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PurposeHashimoto’s thyroiditis (HT) is a significant public health concern, particularly among females. While existing studies have explored the correlation between serum iron levels and HT, limited research has specifically focused on this association in reproductive-age females. Our study aims to investigate the relationship between serum iron and HT.MethodsUsing data from the National Health and Nutrition Examination Survey (NHANES) database (2007–2012), we employed weighted multivariate logistic regression models, an XGBoost model, and smooth curve fitting. We assessed the correlation between serum iron and HT and examined linear and non-linear relationships with thyroid peroxidase antibodies (TPOAb) and thyroglobulin antibodies (TgAb).ResultsAmong 2,356 participants, each unit increase in serum iron was associated with a 43% reduced risk of HT (Odds Ratios (OR) 0.574; 95% Confidence Interval (CI) 0.572, 0.576). Quartile analysis confirmed these effects. The XGBoost model identified serum iron as the most significant variable correlated with HT. Smooth curves revealed a linear association between log2-transformed serum iron and HT. Additionally, log2-transformed serum iron inversely correlated with TPOAb levels (β −15.47; 95% CI -25.01, −5.92), while a non-linear relationship was observed with TgAb.ConclusionOur study reveals that in reproductive-age women, every unit increase in serum iron is associated with a 43% lower risk of HT, demonstrating an inverse relationship. Additionally, serum iron exhibits a negative correlation with TPOAb and a non-linear association with TgAb.

Abstract ETV6::RUNX1 is the most common fusion gene in childhood acute lymphoblastic leukaemia (ALL) and is associated with favorable outcomes, especially in low‐risk children. However, as many as 10% of children relapse within 3 years, and such early relapses have poor survival. Identifying children at risk for early relapse is an important challenge. We interrogated data from 87 children with low‐risk ETV6::RUNX1‐positive B‐cell ALL and with available preserved bone marrow samples (discovery cohort). We profiled somatic point mutations in a panel of 559 genes and genome‐wide transcriptome and single‐nucleotide variants. We found high TIMD4 expression (> 85th‐percentile value) at diagnosis was the most important independent prognostic factor of early relapse (hazard ratio [HR] = 5.07 [1.76, 14.62]; p = 0.03). In an independent validation cohort of low‐risk ETV6::RUNX1‐positive B‐cell ALL (N = 68) high TIMD4 expression at diagnosis had an HR = 4.78 [1.07, 21.36] (p = 0.04) for early relapse. In another validation cohort including 78 children with low‐risk ETV6::RUNX1‐negative B‐cell ALL, high TIMD4 expression at diagnosis had an HR = 3.93 [1.31, 11.79] (p = 0.01). Our results suggest high TIMD4 expression at diagnosis in low‐risk B‐cell ALL in children might be associated with high risk for early relapse.

Not available.

Abstract Background Acute myeloid leukaemia (AML) is characterised by the malignant accumulation of myeloid progenitors with a high recurrence rate after chemotherapy. Blasts (leukaemia cells) exhibit a complete myeloid differentiation hierarchy hiding a wide range of temporal information from initial to mature clones, including genesis, phenotypic transformation, and cell fate decisions, which might contribute to relapse in AML patients. Methods Based on the landscape of AML surface antigens generated by mass cytometry (CyTOF), we combined manifold analysis and principal curve-based trajectory inference algorithm to align myelocytes on a single-linear evolution axis by considering their phenotype continuum that correlated with differentiation order. Backtracking the trajectory from mature clusters located automatically at the terminal, we recurred the molecular dynamics during AML progression and confirmed the evolution stage of single cells. We also designed a ‘dispersive antigens in neighbouring clusters exhibition (DANCE)’ feature selection method to simplify and unify trajectories, which enabled the exploration and comparison of relapse-related traits among 43 paediatric AML bone marrow specimens. Results The feasibility of the proposed trajectory analysis method was verified with public datasets. After aligning single cells on the pseudotime axis, primitive clones were recognized precisely from AML blasts, and the expression of the inner molecules before and after drug stimulation was accurately plotted on the trajectory. Applying DANCE to 43 clinical samples with different responses for chemotherapy, we selected 12 antigens as a general panel for myeloblast differentiation performance, and obtain trajectories to those patients. For the trajectories with unified molecular dynamics, CD11c overexpression in the primitive stage indicated a good chemotherapy outcome. Moreover, a later initial peak of stemness heterogeneity tended to be associated with a higher risk of relapse compared with complete remission. Conclusions In this study, pseudotime was generated as a new single-cell feature. Minute differences in temporal traits among samples could be exhibited on a trajectory, thus providing a new strategy for predicting AML relapse and monitoring drug responses over time scale.

