Mechanistic analysis for the origin of diverse diterpenes in Tripterygium wilfordii

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.

Mutación FTL3-ITD y su relación con variables hematológicas y clínicas en individuos con Leucemia Mieloide Aguda / FTL3-ITD mutation and its relationship with hematological and clinical variables in individuals with Acute Myeloid Leukemia / A mutação FTL3-ITD e sua relação com variáveis hematológicas e clínicas em indivíduos com leucemia mielóide aguda

INTRODUCCIÓN: la Leucemia Mieloide Aguda es la neoplasia hematológica más común, caracterizada por una proliferación incontrolada de células madre hematopoyéticas. La mutación FLT3/ITD se presenta en aproximadamente el 30% de todos los pacientes con ésta patología, se asocia con mayor riesgo de recaída y menor supervivencia. El FLT3-ITD puede usarse como un factor pronóstico de la gravedad de ésta patología, importante para predecir los resultados clínicos en pacientes con LMA. OBJETIVO: el objetivo de este estudio fue relacionar la mutación FLT3/ITD con variables hematológicas y clínicas en pacientes diagnosticados con Leucemia Mieloide Aguda atendidos en la Sociedad de Lucha contra el Cáncer (SOLCA) de la ciudad de Cuenca, periodo 2013 ­2020. MÉTODOS: se obtuvieron los datos a partir de registros secundarios registrados una base de datos del hospital, el universo de la muestra lo constituyeron 63 pacientes, diagnosticados con LMA, se les analizó la mutación FLT3/ITD por PCR Convencional. RESULTADOS: se encontró la presencia de la mutación en un 9.5% y una asociación significativamente estadística con alteraciones hematológicas relacionados con niveles de hemoglobina anormal (p=0,037) y ratio 6,63 y LDH elevada en 1,21 veces (p=0,024); recuento elevado de leucocitos y blastos (p=0,031). Los individuos portadores de la mutación se presentó con mayor incidencia en el sexo masculino y grupo etario adulto mediano (45-64 años). CONCLUSIONES: la literatura internacional afirma que la mutación FLT3/ITD en un importante marcador pronóstico; debido a su baja frecuencia, no se pudo determinar una relación estadísticamente significativa con otras variables clínicas en este estudio.(AU)

INTRODUCTION: acute Myeloid Leukemia is the most common hematological neoplasm, characterized by an uncontrolled proliferation of hematopoietic stem cells. The FLT3 / ITD mutation occurs in approximately 30% of all patients with this pathology, it is associated with a higher risk of relapse and lower survival. FLT3-ITD can be used as a poor prognostic factor, important for predicting clinical outcomes in patients with AML. OBJECTIVE: the objective of this study was to characterize the FLT3 / ITD mutation and its relationship with hematological and clinical variables in patients diagnosed with Acute Myeloid Leukemia treated at SOLCA in the city of Cuenca, period 2013-2020. METHODS: data were obtained from secondary records in a hospital database, the universe of the sample was made up of 63 patients, diagnosed with AML, and the FLT3 / ITD mutation was analyzed by Conventional PCR. RESULTS: the presence of the mutation was found in 9.5% and a statistically significantly association with hematological alterations related to abnormal hemoglobin levels (p = 0.037) and ratio 6.63 and LDH elevated in 1.21 times (p =0.024); Elevated leukocyte and blast count (p = 0.031). Individuals carrying the mutation had a higher incidence in males and in the middle adult age group (45-64 years). CONCLUSIONS: the international literature affirms that the FLT3 / ITD mutation is an important prognostic marker; Due to its low frequency, it was not possible to determine a statistically significant relationship with other clinical variables in our study, for which it is suggested to expand the unirverse of the sample.(AU)

