensitivity to chemotherapeutic agents. Besides, knockdown of DHX9 inactivates the PI3K-AKT and ATR-Chk1 signaling, promotes R-loop accumulation, and R-loop-mediated DNA damage.

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease characterized by hyperglycemia and insulin resistance. Mung bean sprouts are traditionally considered a "folk" hypoglycemic food and their pharmacological effects and underlying mechanisms warrant further investigation.

Cytochrome P450 reductase (CPR) is the redox partner of various P450s involved in primary and secondary metabolism. Here, we identified and characterized two paralogs of cytochrome P450 reductase from Siraitia grosvenorii. There were two full-length CPR isoforms in the S. grosvenorii fruit transcriptome dataset. They had the same open reading frames of 2, 124 bp, encoding 707 amino acids. A phylogenetic analysis characterized both SgCPR1 and SgCPR2 as Class II dicotyledonous CPRs. The recombinant proteins SgCPR1 and SgCPR2 could reduce cytochrome c and ferricyanide in a NADPH-dependent manner. The SgCPR1 and SgCPR2 transcripts were detected in all examined tissues of S. grosvenorii, and in fresh fruit, they had expression patterns similar to several key enzymes that require CPR as a partner during their biosynthesis. The expression levels of the SgCPRs were induced after a methyl jasmonate treatment. The extracts from yeast co-expressing SgCPR1/SgCPR2 and the cytochrome P450 enzyme CYP76AH1 produced ferruginol, indicating the positive effects of SgCPR1/SgCPR2 on the CYP76AH1 activity. A docking analysis confirmed the experimentally deduced functional activities of SgCPR1 and SgCPR2 for NADPH, FAD and FMN. Thus, SgCRP1 and SgCPR2 are both likely to participate in secondary metabolism, especially mogroside biosynthesis in S. grosvenorii.

Tanshinones are important diterpenoid secondary metabolites from Salvia miltiorrhiza, widely used as cardiovascular and cerebrovascular medicines. CYP76AH1 is a membrane-associated cytochrome P450 enzyme and plays a critical role in the biosynthetic pathway of tanshinones. To clarify the relationship between structure and function of CYP76AH1, we recently constructed the expression vector of CYP76AH1 and purified the enzyme. The engineered CYP76AH1 was expressed in E. coli Trans-blue cells and exhibited enhanced expression and solubility. The proper folding of the engineered CYP76AH1 was assessed by CO difference spectrum assay. Functional identification of the recombinant enzyme was performed by conducting enzymatic reaction with the purified CYP76AH1 in presence of substrate, the co-factor NADPH and the purified SmCPR1 (cytochrome P450 reductase from Salvia miltiorrhiza), and by subsequently analyzing the reaction extract through GC-MS. X-ray crystal complex structure of CYP76AH1 with inhibitor 4-phenylimmidazole (4-PI) was determined at the resolution of 2.6 Å. In the ligand-binding cavity of 4-PI bound CYP76AH1, the inhibitor 4-PI forms a hydrogen bound with a water molecule which coordinates with heme at the sixth coordination position. There are two open channels which substrate and product site may access and leave the active site. In the CYP76AH1/4-PI complex structure, the imidazole ring of 4-PI is parallel to helix I instead of perpendicular to helies I in most P450s bound imidazole. 4-PI may be work in the stability of CYP76AH1 crystal structure. These studies provide information on functional expression and purification of CYP76AH1, and overall structure of CYP76AH1 complexed with 4-PI.

To clarify the possible biological differences and implication of the SF3B1 gene for patients with MDS-RS (myelodysplastic syndromes with ring sideroblasts).

The role of mesenchymal stromal cells (MSCs) in the pathogenesis of myelodysplastic syndromes (MDS) has been increasingly addressed, but has yet to be clearly elucidated. In this investigation, we found that MDS cells proliferated to a greater extent on MDS-derived MSCs compared to normal MSCs. Matrix metalloproteinase 1(MMP1), which was downregulated in MDS-MSCs, was identified as an inhibitory factor of MDS cell proliferation, given that treatment with an MMP1 inhibitor or knock-down of MMP1 in normal MSCs resulted in increased MDS cell proliferation. Further investigations indicated that MMP1 induced apoptosis of MDS cells by interacting with PAR1 and further activating the p38 MAPK pathway. Inhibition of either PAR1 or p38 MAPK can reverse the apoptosis-inducing effect of MMP1. Taken together, these data indicate that downregulation of MMP1 in MSCs of MDS patients may contribute to the reduced capacity of MSCs to restrict MDS cell proliferation, which may account for the malignant proliferation of MDS cells.

