Targeting BIRC5 as a therapeutic approach to overcome ASXL1-associated decitabine resistance.

Hypomethylating agents (HMAs) are widely employed in the treatment of myeloid malignancies. However, unresponsive or resistant to HMAs occurs in approximately 50 % of patients. ASXL1, one of the most commonly mutated genes across the full spectrum of myeloid malignancies, has been reported to predict a lower overall response rate to HMAs, suggesting an essential need to develop effective therapeutic strategies for the patients with HMA failure. Here, we investigated the impact of ASXL1 on cellular responsiveness to decitabine treatment. ASXL1 deficiency increased resistance to decitabine treatment in AML cell lines and mouse bone marrow cells. Transcriptome sequencing revealed significant alterations in genes regulating cell cycle, apoptosis, and histone modification in ASXL1 deficient cells that resistant to decitabine. BIRC5 was identified as a potential target for overcoming decitabine resistance in ASXL1 deficient cells. Furthermore, our experimental evidence demonstrated that the small-molecule inhibitor of BIRC5 (YM-155) synergistically sensitized ASXL1 deficient cells to decitabine treatment. This study sheds light on the molecular mechanisms underlying the ASXL1-associated HMA resistance and proposes a promising therapeutic strategy for improving treatment outcomes in affected individuals.

Prognostic Value of Wagner Grade and Platelet Level in Diabetics with Infected Foot Ulcers After Antibiotic Therapy.

Purpose: The aim of the current study was to investigate factors potentially associated with the healing of infected foot ulcers in patients with diabetes after antimicrobial therapy with drugs selected based on antimicrobial susceptibility testing. Patients and Methods: A retrospective study was conducted to analyze clinical data from 99 type 2 diabetes mellitus patients with foot infection admitted to our center from January 2016 to December 2020. Pathogenic characteristics, results of wound discharge testing, and relevant wound surface factors were analyzed. Etiological characteristics and the results of susceptibility testing, wound healing rates, and factors potentially associated with wound healing rates were also analyzed. Results: Baseline data were analyzed via the t-test for independent samples, the Mann-Whitney U-test, and the chi-square test to identify variables significantly associated with prognosis. Least absolute shrinkage and selection operator regression analysis then determined that Wagner grade, essential hypertension, platelets, Gram negative bacteria, and neutrophil-to-lymphocyte ratio were of predictive value. A nomogram plot was built based on these five variables, and it yielded a standard C-index of 0.964, and an internally corrected C-index of 0.931. In multivariate logistic regression analysis Wagner grade (odds ratio [OR] 12.30, 95% confidence interval [CI] 2.471-61.194, p = 0.002) and platelet level (OR 0.978, 95% CI 0.960-0.996, p = 0.018) were significantly associated with wound healing outcomes. Restricted cubic spline analysis indicated that there was a linear relationship between wound healing and platelet levels, and that this relationship was strongest in patients classified as Wagner grade 2 with a platelet count ≤ 200 (p for nonlinearity = 0.442). Conclusion: Wagner grade, essential hypertension, platelet count, Gram negative bacteria, and neutrophil-to-lymphocyte ratio could predict the course of healing of infected foot ulcers in type 2 diabetes mellitus patients. When the Wagner grade was 2 and the platelet level was ≤ 200, platelet level was linearly associated with healing outcome, whereby a lower platelet level predicted a worse wound healing outcome.

STING activation in TET2-mutated hematopoietic stem/progenitor cells contributes to the increased self-renewal and neoplastic transformation.

Somatic loss-of-function mutations of the dioxygenase Ten-eleven translocation-2 (TET2) occur frequently in individuals with clonal hematopoiesis (CH) and acute myeloid leukemia (AML). These common hematopoietic disorders can be recapitulated in mouse models. However, the underlying mechanisms by which the deficiency in TET2 promotes these disorders remain unclear. Here we show that the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway is activated to mediate the effect of TET2 deficiency in dysregulated hematopoiesis in mouse models. DNA damage arising in Tet2-deficient hematopoietic stem/progenitor cells (HSPCs) leads to activation of the cGAS-STING pathway which in turn promotes the enhanced self-renewal and development of CH. Notably, both pharmacological inhibition and genetic deletion of STING suppresses Tet2 mutation-induced aberrant hematopoiesis. In patient-derived xenograft (PDX) models, STING inhibition specifically attenuates the proliferation of leukemia cells from TET2-mutated individuals. These observations suggest that the development of CH associated with TET2 mutations is powered through chronic inflammation dependent on the activated cGAS-STING pathway and that STING may represent a potential target for intervention of relevant hematopoietic diseases.