INTRODUÇÃO: a Leucemia Mielóide Aguda é a malignidade hematológica mais comum, caracterizada pela proliferação descontrolada de células-tronco hematopoiéticas. A mutação FLT3/ITD está presente em aproximadamente 30% de todos os pacientes com esta patologia, e está associada a um maior risco de recaída e menor sobrevida. O FLT3-ITD pode ser usado como um fator prognóstico para a gravidade desta patologia, importante para prever os resultados clínicos em pacientes com LMA. OBJETIVO: o objetivo deste estudo foi relacionar a mutação FLT3/ITD com variáveis hematológicas e clínicas em pacientes diagnosticados com leucemia mielóide aguda tratados na Sociedade de Luta contra o Câncer (SOLCA) na cidade de Cuenca, período 2013 - 202020. Métodos. Os dados foram obtidos de registros secundários registrados em um banco de dados hospitalar, o universo da amostra consistiu de 63 pacientes diagnosticados com AML, eles foram analisados para a mutação FLT3/ITD por PCR convencional. RESULTADOS: a presença da mutação foi encontrada em 9,5% e uma associação estatística significativa com alterações hematológicas relacionadas a níveis anormais de hemoglobina (p=0,037) e relação 6,63 e LDH elevada em 1,21 vezes (p=0,024); contagem elevada de leucócitos e explosões (p=0,031). Os individuos portadores da mutação ocorreram com maior incidência no sexo masculino e na faixa etária média adulta (45-64 anos). CONCLUSÕES: a literatura internacional afirma que a mutação FLT3/ITD em um marcador prognóstico importante; devido a sua baixa freqüência, uma relação estatisticamente significativa com outras variáveis clínicas não pôde ser determinada neste estudo.(AU)

Proiferation inhibtion and pro-apoptotic effects of shikonin on human eukemia MV4-11 clls / 吉林大学学报(医学版)

Objective: To discuss the proliferation inhibition and apoptosis induction of shikonin on the FMS-like tyrosine kinase-3 receptor internal tandem duplication (FLT3-ITD) mutated acute myeloid leukemia (AMD MV4-11 cells, and to preliminarily clarify the molecular mechanisms. Methods: The MV4-11 cells were divided into DMSO group and different concentrations (0.5, 1.0, 2.0, 4.0, and 8.0 μmol · L-1) of shikonin groups, and treated for 24 and 48 h. The inhibitory rate of proliferation was analyzed by CCK-8 assay, and half inhibitory concentration (IC50) was calculated. The MV4-11 cells were divided into blank control group, DMSO group, and different concentrations (0.25, 0.50, and 1. 00 μmol · L-1) of shikonin groups, and treated for 48 and 72 h; the proliferation rate of cells was analyzed by carbox fluorescenceindiacetate succinimidyl este (CFSE). The MV4-11 cells were divided into DMSO group and different concentrations (0.702, 1. 404, and 2. 808 μmol · L-1) of shikonin groups, and treated for 48 h; the apoptotic rate was determined by flow cytometry. The MV4-11 cells were divided into DMSO group and different concentrations (0.351, 0.702, and 1. 404 μmol · L-1) of shikonin groups, and treated for 48 h; the microRNA-155 (miR-155) expression level was detected by Real-time PCR. Results: The results of CCK-8 and CFSE methods indicated that the inhibitory rates of proliferation of MV4-11 cells in different concentrations of shikonin groups were increased compared with DMSO grpup (P<0.05 or P<0.01), and the proliferation rates were decreased (P<0.05 or P<0.01) in a concentration-dependent manner; the IC50 of 24 and 48 h were 1. 743 and 1. 404 μmol · L-1, respectively. The flow cytometry results showed that the apoptotic rates of the cells in different concentrations of shikonin groups were increased compared with DMSO group (P<0.01) in a concentration-dependent manner. The Real-time PCR results showed that the expression levels of miR-155 in the cells in different concentrations of shikonin groups were decreased significantly (P<0.01), and the expression level in 1.404 μmol · L-1 shikonin group was decreased by more than 75%. Conclusion: Shikonin could inhibit the proliferation and promote the apoptosis of FLT3-ITD mutated AML MV4-11 cells, and down-regulate the expression of miR-155, suggesting that shikonin may be one of the potential therapeutic drugs for FLT3-ITD mututed AML.

Comparative Genome Analysis of Scutellaria baicalensis and Scutellaria barbata Reveals the Evolution of Active Flavonoid Biosynthesis / 基因组蛋白质组与生物信息学报·英文版