Hydroxylation at the C-14 position of triptolide is critical for its potent antitumor activity. Here, the authors report two CYP82Ds catalyze the 14-hydroxylation reaction via metabolic grid and achieve heterologous bioproduction of triptolide precursor in engineered Saccharomyces cerevisiae.

Leaf and tuber extracts of Curcuma wenyujin contain a mixture of curcuminoids. However, the curcuminoid constituents and their molecular mechanisms are poorly understood, and the relevant curcumin synthases remain unclear. In this study, we comprehensively compared the metabolite profiles of the leaf and tuber tissues of C. wenyujin. A total of 11 curcuminoid metabolites were identified and exhibited differentially changed contents in the leaf and tuber tissues. An integrated analysis of metabolomic and transcriptomic data revealed the proposed biosynthesis pathway of curcuminoid. Two candidate type Ⅲ polyketide synthases (PKSs) were identified in the metabolically engineering yeasts, indicating that CwPKS1 and CwPKS2 maintained substrate and product specificities. Especially, CwPKS1 is the first type Ⅲ PKS identified to synthesize hydrogenated derivatives of curcuminoid, dihydrocurcumin and tetrehydrocurcumin. Interestingly, the substitution of the glycine at position 219 with aspartic acid (G219D mutant) resulted in the complete inactivation of CwPKS1. Our results provide the first comparative metabolome analysis of C. wenyujin and functionally identified type Ⅲ PKSs, giving valuable information for curcuminoids biosynthesis.

Tripterygium wilfordii is a valuable medicinal plant rich in biologically active diterpenoids, but there are few studies on the origins of these diterpenoids in its secondary metabolism. Here, we identified three regions containing tandemly duplicated diterpene synthase genes on chromosomes (Chr) 17 and 21 of T. wilfordii and obtained 11 diterpene synthases with different functions. We further revealed that these diterpene synthases underwent duplication and rearrangement at approximately 2.3–23.7 million years ago (MYA) by whole-genome triplication (WGT), transposon mediation, and tandem duplication, followed by functional divergence. We first demonstrated that four key amino acids in the sequences of TwCPS3, TwCPS5, and TwCPS6 were altered during evolution, leading to their functional divergence and the formation of diterpene secondary metabolites. Then, we demonstrated that the functional divergence of three TwKSLs was driven by mutations in two key amino acids. Finally, we discovered the mechanisms of evolution and pseudogenization of miltiradiene synthases in T. wilfordii and elucidated that the new function in TwMS1/2 from the terpene synthase (TPS)-b subfamily was caused by progressive changes in multiple amino acids after the WGT event. Our results provide key evidence for the formation of diverse diterpenoids during the evolution of secondary metabolites in T. wilfordii.

Occult breast cancer is an uncommon type of breast cancer and its diagnosis is challenging. It is usually invisible on multiple imaging examines. Metastases to the rectum and inguinal lymph nodes from occult breast lobular cancer are even rarer. 68Ga-DOTA peptides can image neuroendocrine tumors by targeting specific somatostatin receptors. Besides, other tumors, including breast cancer, have been shown to express somatostatin receptors. In this case, we presented a 63-year-old woman who underwent both 18F-FDG and 68Ga-DOTATATE PET/CT due to a rectal polyp. An endoscopic excision biopsy confirmed metastatic carcinoma of suspected breast origin, but subsequent ultrasound and MRI showed no signs of malignancy in the breast and adnexa uteri. PET/CT showed obvious 68Ga-DOTATATE activity in bilateral axillary and right inguinal lymph nodes with mild 18F-FDG uptake. Final histopathology at the left axillary, right inguinal lymph nodes, and rectum indicated metastases from breast cancer while the origin remained radiologically occult. Additionally, one uterine fibroids was found with positive uptake of 68Ga-DOTATATE and negative uptake of 18F-FDG. This case suggested that 68Ga-DOTATAE PET/CT may be an effective supplement in diagnosing OBC lymph node metastases with mild 18F-FDG uptake, and it may provide a new technology for the clinical diagnosis of occult breast cancer.