Stripe rust, caused by the biotrophic fungus Puccinia striiformis f. sp. tritici (Pst), is one of the most widespread and destructive wheat diseases worldwide. Growing resistant cultivars is an effective approach for controlling this disease. However, because host resistance genes were easily overcome by new virulent Pst races, there is a continuous demand for identifying new effective wheat stripe rust resistance genes and develop closely linked markers for marker-assisted selection (MAS). Baidatou, an old Chinese wheat landrace, has been grown for several decades in Longnan region, Gansu Province, where stripe rust epidemics are frequent and severe. In our previous study, a single dominant gene YrBai in Baidatou was identified to control the adult-plant resistance (APR) to Chinese prevalent Pst race CYR33. And the gene was located on wheat chromosome 6DS by four polymorphic simple sequence repeat (SSR) and two sequence-related amplified polymorphism (SRAP) markers, with the genetic distances of two closely linked markers 3.6 and 5.4 cM, respectively. To further confirm the APR gene in Baidatou and construct the high-density map for the resistance gene, adult plants of F1, F2, F3, and F5:6 populations derived from the cross Mingxian169/Baidatou and two parents were inoculated with CYR33 at Yangling field, Shaanxi Province during 2014-2015, 2015-2016, and 2016-2017 crop seasons, respectively. The field evaluation results indicated that a single dominant gene confers the APR to Pst race CYR33 in Baidatou. 92 F3 lines and parents were sequenced using DArTseq technology based on wheat GBS1.0 platform, and 31 genetic maps consisted of 2,131 polymorphic SilicoDArT and 952 SNP markers spanning 4,293.94 cM were constructed. Using polymorphic SilicoDArT, SNP markers and infection types (ITs) data of F3 lines, the gene YrBai was further located in 0.8 cM region on wheat chromosome 6D. These closely linked markers developed in this study should be useful for MAS for Baidatou in crop improvement and map-based clone this gene.

Our previous studies have clarified that red nucleus (RN) interleukin (IL)-6 is involved in the maintenance of neuropathic pain and produces a facilitatory effect by activating JAK2/STAT3 and ERK pathways. In this study, we further explored the immune molecular mechanisms of rubral IL-6-mediated descending facilitation at the spinal cord level. IL-6-evoked tactile allodynia was established by injecting recombinant IL-6 into the unilateral RN of naive male rats. Following intrarubral administration of IL-6, obvious tactile allodynia was evoked in the contralateral hindpaw of rats. Meanwhile, the expressions of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), IL-1β, and IL-6 were elevated in the contralateral spinal dorsal horn (L4-L6), blocking spinal TNF-α, IL-1β, or IL-6 with neutralizing antibodies relieved IL-6-evoked tactile allodynia. Conversely, the levels of anti-inflammatory cytokines transforming growth factor-β (TGF-β) and IL-10 were reduced in the contralateral spinal dorsal horn (L4-L6), an intrathecal supplement of exogenous TGF-β, or IL-10 attenuated IL-6-evoked tactile allodynia. Further studies demonstrated that intrarubral pretreatment with JAK2/STAT3 inhibitor AG490 suppressed the elevations of spinal TNF-α, IL-1β, and IL-6 and promoted the expressions of TGF-β and IL-10 in IL-6-evoked tactile allodynia rats. However, intrarubral pretreatment with ERK inhibitor PD98059 only restrained the increase in spinal TNF-α and enhanced the expression of spinal IL-10. These findings imply that rubral IL-6 plays descending facilitation and produces algesic effect through upregulating the expressions of spinal pro-inflammatory cytokines TNF-α, IL-1β, and IL-6 and downregulating the expressions of spinal anti-inflammatory cytokines TGF-β and IL-10 by activating JAK2/STAT3 and/or ERK pathways, which provides potential therapeutic targets for the treatment of pathological pain.

Novel sequencing designs are necessary to supplement the recognized knowledge of myelodysplastic syndrome (MDS)-related genomic alterations. In this study, we sequenced 28 target genes in 320 Chinese MDS patients but obtained 77.2% of recall factors and 82.8% of genetic abnormalities (including karyotype abnormalities). In addition to known relationships among mutations, some specific chromosomal abnormalities were found to link to specific gene mutations. Trisomy 8 tended to be linked to U2AF1 and ZRSR2 mutations, and 20q- exhibited higher SRSF2/WT1 and U2AF1 mutation frequency. Chromosome 7 involvement accounted for up to 50% of RUNX1 mutations and 37.5% of SETBP1 mutations. Patients carrying a complex karyotype were prone to present TP53 mutations (36.1%). However, individuals with normal karyotypes rarely possessed mutations in the TP53, RUNX1 and U2AF1. Moreover, DNMT3A, TP53, SRSF2, STAG2, ROBO1/2 and WT1 predicted poor survival and high AML transformation. By integrating these predictors into international prognostic scoring system (IPSS) or revised IPSS, we built a set of mutation-based prognostic risk models. These models could layer different degrees of risk in patients more satisfactorily. In summary, this sequencing design was able to detect a number of gene mutations and could be used to stratify patients with varied prognostic risk.

Decitabine (DAC) is considered to be a profound global DNA demethylation, which can induce the re-expression of silenced tumor suppressor genes. Little is known about the function of tumor suppressor gene FOXO1 in myelodysplastic syndromes (MDS). To address this issue, the study firstly investigated differentially expressed genes (DEGs) for DAC treatment in MDS cell lines, then explored the role of FOXO1 through silencing its expression before DAC treatment in MDS. The results showed that FOXO1 exists in a hyperphosphorylated, inactive form in MDS-L cells. DAC treatment both induces FOXO1 expression and reactivates the protein in its low phosphorylation level. Additionally, the results also demonstrated that this FOXO1 activation is responsible for the DAC-induced apoptosis, cell cycle arrest, antigen differentiation, and immunoregulation in MDS-L cells. We also demonstrated DAC-induced FOXO1 activation upregulates anti-tumor immune response in higher-risk MDS specimens. Collectively, these results suggest that DAC induces FOXO1 activation, which plays an important role in anti-MDS tumors.