Reduced nitrogen fertilization from pre-flowering to pre-veraison alters phenolic profiles of Vitis vinifera L. Cv. Cabernet Gernischt wine of Yantai, China.

Nitrogen (N) fertilization is important for grape growth and wine quality. Unreasonable N fertilizer application affects wine growth and has a negative impact on wine quality. Therefore, it is essential to address the mismatch between N application and wine composition. To regulate vine growth and improve grape and wine quality, Cabernet Gernischt (Vitis vinifera L.) grapevines were subjected to lower levels of N, compared to normal N supply treatments, during the grape growing seasons of 2019 and 2020 in the wine region of Yantai, China. The effects of reduced N application from pre-boom to pre-veraison on vine growth, yield and composition of grapes, and dry red wine anthocyanin and non-anthocyanin phenolic compound content were studied. We found that reduced N application significantly decreased dormant shoot fresh mass and yield. However, the effect of N application on fruit ripening depended on the season. Nitrogen-reduction treatment significantly improved wine phenolic parameters, including total phenolics, tannins, and anthocyanins, and enhanced most of the individual anthocyanins and some non-anthocyanin phenolics, especially stilbenes, including piceatannol, trans-resveratrol, and polydatin, regardless of the season. Overall, our findings highlight the importance of reducing N application during the grape growing season in order to modify the wine phenolic profiles.

Modification of wine phenolic profiles by gibberellic acid application in 'Cabernet Gernischt' grapevines before anthesis.

BACKGROUND: Vitis vinifera L. 'Cabernet Gernischt' grapes from the Yantai wine region of China usually form dense clusters and contain low phenolic content. We applied five concentrations (ranged from 5 to 25 mg L-1 ) of gibberellic acid (GA3 ) to 'Cabernet Gernischt' before anthesis to decrease cluster compactness in two consecutive vintages. Yield indices, grape maturity, and wine phenolic compounds were determined. RESULTS: GA3 application significantly reduced cluster compactness, bunch weight, and yield per vine, but it did not significantly improve fruit ripening. The levels of total phenolics, total tannins, and total anthocyanins in wine were enhanced by GA3 application, with 10 and 15 mg L-1 GA3 treatments consistently producing a significant increase in the concentrations of mavidin, cyanidin, and their derivatives. Specifically, trans-resveratrol was consistently significantly increased by 15 mg L-1 GA3 application. Principal component analysis of phenolic compounds demonstrated the differences among wines produced from GA3 treatment groups and the control. CONCLUSION: Overall, wine phenolic profiles could be significantly modified by application of low concentrations of GA3 before anthesis. Application of high levels of GA3 is not recommended due to significant yield decrease. © 2022 Society of Chemical Industry.

A retrospective study on effectiveness of combined recombinant human interferon-α-1b, interleukin-2, and thalidomide for the treatment of acute myeloid leukemia in various disease states.

Background: Interferon-α-1b, interleukin-2 combined with thalidomide (ITI) improved the outcome and prognosis of some acute myeloid leukemia (AML) patients, but the cases was insufficient. This study observed the efficacy and safety of this regimen in the treatment of numbers of AML patients in various disease states. Methods: Starting in January 2014, patients with AML (n=188) were treated with ITI regimen, including 60 refractory/relapses patients in group A, 40 patients in group B remained minimal residual disease-positive (MRD) or changed from negative to positive again after consolidation therapy, and 88 patients in group C with initial complete remission of AML received the ITI treatment after routine consolidation therapy. Bone marrow, fusion gene and MRD were detected to judge the curative effect and the adverse reactions were observed. The remission rate, MRD status and long-term survival of three groups were analyzed. An AML mouse model was constructed to observe the anti-leukemia effect of the three drugs in vivo. Results: Sixty patients with primary AML who were unable to receive chemotherapy, or with relapsed/refractory AML, showed a total response rate of 28.3% (17/60) after receiving the ITI regimen. Forty patients with morphologically complete remission and MRD-positive achieved a response rate of 77.5% (31/40); the MRD converted to negative in 19 patients and was mitigated in 12 patients. Among 88 patients with initial complete remission, 11 failed to maintain the negative MRD, and the relapse rate was 12.5%, which was significantly lower than that of the non-maintenance treatment group (54.3%). In the mouse model, interferon, interleukin-2, and thalidomide exerted an anti-leukemia effect, prolonged the survival time of the mice, and the anti-leukemia effect was further enhanced after administration of the combination ITI regimen. Conclusions: For suitable patients, hematopoietic stem cell transplantation is still strong recommended. The ITI regimen may be an effective option for patients with AML who cannot tolerate conventional chemotherapy, including those with relapsed/refractory disease, those with a complete remission status but are MRD-positive, or those who require maintenance treatment after consolidation therapy. However, a rigorous clinical randomized controlled trial and more in-depth mechanism exploration are still needed to verify this conclusion.