This study investigated the effects of three extraction methods, including cold pressing (CP), microwave pretreatment pressing (MP), and supercritical fluid extraction (SFE), on the yield, physicochemical properties, bioactive compounds content, and antioxidant properties of pumpkin seed oil (PSO). Furthermore, the correlation between bioactive compounds and the antioxidant properties of PSO was determined. The results revealed that the yield of PSO extracted using the three methods was in the order of SFE > MP > CP. Additionally, the PSO generated by SFE showed the highest unsaturated fatty acid content, followed by MP and CP. Additionally, MP-PSO exhibited the highest acid value and saponification value, while SFE-PSO displayed the highest moisture content, peroxide value, and iodine value. Moreover, the PSO generated by MP demonstrated superior antioxidant properties compared to that of PSOs from CP and SFE in the oxidation induction, DPPH, FRAP, and ABTS tests. Finally, the correlation analysis revealed that specific types of bioactive compounds, such as β-sitosterol and γ-tocopherol, were highly correlated with the antioxidant properties of PSOs. Consequently, this study provides comprehensive knowledge regarding PSO extraction, physicochemical properties, bioactive compound extraction, and the correlated antioxidant properties.

Germacrene A (GA) is a key intermediate for the synthesis of medicinal active compounds, especially for β-elemene, which is a broad-spectrum anticancer drug. The production of sufficient GA in the microbial platform is vital for the precursors supply of active compounds. In this study, Escherichia coli BL21 Star (DE3) was used as the host and cultivated in SBMSN medium, obtaining a highest yield of FPP. The GA synthase from Lactuca sativa (LTC2) exhibited the highest level of GA production. Secondly, two residues involved in product release (T410 and T392) were substituted with Ser and Ala, respectively, responsible for relatively higher activities. Next, substitution of selected residues S243 with Asn caused an increase in activity. Furthermore, I364K-T410S and T392A-T410S were created by combination with the beneficial mutation, and they demonstrated dramatically enhanced titers with 1.90-fold and per-cell productivity with 5.44-fold, respectively. Finally, the production titer of GA reached 126.4 mg/L, and the highest productivity was 7.02 mg/L.h by the I364K-T410S mutant in a shake-flask batch culture after fermentation for 18 h. To our knowledge, the productivity of the I364K-T410S mutant is the highest level ever reported. These results highlight a promising method for the industrial production of GA in E. coli, and lay a foundation for pathway reconstruction and the production of valuable natural sesquiterpenes.

Squalene synthase (SQS), squalene epoxidase (SE), and oxidosqualene cyclase (OSC) are encoding enzymes in downstream biosynthetic pathway of triterpenoid in plants, but the relationship between three genes and celastrol accumulation in Tripterygium wilfordii still remains unknown. Gene transformation system in plant can be used for studying gene function rapidly. However, there is no report on the application of cambial meristematic cells (CMCs) and dedifferentiated cells (DDCs) in genetic transformation systems. Our aim was to study the effects of individual overexpression of TwSQS, TwSE, and TwOSC on terpenoid accumulation and biosynthetic pathway related gene expression through CMCs and DDCs systems. Overexpression vectors of TwSQS, TwSE, and TwOSC were constructed by Gateway technology and transferred into CMCs and DDCs by gene gun. After overexpression, the content of celastrol was significantly increased in CMCs compared with the control group. However, there was no significant increment of celastrol in DDCs. Meanwhile, the relative expression levels of TwSQS, TwSE, TwOSC, and terpenoid biosynthetic pathway related genes were detected. The relative expression levels of TwSQS, TwSE, and TwOSC were increased compared with the control group in both CMCs and DDCs, while the pathway-related genes displayed different expression trends. Therefore, it was verified in T. wilfordii CMCs that overexpression of TwSQS, TwSE, and TwOSC increased celastrol accumulation and had different effects on the expression of related genes in terpenoid biosynthetic pathway, laying a foundation for further elucidating the downstream biosynthetic pathway of celastrol through T. wilfordii CMCs system.