Vaccinia virus induces EMT-like transformation and RhoA-mediated mesenchymal migration.

The emerging outbreak of monkeypox is closely associated with the viral infection and spreading, threatening global public health. Virus-induced cell migration facilitates viral transmission. However, the mechanism underlying this type of cell migration remains unclear. Here we investigate the motility of cells infected by vaccinia virus (VACV), a close relative of monkeypox, through combining multi-omics analyses and high-resolution live-cell imaging. We find that, upon VACV infection, the epithelial cells undergo epithelial-mesenchymal transition-like transformation, during which they lose intercellular junctions and acquire the migratory capacity to promote viral spreading. After transformation, VACV-hijacked RhoA signaling significantly alters cellular morphology and rearranges the actin cytoskeleton involving the depolymerization of robust actin stress fibers, leading-edge protrusion formation, and the rear-edge recontraction, which coordinates VACV-induced cell migration. Our study reveals how poxviruses alter the epithelial phenotype and regulate RhoA signaling to induce fast migration, providing a unique perspective to understand the pathogenesis of poxviruses.

KDM5A suppresses PML-RARα target gene expression and APL differentiation through repressing H3K4me2.

Epigenetic abnormalities are frequently involved in the initiation and progression of cancers, including acute myeloid leukemia (AML). A subtype of AML, acute promyelocytic leukemia (APL), is mainly driven by a specific oncogenic fusion event of promyelocytic leukemia-RA receptor fusion oncoprotein (PML-RARα). PML-RARα was reported as a transcription repressor through the interaction with nuclear receptor corepressor and histone deacetylase complexes leading to the mis-suppression of its target genes and differentiation blockage. Although previous studies were mainly focused on the connection of histone acetylation, it is still largely unknown whether alternative epigenetics mechanisms are involved in APL progression. KDM5A is a demethylase of histone H3 lysine 4 di- and tri-methylations (H3K4me2/3) and a transcription corepressor. Here, we found that the loss of KDM5A led to APL NB4 cell differentiation and retarded growth. Mechanistically, through epigenomics and transcriptomics analyses, KDM5A binding was detected in 1889 genes, with the majority of the binding events at promoter regions. KDM5A suppressed the expression of 621 genes, including 42 PML-RARα target genes, primarily by controlling the H3K4me2 in the promoters and 5' end intragenic regions. In addition, a recently reported pan-KDM5 inhibitor, CPI-455, on its own could phenocopy the differentiation effects as KDM5A loss in NB4 cells. CPI-455 treatment or KDM5A knockout could greatly sensitize NB4 cells to all-trans retinoic acid-induced differentiation. Our findings indicate that KDM5A contributed to the differentiation blockage in the APL cell line NB4, and inhibition of KDM5A could greatly potentiate NB4 differentiation.

Lysobacter gilvus sp. nov., isolated from activated sludge.

Strain HX-5-24T was isolated from the sludge collected from the outlet of the biochemical treatment facility of an agricultural chemical plant in Maanshan city, Anhui province, PR China (118° 28' N, 31° 47' E). Cells were observed to be Gram-reaction-negative, rod-shaped, non-motile and aerobic. Strain HX-5-24T shared 99.1% 16S rRNA gene sequence similarity with Lysobacter dokdonensis DS-58T and less than 97% similarities with other type strains. The phylogenetic analysis based on 16S rRNA indicated that strain HX-5-24T belonged to the genus Lysobacter and formed a subclade with L. dokdonensis DS-58T. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain HX-5-24T and L. dokdonensis DS-58T were 87.5% and 35.3%, respectively. The genomic DNA G + C content of the strain was 66.4%. The major fatty acids (> 5%) were iso-C15:0, anteiso-C15:0, iso-C16:0, C16:0 and summed feature 9 (iso-C17:1 ω9c and/or C16:0 10-methyl). The predominant quinone was ubiquinone Q-8. The polar lipid profile consisted of diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and phospholipids (PL). On the basis of phenotypic and phylogenetic evidences, strain HX-5-24T is considered as a novel species in the genus Lysobacter, for which the name Lysobacter gilvus sp. nov. is proposed. The type strain is HX-5-24T (= KCTC 72470T = CCTCC AB 2019228T).