HMGA2 regulates acute myeloid leukemia progression and sensitivity to daunorubicin via Wnt/ß-catenin signaling.

Acute myeloid leukemia (AML) is a malignant disease with an increasing prevalence in adults and children. However, valuable molecular diagnostic research is rare. In the present study, plasmids silencing and overexpressing high­mobility group AT­hook 2 (HMGA2) were respectively transfected in HL60 and NB4 cells. The effects of HMGA2 on AML cell viability, apoptosis, migration and invasion were determined by preforming MTT, flow cytometry, wound scratch and Transwell assays, respectively. Genes associated with apoptosis and Wnt signaling were evaluated by reverse transcription­quantitative (RT­q)­PCR and western blotting. AML cell sensitivity to daunorubicin (DNR) and the regulatory effects of the Wnt signaling pathway via HMGA2 following treatment with the agonist LiCl or antagonist XAV939 were detected by MTT, RT­qPCR and western blot analysis. The results revealed that the expression of HMGA2 was elevated more so in HL60, KG1, U937, Kasumi­1, THP­1 and K562 cells than in NB4 cells. Silencing HMGA2 suppressed cell viability, migration and invasion, enhanced cell apoptosis and sensitivity to DNR, and almost restored the DNR inhibitory function that was promoted by LiCl treatment. In addition, low expression of HMGA2 attenuated X­linked inhibitor of apoptosis and Bcl­2 mRNA and protein levels, and upregulated the expression of Bax and cleaved­caspase­3. Furthermore, silencing HMGA2 not only decreased Wnt and non­phospho­ß­catenin expressions, but also partially reversed the increased expressions of these proteins induced by LiCl treatment. On the other hand, overexpression of HMGA2 exhibited the opposite results after transfection in NB4 cells. The results of the present study demonstrated that HMGA2 played important roles in driving AML progression and chemosensitivity in HL60 and NB4 cells, potentially by activating the Wnt/ß­catenin signaling pathway. Therefore, it was suggested that HMGA2 may be a promising molecular marker for AML diagnosis.

PML/RARa blocks the differentiation and promotes the proliferation of acute promyelocytic leukemia through activating MYB expression by transcriptional and epigenetic regulation mechanisms.

The promyelocytic leukemia (PML)/retinoic acid receptor-alpha (RARα) onco-fusion protein that is generated from t(15;17) chromosome translocation is crucial for the leukemogenesis of acute promyelocytic leukemia (APL) and is well documented as a transcriptional repressor. To understand the relationship between PML/RARα and the oncogene in the development of APL, we investigate the regulation mechanism of PML/RARα to MYB proto-oncogene and the role of this regulation on the proliferation and differentiation of APL cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays show that MYB expression was significantly higher in PML/RARα positive cell lines. Microarray data verify that the MYB expression was significantly higher in APL patient samples than in normal promyelocyte samples. Further expression analysis from RT-qPCR and microarray data verifies that the expression of MYB is upregulated by PML/RARα. Transcriptional factor binding analysis shows that MYB is directly bound by PML/RARα and its cofactors. Luciferase assays show that PML/RARα transactivated MYB promoter activity through the RARα binding site and the coexistence of CCAAT enhancer binding protein ε. We also find that PML/RARα increases the acetylation level of the promoter region of MYB. Further evidence demonstrates that PML/RARα regulates MYB expression through long-range interaction. Functionally, PML/RARα increases the cell proliferation and blocks the differentiation through activating MYB expression. Collectively, this study uncovers a novel mechanism of PML/RARα-mediated transcriptional activation and enriches our knowledge of the onco-fusion protein-mediated transcription activation.

c-Myc, RMRP, and miR-34a-5p form a positive-feedback loop to regulate cell proliferation and apoptosis in multiple myeloma.