MYB superfamily is one of the most abundant families in plants, and plays critical role in plant growth, development, metabolism regulation, and stress response. Curcuma wenyujin is the main source plant of three traditional Chinese medicines, which are widely used in clinical treatment due to its diverse pharmacological activities. In present study, 88 CwMYBs were identified and analyzed in C. wenyujin, including 43 MYB-related genes, 42 R2R3-MYB genes, two 3R-MYB genes, and one 4R-MYB gene. Forty-three MYB-related proteins were classified into several types based on conserved domains and specific motifs, including CCA1-like type, R-R type, Myb-CC type, GARP-like type, and TBR-like type. The analysis of motifs in MYB DBD and no-MYB regions revealed the relevance of protein structure and function. Comparative phylogeny analysis divided 42 R2R3-MYB proteins into 19 subgroups and provided a reference for understanding the functions of some CwMYBs based on orthologs of previously characterized MYBs. Expression profile analysis of CwMYB genes revealed the differentially expressed genes responding to various abiotic stresses. Four candidate MYB genes were identified by combining the results of phylogeny analysis and expression analysis. CwMYB10, CwMYB18, CwMYB39, and CwMYB41 were significantly induced by cold, NaCl, and MeJA stress treatments. CwMYB18 and CwMYB41 were proved as regulators with activity of transcriptional activation, whereas CwMYB39 and CwMYB10 were not. They may participate in the response to abiotic stresses through different mechanisms in C. wenyujin. This study was the first step toward understanding the CwMYB family and the response to abiotic stresses in C. wenyujin.

Understanding the evolutionary trajectory of cancer samples may enable understanding resistance to treatment. Here, the authors used single cell sequencing of a cohort of acute myeloid leukemia tumours and identify features of linear and branching evolution in tumours.

Tripterygium wilfordii is a medical plant that can produce antitumor activity compound triptolide. Here, the authors assemble its genome and identify a cytochrome P450 that can catalyze oxidation of a methyl to the acid moiety of dehydroabietic acid in triptolide biosynthesis by integrating multi-omics data.

Curcuma wenyujin is the source plant of three traditional Chinese medicines, which have been widely used in clinical treatment over 1000 years. The content of terpenes, the major medicinal active ingredients, is relatively low in this plant. Studies have shown that MeJA can promote terpenes biosynthesis in plants. However, the mechanism underlying the effect of MeJA in C. wenyujin remains unclear. In this work, the transcriptome of C. wenyujin leaves with MeJA treatment was analyzed to elucidate the regulation mechanism of MeJA-mediated terpene biosynthesis. Based on the RNA-seq data, 7,246 unigenes were differentially expressed with MeJA treatment. Expression pattern clustering of DEGs revealed that unigenes, related to JA biosynthesis and signal transduction, responded to exogenous MeJA stimulation on the early stage and maintained throughout the process. Subsequently, unigenes related to terpene biosynthesis pathway showed a significant up-regulation with 6 h treatment. The analysis results suggested that MeJA induced the expression of JA biosynthesis genes (such as LOXs, AOSs, AOCs, OPRs, and MFPs) and JA signal transduction core genes (JAZs and MYCs) to activate JA signaling pathway. Meanwhile, downstream JA-responsive genes presented up-regulated expression levels such as AACT, HMGSs, HMGRs, DXSs, DXRs, MCTs, HDSs, and HDRs, thus promoting terpenes biosynthesis. The transcriptional expressions of these genes were validated by qRT-PCR. In addition, six CwTPS genes in response to MeJA were identified. With MeJA treatment, the expression levels of CwTPSs were increased as well as those of the transcription factors MYB, NAC, bZIP, WRKY, AP2/ERF, and HLH. These TFs might potentially regulate terpenes biosynthesis. These results provide insights for regulation mechanism of terpenes biosynthesis.

Relapse of childhood AML1-ETO (AE) acute myeloid leukemia is the most common cause of treatment failure. Optimized minimal residual disease monitoring methods is required to prevent relapse. In this study, we used next-generation sequencing to identify the breakpoints in the fusion gene and the DNA-based droplet digital PCR (ddPCR) method was used for dynamic monitoring of AE-DNA. The ddPCR technique provides more sensitive and precise quantitation of the AE gene during disease progression and relapse. Quantification of the AE fusion gene by ddPCR further contributes to improved prognosis. Our study provides valuable methods for dynamic surveillance of AE fusion DNA and assistance in determining the prognosis.