Crenobacter caeni sp. nov. Isolated from Sludge.

Strain HX-7-9T was isolated from the activated sludge collected from the outlet of the biochemical treatment facility of agricultural chemical plant in Nanjing, Jiangsu province, PR China. Strain HX-7-9T is Gram staining-negative, rod-shaped, non-spore-forming and flagellated. The 16S rRNA gene-based phylogenetic analysis indicate that strain HX-7-9T belongs to the genus Crenobacter, moderately related to Crenobacter luteus YIM-78141T (94.8% similarity). The genomic DNA G+C content of the strain was 67.5 mol%. Strain HX-7-9T was able to grow at 16-45 °C (optimum at 37 °C), at pH 6.0-8.0 (optimum at pH 7.0) and with 0-1% (w/v) NaCl (optimum at 0). Predominant fatty acid constituents were C16:0 and summed feature 3 (C16:1ω7c and/or C16:1ω6c). The respiratory ubiquinone was Q-8. The polar lipid profile is composed of diphosphatidylglycerol (DPG), phosphatidylmonomethylethanolamine (PME), phosphatidylethanolamine (PE), phospholipids (PL), glycolipid (GL) and aminophospholipid (APL). The ANI and dDDH values obtained between the genomes of HX-7-9T and C. luteus YIM-78141T were 79.8 and 19.1%, respectively. On the basis of data from phenotypic, chemotaxonomic and genotypic analysis, strain HX-7-9T represents a novel species of the genus Crenobacter, for which the name Crenobacter caeni sp. nov. is proposed. The type strain is HX-7-9T (= KCTC 72654T = CCTCC AB 2019349T).

Pedobacter puniceum sp. nov. Isolated from Sludge.

Strain HX-22-1T was isolated from the sludge collected from the outlet of the biochemical treatment facility of an agricultural chemical factory in Nanjing city, Jiangsu province, PR China. Strain HX-22-1T is Gram-staining-negative, rod-shaped, non-spore-forming and non-flagellated. The 16S rRNA gene-based phylogenetic analysis indicates that the strain HX-22-1T belongs to the genus Pedobacter, closely related to Pedobacter glucosidilyticus KCTC 22438T (98.63% similarity). The genomic DNA G+C content of the strain was 34.4 mol%. Strain HX-22-1T was able to grow at 16-37 °C (optimum at 30 °C), at pH 6.0-8.0 (optimum at pH 7.0), and with 0-1% (w/v) NaCl (optimum at 0). Predominant fatty acid constituents were iso-C15:0 and summed feature 3 (iso-C16:1ω7c and/or C16:1ω6c). The predominant respiratory ubiquinone was MK-7. The polar lipid profile is composed of phosphatidylethanolamine (PE), aminophospholipid (APL), aminolipid (AL), and phospholipids (PL). The ANI and dDDH values obtained between the genomes of HX-22-1T and P. glucosidilyticus KCTC 22438T were 89.6 and 38.8%, respectively. On the basis of data from phenotypic, chemotaxonomic, and genotypic analysis, strain HX-22-1T represents a novel species of the genus Pedobacter, for which the name Pedobacter puniceum sp. nov. is proposed. The type strain is HX-22-1T (= KCTC 72655T = CCTCC AB 2019348T).

Cloning of a novel topramezone-resistant 4-hydroxyphenylpyruvate dioxygenase gene and improvement of its resistance through pressure acclimation.