Scutellaria baicalensis (S. baicalensis) and Scutellaria barbata (S. barbata) are common medicinal plants of the Lamiaceae family. Both produce specific flavonoid compounds, including baicalein, scutellarein, norwogonin, and wogonin, as well as their glycosides, which exhibit antioxidant and antitumor activities. Here, we report chromosome-level genome assemblies of S. baicalensis and S. barbata with quantitative chromosomal variation (2n = 18 and 2n = 26, respectively). The divergence of S. baicalensis and S. barbata occurred far earlier than previously reported, and a whole-genome duplication (WGD) event was identified. The insertion of long terminal repeat elements after speciation might be responsible for the observed chromosomal expansion and rearrangement. Comparative genome analysis of the congeneric species revealed the species-specific evolution of chrysin and apigenin biosynthetic genes, such as the S. baicalensis-specific tandem duplication of genes encoding phenylalanine ammonia lyase and chalcone synthase, and the S. barbata-specific duplication of genes encoding 4-CoA ligase. In addition, the paralogous duplication, colinearity, and expression diversity of CYP82D subfamily members revealed the functional divergence of genes encoding flavone hydroxylase between S. baicalensis and S. barbata. Analyzing these Scutellaria genomes reveals the common and species-specific evolution of flavone biosynthetic genes. Thus, these findings would facilitate the development of molecular breeding and studies of biosynthesis and regulation of bioactive compounds.

Progress of FMS-like tyrosine kinase 3 internal tandem duplication-positive acute myeloid leukemia / 白血病·淋巴瘤

FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) mutation is a commonly found type of mutation in acute myeloid leukemia (AML), and this mutation is closely related to the development of AML and affects the prognosis of patients. FLT3-ITD-positive AML patients have poor prognosis and high relapse rate after treated with chemotherapy alone. The efficacy of allogeneic hematopoietic stem cell transplantation for this disease is controversial. Targeted therapies such as sorafenib and the combination of different type of therapies have been carried out and have shown preliminary effect in preclinical or clinical trials. In this paper, the clinical effect of FLT3-ITD in AML patients and the latest progress of its treatment were reviewed.

Clinical characteristics and prognosis of 34 cases of acute myeloid leukemia with FLT3 internal tandem duplication and MLL gene rearrangement / 中华血液学杂志

Objective@#To analyze the clinical characteristics and prognosis of 34 cases of acute myeloid leukemia (AML) with FLT3 internal tandem duplication (FLT3-ITD) and MLL gene rearrangement.@*Methods@#The clinical data of 34 AML patients with FLT3-ITD and MLL gene rearrangement was compared and analyzed for the therapeutic efficacy, prognostic factors when treated with chemotherapy, chemotherapy combined with targeted therapy or allogenic hematopoietic stem cell transplantation (allo-HSCT).@*Results@#Of the thirty-four cases with median age 41 (4-71) years old, 63.6% presented with white blood cells (WBC) greater than 30×109/L, 39.4% greater than 50 × 109/L respectively on admission. M5 (35.3%) made up the highest proportion. The cytogenetic abnormality reached 61.8%, of which the complex cytogenetic abnormality accounted for 11.8%. Eleven patients (32.35%) had both FLT3-ITD and MLL gene abnormalities. In addition to FLT3 and MLL abnormalities, 23 patients (67.6%) had one or more other gene abnormalities (multiple gene abnormalities). Of the 34 cases, 29.4% patients went into complete remission (CR) after two courses of chemotherapy. 20.6% (7 patients) went into CR after 3 or more courses of chemotherapy. The rate of early relapse in the CR group was 52.9%. Patients with WBC>50×109/L or multiple gene abnormalities had a lower remission rate (7.7%, 5.4%) after two courses of chemotherapy. CR rate for the patients with more than three gene abnormalities was 0. The total 2-year overall survival (OS) in the 34 patients was 28.8% (95% CI 13.5%-46.0%) and the disease-free survival (DFS) was 27.1% (95% CI 12.5%-44.0%). Of the 18 patients treated with chemotherapy alone or chemotherapy combined with targeted therapy, 17 cases died within 2 years and 1 lost follow-up after giving up treatment. For the 16 patients received allo-HSCT, the 3-year OS was 43.4% (95% CI 13.7%-70.4%) and DFS 42.7% (95% CI 13.4%-69.7%).@*Conclusion@#AML patients with FLT3-ITD and MLL gene rearrangement often presented with M5, accompanied by hyperleukocytosis, cytogenetic or multiple gene abnormalities. Those patients were observed to have low response rate and high early relapse when treated with chemotherapy without allo-HSCT. Patients had multiple gene abnormalities may be an important poor prognostic factor. Allo-HSCT is an effective treatment which could significantly improve the prognosis and survival of AML patients with FLT3-ITD and MLL gene abnormalities.