Abstract Background Tripterygium wilfordii Hook. f. (T. wilfordii) is an important medicinal plant with anti-inflammatory, immunosuppressive and anti-tumor activities. The main bioactive ingredients are diterpenoids and triterpenoids, such as triptolide, triptophenolide and celastrol. However, the production of terpenoids from original plants, hairy roots and dedifferentiated cells (DDCs) are not satisfactory for clinical applications. To find a new way to further improve the production of terpenoids, we established a new culture system of cambial meristematic cells (CMCs) with stem cell-like properties, which had strong vigor and high efficiency to produce large amounts of terpenoids of T. wilfordii. Results CMCs of T. wilfordii were isolated and cultured for the first time. CMCs were characterized consistent with stem cell identities based on their physiological and molecular analysis, including morphology of CMCs, hypersensitivity to zeocin, thin cell wall and orthogonal partial least square-discriminant analysis, combination of transcriptional data analysis. After induction with methyl jasmonate (MJ), the maximal production of triptolide, celastrol and triptophenolide in CMCs was 312%, 400% and 327% higher than that of control group, respectively. As for medium, MJ-induced CMCs secreted 231% triptolide and 130% triptophenolide at the maximum level into medium higher than that of control group. Maximal celastrol production of induced CMCs medium was 48% lower than that of control group. Long-term induction significantly enhanced the production of terpenoids both in cells and medium. The reason for increasing the yield of terpenoids was that expression levels of 1-deoxy-d-xylulose-5-phosphate synthase (DXS), 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR) and hydroxymethylglutaryl-CoA synthase (HMGS) were upregulated in CMCs after induction. Conclusions For the first time, CMCs of T. wilfordii were isolated, cultured, characterized and applied. Considering the significant enrichment of terpenoids in CMCs of T. wilfordii, CMCs could provide an efficient and controllable platform for sustainable production of terpenoids, which can be a better choice than DDCs.

Summary: Bone marrow stromal cells (BMSCs) that express high levels of stem cell factor (SCF) and CXC chemokine ligand 12 (CXCL12) are one crucial component of the hematopoietic stem cell (HSC) niche. While the secreted factors produced by BMSCs to support HSCs have been well described, little is known regarding the transcriptional regulators controlling the cell fate of BMSCs and thus indirectly maintaining HSCs. BMI1 is a polycomb group protein that regulates HSCs both cell intrinsically and extrinsically, but it is unknown in which cell type and how BMI1 functions to maintain HSCs extrinsically. Here we show that Bmi1 maintains HSCs by preventing adipogenic differentiation of BMSCs. Bmi1 is highly expressed in BMSCs but becomes downregulated upon adipogenic differentiation and during aging. Deleting Bmi1 from BMSCs increased marrow adipocytes, induced HSC quiescence and depletion, and impaired hematopoiesis. We found that BMI1 repressed multiple developmental programs in BMSCs by safeguarding the repressive epigenetic marks histone H2A ubiquitylation and H3 lysine 27 trimethylation. We identified a novel adipogenic program governed by Pax3, which BMI1 repressed in BMSCs. Our results establish Bmi1 as a critical regulator of BMSC cell fate that suppresses marrow adipogenesis to create a supportive niche for HSCs. : Nakada and colleagues show that Bmi1 is highly expressed in bone marrow stromal cells, loss of which caused marrow adipocyte expansion and hematopoietic stem cell depletion. Bmi1 deletion diminished repressive histone marks on Pax3, leading to its expression and adipogenesis. Reduced Bmi1 expression with age may contribute to age-associated increase of marrow adipocytes. Keywords: BMI1, mesenchymal stromal cells, hematopoietic stem cells, niche, epigenetics, polycomb, PAX3, adipocytes

Panax notoginseng, a perennial herb of the genus Panax in the family Araliaceae, has played an important role in clinical treatment in China for thousands of years because of its extensive pharmacological effects. Here, we report a high-quality reference genome of P. notoginseng, with a genome size up to 2.66 Gb and a contig N50 of 1.12 Mb, produced with third-generation PacBio sequencing technology. This is the first chromosome-level genome assembly for the genus Panax. Through genome evolution analysis, we explored phylogenetic and whole-genome duplication events and examined their impact on saponin biosynthesis. We performed a detailed transcriptional analysis of P. notoginseng and explored gene-level mechanisms that regulate the formation of characteristic tubercles. Next, we studied the biosynthesis and regulation of saponins at temporal and spatial levels. We combined multi-omics data to identify genes that encode key enzymes in the P. notoginseng terpenoid biosynthetic pathway. Finally, we identified five glycosyltransferase genes whose products catalyzed the formation of different ginsenosides in P. notoginseng. The genetic information obtained in this study provides a resource for further exploration of the growth characteristics, cultivation, breeding, and saponin biosynthesis of P. notoginseng.