Topramezone is a new 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicide that is widely used on corn to control annual grass weeds and broadleaf weeds. Due to its broad-spectrum weed control capacity, improved activity, excellent crop selectivity, low mammalian toxicity and high environmental safety, topramezone is considered an ideal target herbicide for transgenic engineering of herbicide tolerance. In this study, a topramezone-resistant strain, Burkholderia sp. BW-1, was screened from soil, and a novel topramezone-resistant HPPD gene (Bkhppd) was cloned from this strain. Purified BkHPPD displayed relatively high HPPD activity and topramezone resistance with a half-maximal inhibitory concentration (IC50) of 572.0 nM. Two BkHPPD mutants designated as BkHPPDt31 and BkHPPDt76 were screened through pressure acclimation. BkHPPDt31 contained three amino acid substitutions (H65D, N160 T and N258S), whereas BkHPPDt76 contained four amino acid substitutions (H65D, N160 T, N258S and N343 T). The topramezone IC50 values of BkHPPDt31 and BkHPPDt76 were 1.1- and 2.3-fold higher, respectively, than that of wild-type BkHPPD. In addition, site-directed mutagenesis indicated that the increased resistance conferred by BkHPPDt31 resulted from the synergistic effects of the three site mutations rather than a single site mutation, and that substitution of asparagine 343 with threonine significantly decreased catalytic efficiency and affinity but increased topramezone resistance. In summary, this study provides a novel topramezone-resistant HPPD gene for the engineering of genetically modified herbicide-resistant crops and facilitates further elucidation of the resistance mechanism of BkHPPD and improvement of resistance through directed evolution.

Caenimonas sedimenti sp. nov., Isolated from Sediment of the Wastewater Outlet of an Agricultural Chemical Plant.

A novel bacterial strain, designated HX-9-20T, was isolated from the sediment collected from the wastewater outlet of an agricultural chemical plant in Maanshan city, Anhui province, PR China. Cells of strain HX-9-20T were Gram-staining-negative, rod-shaped, translucent, non-motile, and strictly aerobic. Growth was observed between 15 and 35 °C (optimum 30 °C), at pH 6.0-8.0 (optimum pH 7.0), and in the presence of 0-0.4% (w/v) NaCl (optimum 0.2%). The predominant cellular fatty acids were summed feature 3 (C16:1 ω7c and/or C16:1 ω6c), C16:0 and summed feature 8 (C18:1 ω6c and/or C18:1 ω7c). The major quinone was ubiquinone Q-8. The major polar lipids were phosphatidylglycerol, phosphatidylethanolamine, and an aminophospholipid. Strain HX-9-20T contains 2-hydroxyputrescine and putrescine as the major polyamine. A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain HX-9-20T was affiliated with the genus Caenimonas, exhibiting the highest sequence similarities with Caenimonas koreensis EMB320T (97.3% similarity) and Ramlibacter humi 18×22-1T (97.0%), and less than 97.0% similarity with other type strains. The average nucleotide identity (ANI) and digital DNA-DNA hybridization values (dDDH) between HX-9-20T and C. koreensis EMB320T were 76.9% and 23.5% respectively. The ANI and dDDH between HX-9-20T and R. humi 18×22-1T were 80.3% and 23.6%, respectively. The G + C content of the genomic DNA was 67.5 mol%. On the basis of the polyphasic taxonomic data, strain HX-9-20T represents a novel species of the genus Caenimonas, for which the name Caenimonas sedimenti sp. nov. is proposed. The type strain is HX-9-20T (=KCTC 72473T=CCTCC AB 2019266T).

Niastella caeni sp. nov., isolated from activated sludge.

A Gram-stain-negative, aerobic, non-flagellated and filamentous-shaped bacterium, HX-16-21T, was isolated from activated sludge. Strain HX-16-21T was able to degrade gentisate, protocatechuic acid and p-hydroxybenzoic acid and herbicides quizalofop-p-ethyl and diclofop-methyl. The strain shared 97.2 % 16S rRNA gene sequence similarity to Niastella vici CCTCC AB 2015052T and less than 97 % similarities to other type strains. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain HX-16-21T belonged to the genus Niastella and formed a subclade with N. vici CCTCC AB 2015052T. The major polar lipids were phosphatidylethanolamine, phosphatidylcholine and six unidentified lipids. The major fatty acids were iso-C15:0, iso-C15:1 G and iso-C17:0 3-OH. The predominant respiratory quinone was menaquinone 7 (MK-7). The draft genome of strain HX-16-21T was 8.1 Mb, and the G+C content was 43.5 mol%. The average nucleotide identity and digital DNA-DNA hybridization values between strain HX-16-21T and N. vici CCTCC AB 2015052T were 80.6 and 26.8 %, respectively. Based on both phenotypic and phylogenetic evidence, strain HX-16-21T is considered to represent a novel species in the genus Niastella, for which the name Niastella caeni sp. nov. is proposed. The type strain is HX-16-21T (=KCTC 72288T=ACCC 61580T).