Clinical feature between FLT3-ITD mutation type and wild type de novo primary AML / 实用医学杂志

Objective To investigate the difference of clinical features between FLT3-ITDmt and FLT3-IT-Dwt de novo primary acute myeloid leukemia(AML). Methods Clinical data of 31 FLT3-ITDmt and 113 FLT3-ITDwt de novo primary AML patients from January 2015 to December 2016 were retrospectively reviewed and ana-lyzed by Student′s test,chi-square test or rank sum test according to the types of clinical data. Results There were statistically significant differences in WBC,RBC,HGB of peripheral blood and the mutation of DNMT3A gene(statistical values:705.000;-2.535;-2.290 and 5.715 respectively,all P < 0.05)in 2 types of AML. Conclusion When compared with FLT3-ITDwt de novo primary AML patients,FLT3-ITDmt ones have the fea-tures of higher WBC,lower RBC and HGB of peripheral blood,and are more likely to be associated with mutation in the DNMT3A gene.

Prognostic value of FLT3-ITD detection on acute myelogenous leukemia by DNA extracted from stored bone marrow slides by chemical method / 白血病·淋巴瘤

Objective To investigate the prognostic value of Fms-like tyrosine kinase3, intenal tandem duplication (FLT3-ITD) detection by DNA extracted from stored bone marrow slides in chemical method. Methods Trace DNA was extracted from 58 bone marrow slides which were stored for 1-5 years below 20 ℃, including 48 patients with de novo acute myeloid leukemia (AML) and 10 controls without hematologic malignancies. Polymerase chain reaction (PCR) was used to detect the FLT3-ITD of these bone marrow slides samples. Results There were 6 patients of FLT3-ITD+ detected in these 48 AML patients (12.5 %, 6/48). No FLT3-ITD was found in 10 healthy controls. AML patients with FLT3-ITD+ had low complete time compared with FLT3-ITD-patients (x2= 7.274, P= 0.007). Splenohepatomegalia and FLT3 mutation were the risk factors affecting AML patients with CR after the first chemotherapy (OR= 7.2, P=0.12; OR=36.3, P=0.10). FLT3-ITD was a risk factor of poor prognosis in patients with newly diagnosed AML (RR=9.088, P= 0.029). Conclusion Extraction of AML bone marrow slides trace DNA by using chemical method can be widely applied in clinic and is a key experimental way to study the molecular biology retrospectively. Furthermore, the detection of FLT3-ITD by trace DNA extracted from stored bone marrow slides can be used to predict the prognosis of AML.

Prevalence and Clinical Implication of Partial Tandem Duplication of the Mixed Lineage Leukemia Gene in Pediatric Acute Leukemia / 임상소아혈액종양

BACKGROUND: The mixed lineage leukemia (MLL) gene may induce hematopoiesis and leukemia. Partial tandem duplication of MLL (MLL-PTD) is associated with poor prognosis in acute myeloid leukemia (AML); however, the significance of MLL-PTD in acute lymphoblastic leukemia (ALL) has not been thoroughly studied. We evaluated the incidence, relationship with other cytogenetic abnormalities, and the prognostic role of MLL-PTD in ALL.METHODS: We reviewed medical records from pediatric patients diagnosed with ALL in Severance Hospital, Yonsei University Health System, South Korea from 2002 to 2008. MLL-PTD was detected by nested reverse transcriptase polymerase chain reaction.RESULTS: In ALL patients, 50.0% (42/84) were positive for MLL-PTD. There was no significant difference in the 10-year overall survival (10Y OS) and event-free survival (EFS) between MLL-PTD-positive (+) and MLL-PTD-negative (-) groups (69.4% vs. 76.2%, P=0.609, and 62.6% vs. 66.7%, P=0.818, respectively). The combination of high level of lactate dehydrogenase (>1,100 IU/L) and MLL-PTD(+) [MLL-PTD(+)/High LDH] was a statistically significant negative prognostic factor for 10Y OS and EFS (P=0.0226 and P=0.0230, respectively). In multivariate analysis, National Cancer Institute risk stratification and very high risk features were independent significant prognostic factors but MLL-PTD (+)/High LDH was not.CONCLUSION: MLL-PTD was observed frequently in pediatric ALL patients. MLL-PTD was not an independent prognostic factor. MLL-PTD (+)/High LDH should be evaluated further for its prognostic potential in ALL.