PDK1 (phosphoinositide dependent kinase-1) plays an important regulatory role in B cells, T cells and platelets. Less is known about how PDK1 acts in hematopoietic stem cells (HSCs), especially in the fetal liver (FL) during embryonic hematopoiesis, as the FL is the primary fetal hematopoietic organ and the main site of HSC expansion and differentiation. Here, we deleted the PDK1 gene in hematopoietic cells by crossing Vav-Cre transgenic mice with PDK1f/f mice. Using a transplantation assay, we found that HSCs from the E15.5 FL of Vav-Cre;PDK1f/f embryos are severely impaired compared when compared with HSCs from PDK1f/f or PDK1f/+ FLs. Additionally, we found that there were more FL HSCs in an apoptotic state and active cell cycle in PDK1-deficient embryos than in control embryos. By comparing the expression profiles of FL-derived LSKs in Vav-Cre;PDK1f/f embryos to the controls, we found that the BH3-only protein PUMA and the cyclin family proteins were expressed higher in the Vav-Cre;PDK1f/f group, which may account for the increased apoptosis and activated cell cycle in the deficient HSCs. Furthermore, we demonstrated that the expression of FoxO3a was higher in PDK1-deficient LSKs, indicating that the Akt-FoxO3a-PUMA axis may participate in regulating LSKs apoptosis in the E15.5 FL. In contrast, FoxO1 expression was lower in PDK1-deficient LSK cells, suggesting that Akt-FoxO1-CCND may regulate the HSC cell cycle. Taken together, our findings support a critical role for PDK1 in maintaining FL hematopoiesis via regulating apoptosis and cell cycle of definitive hematopoiesis by the Akt-FOXO signaling pathways. Keywords: PDK1, Akt, HSC, FOXO, Fetal liver, Embryonic hematopoiesis

Abstract: Characterization of hematopoietic stem cells (HSCs) has advanced largely owing to transplantation assays, in which the developmental potential of HSCs is assessed generally in nonhomeostatic conditions. These studies established that adult HSCs extensively contribute to multilineage hematopoietic regeneration upon transplantation. On the contrary, recent studies performing lineage tracing of HSCs under homeostatic conditions have shown that adult HSCs may contribute far less to steady-state hematopoiesis than would be anticipated based on transplantation assays. Here, we used 2 independent HSC-lineage–tracing models to examine the contribution of adult HSCs to steady-state hematopoiesis. We show that adult HSCs contribute robustly to steady-state hematopoiesis, exhibiting faster efflux toward the myeloid lineages compared with lymphoid lineages. Platelets were robustly labeled by HSCs, reaching the same level of labeling as HSCs by 1 year of chase. Our results support the view that adult HSCs contribute to the continuous influx of blood cells during steady-state hematopoiesis.

Sterol C24-methyltransferase (SMT) plays multiple important roles in plant growth and development. SMT1, which belongs to the family of transferases and transforms cycloartenol into 24-methylene cycloartenol, is involved in the biosynthesis of 24-methyl sterols. Here, we report the cloning and characterization of a cDNA encoding a sterol C24-methyltransferase from Tripterygium wilfordii (TwSMT1). TwSMT1 (GenBank access number KU885950) is a 1530 bp cDNA with a 1041 bp open reading frame predicted to encode a 346-amino acid, 38.62 kDa protein. The polypeptide encoded by the SMT1 cDNA was expressed and purified as a recombinant protein from Escherichia coli (E. coli) and showed SMT activity. The expression of TwSMT1 was highly up-regulated in T. wilfordii cell suspension cultures treated with methyl jasmonate (MeJA). Tissue expression pattern analysis showed higher expression in the phellem layer compared to the other four organs (leaf, stem, xylem and phloem), which is about ten times that of the lowest expression in leaf. The results are meaningful for the study of sterol biosynthesis of T. wilfordii and will further lay the foundations for the research in regulating both the content of other main compounds and growth and development of T. wilfordii.

Abstract 3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a pivotal regulator in the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway that have been shown to play key roles in the functional development of B and T cells via activation of AGC protein kinases during hematopoiesis. However, the role of PDK1 in HSCs has not been fully defined. Here we specifically deleted the PDK1 gene in the hematopoietic system and found that PDK1-deficient HSCs exhibited impaired function and defective lineage commitment abilities. Lack of PDK1 caused HSCs to be less quiescent and to produce a higher number of phenotypic HSCs and fewer progenitors. PDK1-deficient HSCs were also unable to reconstitute the hematopoietic system. Notably, HSC function was more dependent on PDK1 than on mTORC2, which indicates that PDK1 plays a dominant role in the Akt-mediated regulation of HSC function. PDK1-deficient HSCs also exhibited reduced ROS levels, and treatment of PDK1-deficient HSCs with L-butathioninesulfoximine in vitro elevated the low ROS level and promoted colony formation. Therefore, PDK1 appears to contribute to HSC function partially via regulating ROS levels.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-19902-7