Extensimonas perlucida sp. nov., a Novel Bacterium Isolated from Sludge.

A bacterium, designated HX2-24 T, was isolated from activated sludge treating pesticide-manufacturing wastewater. Colonies of the strain on nutrient agar were circular, transparent, and colorless. Strain HX2-24 T shared 98.1% 16S rRNA gene sequence similarity with Extensimonas vulgaris S4T, and less than 97% similarities with other type strains. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain formed a clade with E. vulgaris S4T. The major cellular fatty acids were C16:0, summed feature 3 (C16:1ω7c and/or C16:1ω6c) and C17:0 cyclo, the major polar lipids were phosphatidylethanolamine (PE), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), aminophospholipid (APL), glycophospholipid (GPL), and aminoglycolipid (AGL). The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between HX2-24 T and E. vulgaris S4T were 92% and 41%, respectively. The G + C content of strain HX2-24 T was 64.4 mol%. Thus, based on the phenotypic, chemotaxonomic, and genotypic characteristics, strain HX2-24 T represents a novel species in the genus Extensimonas, for which the name Extensimonas perlucida HX2-24 T sp. nov. is proposed. The type strain is HX2-24 T (= KCTC 72472 T = CCTCC AB 2019178 T).

Sphingobacterium olei sp. nov., isolated from oil-contaminated soil.

A Gram-stain-negative, rod-shaped, non-motile and non-spore-forming bacterium, designated HAL-9T, was isolated from oil-contaminated soil in Daqing oilfield, Heilongjiang Province, PR China. Strain HAL-9T was able to degrade quizalofop-p-ethyl and diclofop-methyl. Growth was observed at 10-35 °C (optimum, 30 °C), pH 6.0-10.0 (optimum, pH 7.0) and salinity of 0 %-5.0 % (w/v; optimum 1.0 %). The results of phylogenetic analysis based on the 16S rRNA gene indicated that strain HAL-9T belongs to the genus Sphingobacterium and showed the highest sequence similarity (98.3 %) to Sphingobacterium alkalisoli Y3L14T, followed by Sphingobacterium mizutaii DSM 11724T (95.1 %) and Sphingobacterium lactis DSM 22361T (95.1 %). Menaquinone-7 (MK-7) was the only isoprenoid quinone. The predominant cellular fatty acids were summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c), iso-C15: 0 and iso-C17 : 0 3-OH. The major polar lipids were phosphatidylethanolamine, three phosphoglycolipids and three unidentified lipids. The draft genome of strain HAL-9T was 5.41 Mb. The G+C content of strain HAL-9T was 40.6 mol%. Furthermore, the average nucleotide identity and in silico DNA-DNA hybridization values between strain HAL-9T and S. alkalisoli Y3L14T were 86.2 % and 32.8 %, respectively, which were below the standard thresholds for species differentiation. On the basis of phenotypic, genotypic and phylogenetic evidence, strain HAL-9T represents a novel species in the genus Sphingobacterium, for which the name Sphingobacterium olei sp. nov. is proposed. The type strain is HAL-9T (=ACCC 61581T=CCTCC AB 2019176T=KCTC 72287T).

Isolation and Characterization of a Topramezone-Resistant 4-Hydroxyphenylpyruvate Dioxygenase from Sphingobium sp. TPM-19.