Prevalence and Clinical Implication of Partial Tandem Duplication of the Mixed Lineage Leukemia Gene in Pediatric Acute Leukemia / 임상소아혈액종양

BACKGROUND: The mixed lineage leukemia (MLL) gene may induce hematopoiesis and leukemia. Partial tandem duplication of MLL (MLL-PTD) is associated with poor prognosis in acute myeloid leukemia (AML); however, the significance of MLL-PTD in acute lymphoblastic leukemia (ALL) has not been thoroughly studied. We evaluated the incidence, relationship with other cytogenetic abnormalities, and the prognostic role of MLL-PTD in ALL. METHODS: We reviewed medical records from pediatric patients diagnosed with ALL in Severance Hospital, Yonsei University Health System, South Korea from 2002 to 2008. MLL-PTD was detected by nested reverse transcriptase polymerase chain reaction. RESULTS: In ALL patients, 50.0% (42/84) were positive for MLL-PTD. There was no significant difference in the 10-year overall survival (10Y OS) and event-free survival (EFS) between MLL-PTD-positive (+) and MLL-PTD-negative (-) groups (69.4% vs. 76.2%, P=0.609, and 62.6% vs. 66.7%, P=0.818, respectively). The combination of high level of lactate dehydrogenase (>1,100 IU/L) and MLL-PTD(+) [MLL-PTD(+)/High LDH] was a statistically significant negative prognostic factor for 10Y OS and EFS (P=0.0226 and P=0.0230, respectively). In multivariate analysis, National Cancer Institute risk stratification and very high risk features were independent significant prognostic factors but MLL-PTD (+)/High LDH was not. CONCLUSION: MLL-PTD was observed frequently in pediatric ALL patients. MLL-PTD was not an independent prognostic factor. MLL-PTD (+)/High LDH should be evaluated further for its prognostic potential in ALL.

Prognostic Significance of FLT3 Internal Tandem Duplication in Acute Myeloid Leukemia with Normal Karyotype / 대한혈액학회지

BACKGROUND: Activating mutations of the fms-like tyrosine kinase 3 gene (FLT3) by internal tandem duplication (ITD) in the juxtamembrane region are found in 20~30% of the adults with acute myeloid leukemia (AML). The allelic ratio of FLT3/ITD (ITD-AR), as assessed by Genescan analysis, has been reported to carry prognostic significance in AML patients who have normal karyotype. METHODS: FLT3/ITD was studied by PCR in 113 adults with AML including 55 patients with normal karyotype. Genescan analysis was performed for the PCR products to determine the allelic distribution. The results were correlated with the prognostic factors. RESULTS: FLT3/ITD was found in 23% of the total AML patients and in 32.7% of those patients with normal karyotype. The mutation was related to a high WBC count, a high serum LD level and a low percentage of CD34 positive cells. The ITD-AR ranged from 0.05 to 8.27 (median, 0.61). The patients with a high ITD-AR (> or =0.7) had significantly higher WBC count and LD level than those without FLT3/ITD. On multivariate analysis, a high ITD-AR as well as FLT3/ITD were confirmed to be independent adverse prognostic factors for AML patients with normal karyotype. CONCLUSION: This study demonstrated that a high ITD-AR and FLT3/ITD had major adverse impacts on the prognostic relevance for AML patients with normal karyotype.

Low Frequency and Variability of FLT3 Mutations in Korean Patients with Acute Myeloid Leukemia

FLT3 mutations are common genetic changes, and are reported to have prognostic significance in acute myeloid leukemia (AML). The FLT3 internal tandem duplication (ITD) and the D835 activating mutation in the tyrosine kinase domain (TKD) were analyzed by polymerase chain reaction (PCR) in the genomic DNA of Korean patients with AML at diagnosis and during follow-up. There were 226 patients with AML enrolled between March 1996 and August 2005. The incidence of ITD and TKD at diagnosis was 13% (29/226) and 3% (6/226). When compared to Western and other Asian patients with AML, Korean patients had a lower frequency by about two-thirds of ITD and TKD. Among the non-M3 cases (N=203), the patients with an ITD had a significantly shorter event-free survival when compared with those without an ITD (p=0.0079). Among 54 relapsed patients, 9 patients had the FLT3 ITD at diagnosis. Six patients demonstrated a reappearance of the ITD and 3 patients remained negative at relapse. One patient, among 45 patients who relapsed, had a negative baseline ITD but acquired a de novo ITD at relapse. There were 101 samples from 93 patients in remission; they were all negative for an ITD. Among 34 patients who failed to achieve a remission, five patients had a persistent ITD and one patient had a de novo ITD. These results support the concept of resistance of FLT3 ITD leukemic clones to chemotherapy. Therefore, effective therapy with FLT3 targeting agents may improve the prognosis of non-M3 AML patients with the FLT3 mutation.