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Activation of the unfolded protein response (UPR) sustains protein homeostasis (proteostasis) and plays a fundamental role in tissue maintenance and longevity of organisms. Long-range control of UPR activation has been demonstrated in invertebrates, but such mechanisms in mammals remain elusive. Here, we show that the female sex hormone estrogen regulates the UPR in hematopoietic stem cells (HSCs). Estrogen treatment increases the capacity of HSCs to regenerate the hematopoietic system upon transplantation and accelerates regeneration after irradiation. We found that estrogen signals through estrogen receptor α (ERα) expressed in hematopoietic cells to activate the protective Ire1α-Xbp1 branch of the UPR. Further, ERα-mediated activation of the Ire1α-Xbp1 pathway confers HSCs with resistance against proteotoxic stress and promotes regeneration. Our findings reveal a systemic mechanism through which HSC function is augmented for hematopoietic regeneration.

Leukemia is characterized by the uncontrolled production of leukemic cells and impaired normal hematopoiesis. Although the combination of chemotherapies and hematopoietic stem cell transplantation has significantly improved the outcome of leukemia patients, a proportion of patients still suffer from relapse after treatment. Upon relapse, a phenomenon termed “lineage switch” is observed in a subset of leukemia patients, in which conversion of lymphoblastic leukemia to myeloid leukemia or vice versa is observed. A rare entity of leukemia called mixed-phenotype acute leukemia exhibits co-expression of markers representing two or three lineages. These two phenotypes regarding the lineage ambiguity suggest that the fate of some leukemia retain or acquire a certain degree of plasticity. Studies using animal models provide insight into how lineage specifying transcription factors can enforce or convert a fate in hematopoietic cells. Modeling lineage conversion in normal hematopoietic progenitor cells may improve our current understanding of how lineage switch occurs in leukemia. In this review, we will summarize the role of transcription factors and microenvironmental signals that confer fate plasticity to normal hematopoietic progenitor cells, and their potential to regulate lineage switching in leukemias. Future efforts to uncover the mechanisms contributing to lineage conversion in both normal hematopoiesis and leukemia may pave the way to improve current therapeutic strategies.

Tripterygium wilfordii produces not only ent-kaurene, which is an intermediate of gibberellin (GA) biosynthesis in flowering plants, but also 16α-hydroxy-ent-kaurane, whose physiological role has not been characterized. The two compounds are biosynthesized from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) by diterpene synthases, which have been discovered and functionally characterized in T. wilfordii. Here, we described the functional characterization of four cytochrome P450 reductases (TwCPR) and one ent-kaurene oxidase (TwKO). Four TwCPRs were found to have relatively ubiquitous expression in T. wilfordii root, stem, leaf, and flower tissues. Co-expression of both a TwCPR and TwKO in yeast showed that TwCPR3 has a slightly better activity for providing the electrons required for these reactions, indicating that TwCPR3 is more suitable for use in the functional analysis of other cytochrome P450 monooxygenases. TwKO catalyzed the three-step oxidation of the C4α methyl of the tetracyclic diterpene intermediate ent-kaurene to form ent-kaurenoic acid as an early step in GA biosynthesis. Notably, TwKO could also convert 16α-hydroxy-ent-kaurane to 16α-hydroxy-ent-kaurenoic acid, indicating an important function of 16α-hydroxy-ent-kaurane in the anti-HIV principle tripterifordin biosynthetic pathway in planta. Homology modeling and molecular docking were used to investigate the unknown crucial active amino acid residue involved in the catalytic reaction of TwKO, and one key residue (Leu387) contributed to the formation of 16α-hydroxy-ent-kaurenoic acid, most likely by forming hydrogen bonds with the hydroxyl group (-OH) of 16α-hydroxy-ent-kaurane, which laid the basis for further investigation of the multifunctional nature of KO catalysis. Also, our findings paved the way for the complete biosynthesis of the anti-HIV principle tripterifordin.