Topramezone is a 4-hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor. Due to its broad-spectrum, high efficiency, and low toxicity, topramezone is a candidate herbicide for the construction of genetically modified (GM) herbicide-resistant crops. In the present study, we screened a topramezone-resistant isolate Sphingobium sp. TPM-19 and cloned a topramezone-resistant HPPD gene (SphppD) from this isolate. SpHPPD shared the highest similarity (53%) with an HPPD from Vibrio vulnificus CMCP6. SpHPPD was synthesized in Escherichia coli BL21(DE3) and purified to homogeneity using Co2+-affinity chromatography. SpHPPD was found to be a monomer. The Km and kcat of SpHPPD for 4-hydroxyphenylpyruvate (4-HPP) were 82.8 µM and 15.0 s-1, respectively. SpHPPD showed high resistance to topramezone with half maximal inhibitory concentration (IC50) and Ki values of 5.2 and 2.5 µM, respectively. Additionally, SpHPPD also showed high resistance to isoxaflutole (DKN) (IC50: 8.7 µM; Ki: 6.0 µM) and mesotrione (IC50: 4.2 µM; Ki: 1.3 µM) and moderate resistance to tembotrione (IC50: 2.5 µM; Ki: 1.0 µM). The introduction of the SphppD gene into Arabidopsis thaliana enhanced obvious resistance against topramezone. In conclusion, this study provides a novel topramezone-resistant HPPD gene for the genetic engineering of GM herbicide-resistant crops.

Chitiniphilus eburneus sp. nov., a novel chitinolytic bacterium isolated from sludge.

A novel Gram-stain-negative, curved rod-shaped, motile and non-endospore-forming strain, designated HX-2-15T, was isolated from activated sludge of agricultural chemical plant in Nanjing, Jiangsu province, PR China (32° 03' N, 118° 46' E) . Growth was observed at 15-37 °C (optimum between 25 and 30 °C), at pH 6.0-8.0 (optimum at pH 7.0) and with 0-3.0 % (w/v) NaCl (optimum at 0.5 %). Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strain showed closest affiliation to Chitiniphilus shinanonensis SAY3T, with a sequence similarity of 99.0 %. The predominant cellular fatty acids were C16:0, C17:0 cyclo and summed feature 3 (C16:1 ω7c and/or C16:1 ω6c). The major quinone was ubiquinone Q-8 . The polar lipid profile was composed of phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, three unidentified phospholipids, one unidentified lipid and one unidentified aminophosphoglycolipid . The genomic DNA G+C content of the strain was 63.6 mol%. The ANI and dDDH values obtained between the genomes of HX-2-15T and C. shinanonensis SAY3T were 85.3 and 29.3 % respectively. On the basis of data from phenotypic, chemotaxonomic and genotypic analysis, strain HX-2-15T represents a novel species of the genus Chitiniphilus, for which the name Chitiniphilus eburneus sp. nov. is proposed. The type strain is HX-2-15T (=KCTC 72286T=CCTCC AB 2019178T).

Humidity Control Strategies for Solid-State Fermentation: Capillary Water Supply by Water-Retention Materials and Negative-Pressure Auto-controlled Irrigation.

Solid-state fermentation (SSF) has regained interest owing to its advantages in solid waste treatment and fermentation industries. However, heterogeneous heat and mass transfer are often caused by the absence of free water and noticeable water loss from microbial utilization and moisture evaporation in SSF. It is necessary to explore more effective ways to solve issues of water loss and water supplement in SSF based on online capillary water monitoring, because capillary water is the dominant form of water that is present and lost in substrate. Two novel capillary-water supply strategies were proposed, established and evaluated using three selected reference strains, including water-retention materials and negative-pressure auto-controlled irrigation (NPACI). This study employed superabsorbent polymer, a kind of water-retention material to enhance enzyme productivity with the most significant increase of 2.47 times. Moreover, the combination of NPACI and 0.1% superabsorbent polymers increased productivity by 2.80-fold, together with lowered gradients of temperature, moisture and products. Furthermore, a modified liquid-supply SSF was constructed through successful capillary water control by proposed humidity control strategies. This modified SSF system could address the shortcomings of inhomogeneous culture of traditional SSF.

Directed Evolution of Sulfonylurea Esterase and Characterization of a Variant with Improved Activity.