Long non-coding RNA (lncRNA) component of mitochondrial RNA processing endoribonuclease (RMRP) has been demonstrated to be implicated in human cancer processes. However, the role of lncRNA RMRP in multiple myeloma (MM) remains unknown. In this paper, we proved that RMRP and c-Myc were upregulated, while miR-34a-5p was downregulated in MM cell lines and bone marrows of MM patients. High RMRP expression significantly correlated with worse disease-free survival and overall survival in MM patients. c-Myc promoted RMRP transcription by directly binding to its promoter region. Knockdown of RMRP inhibited proliferation and promoted apoptosis of OPM2 and RPMI-8226 cells. Negative correlation between RMRP, and miR-34a-5p was discovered in bone marrows of MM patients. c-Myc expression was inversely correlated with miR-34a-5p in bone marrows of MM patients. Additionally, silencing of RMRP led to a marked reduction in c-Myc expression in OPM2 and RPMI-8226 cells, and this action was obviously blocked by miR-34a-5p knockdown. Moreover, upregulation of miR-34a-5p repressed proliferation and promoted apoptosis of OPM2 and RPMI-8226 cells. However, RMRP overexpression blocked these changes triggered by miR-34a-5p mimic. Besides, RMRP knockdown repressed MM tumor growth in vivo. Conclusions, RMRP functions as a miR-34a-5p sponge to promote cell proliferation and repress cell apoptosis through upregulation of c-Myc in MM.

[Effect of SHIP-1 on Invasion, Migration and PI3K-AKT Signaling Pathway of Leukemic Cells].

OBJECTIVE: To investigate the effect of SH2-containing inositol phosphatase-1 (SHIP-1) on the proliferation, invasion and migration of human leukemia cells as well as phosphatidylinositol-3 kinase (PI3K) / protein kinase B (AKT) signaling pathway. METHODS: The overexpression vector pCDNA3.1-SHIP1 was transfected into THP-1 cells by Lipofectamine 2000. The experiment was divided into 3 groups: control group (untreated cells) and empty vector group (transfected with empty vector pCDNA3.1-NC) and overexpression group (transfected with overexpression vector pCDNA3.1-SHIP1). The cell proliferation was tected by CCK-8 assay, Transwell assay was used to evaluate the cell invasion and migration capabilities. The expressions of SHIP-1, AKT, phosphorylated AKT (pAKT), matrix metalloproteinase-9 (MMP-9) protein were analyzed by Western blot. RESULTS: The expression of SHIP-1 in overexpression group was significantly higher than that in the control group(P<0.05). Compared with the control group, the absorbance of the cells in the empty vector group was not statistically different (P>0.05), and the absorbance in overexpression group decreased significantly(P<0.05). The cell numbers of invasion and migration were not significantly different between empty and control groups(P>0.05), but those in overexpression group were significantly lower than those in the control group(P<0.05). Compared with the control group, the expression of AKT, pAKT and MMP-9 in the empty vector group was not statistically different (P>0.05); the AKT protein in overexpression group was not significantly different (P>0.05), but the pAKT and MMP-9 significantly decreased(P<0.05). CONCLUSION: SHIP-1 plays a role in inhibiting the proliferation, invasion and migration of leukemia cells, the mechanism probably relates with supressing the expression of MMP-9 by regulating PI3K/AKT signaling pathway.

AML1/ETO trans-activates c-KIT expression through the long range interaction between promoter and intronic enhancer.

The AML1/ETO onco-fusion protein is crucial for the genesis of t(8;21) acute myeloid leukemia (AML) and is well documented as a transcriptional repressor through dominant-negative effect. However, little is known about the transactivation mechanism of AML1/ETO. Through large cohort of patient's expression level data analysis and a series of experimental validation, we report here that AML1/ETO transactivates c-KIT expression through directly binding to and mediating the long-range interaction between the promoter and intronic enhancer regions of c-KIT. Gene expression analyses verify that c-KIT expression is significantly high in t(8;21) AML. Further ChIP-seq analysis and motif scanning identify two regulatory regions located in the promoter and intronic enhancer region of c-KIT, respectively. Both regions are enriched by co-factors of AML1/ETO, such as AML1, CEBPe, c-Jun, and c-Fos. Further luciferase reporter assays show that AML1/ETO trans-activates c-KIT promoter activity through directly recognizing the AML1 motif and the co-existence of co-factors. The induction of c-KIT promoter activity is reinforced with the existence of intronic enhancer region. Furthermore, ChIP-3C-qPCR assays verify that AML1/ETO mediates the formation of DNA-looping between the c-KIT promoter and intronic enhancer region through the long-range interaction. Collectively, our data uncover a novel transcriptional activity mechanism of AML1/ETO and enrich our knowledge of the onco-fusion protein mediated transcription regulation.