We describe a multiple combined strategy to discover novel aptamers specific for clenbuterol (CBL). An immobilized ssDNA library was used for the selection of specific aptamers using the systematic evolution of ligands by exponential enrichment (SELEX). Progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing. Candidate aptamers were picked and preliminarily identified using a gold nanoparticles (AuNPs) biosensor. Bioactive aptamers were characterized for affinity, circular dichroism (CD), specificity and sensitivity. The Q-PCR amplification curve increased and the retention rate was about 1% at the eighth round. Use of the AuNPs biosensor and CD analyses determined that six aptamers had binding activity. Affinity analysis showed that aptamer 47 had the highest affinity (Kd = 42.17 ± 8.98 nM) with no cross reactivity to CBL analogs. Indirect competitive enzyme linked aptamer assay (IC-ELAA) based on a 5′-biotin aptamer 47 indicated the limit of detection (LOD) was 0.18 ± 0.02 ng/L (n = 3), and it was used to detect pork samples with a mean recovery of 83.33–97.03%. This is the first report of a universal strategy including library fixation, Q-PCR monitoring, high-throughput sequencing, and AuNPs biosensor identification to select aptamers specific for small molecules.

ATP-dependent SWI/SNF chromatin remodeling complexes utilize ATP hydrolysis to non-covalently change nucleosome-DNA interactions and are essential in stem cell development, organogenesis, and tumorigenesis. Biochemical studies show that SWI/SNF in mammalian cells can be divided into two subcomplexes BAF and PBAF based on the subunit composition. ARID2 or BAF200 has been defined as an intrinsic subunit of PBAF complex. However, the function of BAF200 in vivo is not clear. To dissect the possible role of BAF200 in regulating embryogenesis and organ development, we generated BAF200 mutant mice and found they were embryonic lethal. BAF200 mutant embryos exhibited multiple cardiac defects including thin myocardium, ventricular septum defect, common atrioventricular valve, and double outlet right ventricle around E14.5. Moreover, we also detected reduced intramyocardial coronary arteries in BAF200 mutants, suggesting that BAF200 is required for proper migration and differentiation of subepicardial venous cells into arterial endothelial cells. Our work revealed that PBAF complex plays a critical role in heart morphogenesis and coronary artery angiogenesis.

The development of early B cells, which are generated from hematopoietic stem cells (HSCs) in a series of well-characterized stages in bone marrow (BM), represents a paradigm for terminal differentiation processes. Akt is primarily regulated by phosphorylation at Thr308 by PDK1 and at Ser473 by mTORC2, and Akt signaling plays a key role in hematopoiesis. However, the role of mTORC2 in the development of early B cells remains poorly understood. In this study, we investigated the functional role of mTORC2 by specifically deleting an integral component, Rictor, in a hematopoietic system. We demonstrated that the deletion of Rictor induced an aberrant increase in the FoxO1 and Rag-1 proteins in BM B cells and that this increase was accompanied by a significant decrease in the abundance of B cells in the peripheral blood (PB) and the spleen, suggesting impaired development of early B cells in adult mouse BM. A BM transplantation assay revealed that the B cell differentiation defect induced by Rictor deletion was not affected by the BM microenvironment, thus indicating a cell-intrinsic mechanism. Furthermore, the knockdown of FoxO1 in Rictor-deleted HSCs and hematopoietic progenitor cells (HPCs) promoted the maturation of B cells in the BM of recipient mice. In addition, we revealed that treatment with rapamycin (an mTORC1 inhibitor) aggravated the deficiency in B cell development in the PB and BM. Taken together, our results provide further evidence that Rictor regulates the development of early B cells in a cell-intrinsic manner by modifying the expression of FoxO1 and Rag-1.

Avian embryo experiments proved an ingrowth model for the coronary artery connections with the aorta. However, whether a similar mechanism applies to the mammalian heart still remains unclear. Here we analyzed how the main coronary arteries and their orifices form during murine heart development. Apelin (Apln) is expressed in coronary vascular endothelial cells including peritruncal endothelial cells. By immunostaining, however, we did not find Apln expression in endothelial cells of the aorta during the period of coronary vessel development (E10.5 to E15.5). As a result of this unique expression difference, Apln(CreERT2/+) genetically labels nascent coronary vessels forming on the heart, but not the aorta endothelium when pulse activated by tamoxifen injection at E10.5. This allowed us to define the temporal contribution of these distinct endothelial cell populations to formation of the murine coronary artery orifice. We found that the peritruncal endothelial cells were recruited to form the coronary artery orifices. These cells penetrate the wall of aorta and take up residence in the aortic sinus of valsalva. In conclusion, main coronary arteries and their orifices form through the recruitment and vascular remodeling of peritruncal endothelial cells in mammalian heart.