Esterase SulE detoxicates a variety of sulfonylurea herbicides through de-esterification. SulE exhibits high activity against thifensulfuron-methyl but low activity against other sulfonylureas. In this study, two variants, m2311 (P80R) and m0569 (P80R and G176A), with improved activity were screened from a mutation library constructed by error-prone PCR. Variant m2311 showed a higher activity against sulfonylureas in comparison variant m0569 and was further investigated. The kcat/ Km value of variant m2311 for metsulfuron-methyl, sulfometuron-methyl, chlorimuron-ethyl, tribenuron-methyl, and ethametsulfuron-methyl increased by 3.20-, 1.72-, 2.94-, 2.26- and 2.96-fold, respectively, in comparison with the wild type. Molecular modeling suggested that the activity improvement of variant m2311 is due to the substitution of Pro80 by arginine, leading to the formation of new hydrogen bonds between the enzyme and substrate. This study facilitates further elucidation of the structure and function of SulE and provides an improved gene resource for the detoxification of sulfonylurea residues and the genetic engineering of sulfonylurea-resistant crops.

Chromatin regulator Asxl1 loss and Nf1 haploinsufficiency cooperate to accelerate myeloid malignancy.

ASXL1 is frequently mutated in myeloid malignancies and is known to co-occur with other gene mutations. However, the molecular mechanisms underlying the leukemogenesis associated with ASXL1 and cooperating mutations remain to be elucidated. Here, we report that Asxl1 loss cooperated with haploinsufficiency of Nf1, a negative regulator of the RAS signaling pathway, to accelerate the development of myeloid leukemia in mice. Loss of Asxl1 and Nf1 in hematopoietic stem and progenitor cells resulted in a gain-of-function transcriptional activation of multiple pathways such as MYC, NRAS, and BRD4 that are critical for leukemogenesis. The hyperactive MYC and BRD9 transcription programs were correlated with elevated H3K4 trimethylation at the promoter regions of genes involving these pathways. Furthermore, pharmacological inhibition of both the MAPK pathway and BET bromodomain prevented leukemia initiation and inhibited disease progression in Asxl1Δ/Δ Nf1Δ/Δ mice. Concomitant mutations of ASXL1 and RAS pathway genes were associated with aggressive progression of myeloid malignancies in patients. This study sheds light on the effect of cooperation between epigenetic alterations and signaling pathways on accelerating the progression of myeloid malignancies and provides a rational therapeutic strategy for the treatment of myeloid malignancies with ASXL1 and RAS pathway gene mutations.

Therapeutic potential of GSK-J4, a histone demethylase KDM6B/JMJD3 inhibitor, for acute myeloid leukemia.

PURPOSE: Acute myeloid leukemia (AML) is a heterogeneous disease with poor outcomes. Despite increased evidence shows that dysregulation of histone modification contributes to AML, specific drugs targeting key histone modulators are not applied in the clinical treatment of AML. Here, we investigated whether targeting KDM6B, the demethylase of tri-methylated histone H3 lysine 27 (H3K27me3), has a therapeutic potential for AML. METHODS: A KDM6B-specific inhibitor, GSK-J4, was applied to treat the primary cells from AML patients and AML cell lines in vitro and in vivo. RNA-sequencing was performed to reveal the underlying mechanisms of inhibiting KDM6B for the treatment of AML. RESULTS: Here we observed that the mRNA expression of KDM6B was up-regulated in AML and positively correlated with poor survival. Treatment with GSK-J4 increased the global level of H3K27me3 and reduced the proliferation and colony-forming ability of primary AML cells and AML cell lines. GSK-J4 treatment significantly induced cell apoptosis and cell-cycle arrest in Kasumi-1 cells, and displayed a synergistic effect with cytosine arabinoside. Notably, injection of GSK-J4 attenuated the disease progression in a human AML xenograft mouse model in vivo. Treatment with GSK-J4 predominantly resulted in down-regulation of DNA replication and cell-cycle-related pathways, as well as abrogated the expression of critical cancer-promoting HOX genes. ChIP-qPCR validated an increased enrichment of H3K27me3 in the transcription start sites of these HOX genes. CONCLUSIONS: In summary, our findings suggest that targeting KDM6B with GSK-J4 has a therapeutic potential for the treatment of AML.