Current application and future perspective of CRISPR/cas9 gene editing system mediated immune checkpoint for liver cancer treatment.

Liver cancer, which is well-known to us as one of human most prevalent malignancies across the globe, poses a significant risk to live condition and life safety of individuals in every region of the planet. It has been shown that immune checkpoint treatment may enhance survival benefits and make a significant contribution to patient prognosis, which makes it a promising and popular therapeutic option for treating liver cancer at the current time. However, there are only a very few numbers of patients who can benefit from the treatment and there also exist adverse events such as toxic effects and so on, which is still required further research and discussion. Fortunately, the clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9 (CRISPR/Cas9) provides a potential strategy for immunotherapy and immune checkpoint therapy of liver cancer. In this review, we focus on elucidating the fundamentals of the recently developed CRISPR/Cas9 technology as well as the present-day landscape of immune checkpoint treatment which pertains to liver cancer. What's more, we aim to explore the molecular mechanism of immune checkpoint treatment in liver cancer based on CRISPR/Cas9 technology. At last, its encouraging and powerful potential in the future application of the clinic is discussed, along with the issues that already exist and the difficulties that must be overcome. To sum up, our ultimate goal is to create a fresh knowledge that we can utilize this new CRISPR/Cas9 technology for the current popular immune checkpoint therapy to overcome the treatment issues of liver cancer.

Unraveling Mechanism for Microstructure Engineering toward High-Capacity Nickel-Rich Cathode Materials.

Microstructural engineering on nickel-rich layered oxide (NRLO) cathode materials is considered a promising approach to increase both the capacity and lifespan of lithium-ion batteries by introducing high valence-state elements. However, rational regulation on NRLO microstructures based on a deep understanding of its capacity enhancement mechanism remains challenging. Herein for the first time, it is demonstrated that an increase of 14 mAh g-1 in reversible capacity at the first cycle can be achieved via tailoring the micro and nano structure of NRLO through introducing tungsten. Aberration-corrected scanning transmission electron microscopy (STEM) characterization reveals that the formation of a modified microstructure featured as coherent spinel twin boundaries. Theoretical modeling and electrochemical investigations further demonstrate that the capacity increase mechanism is related to such coherent spinel twin boundaries, which can lower the Li+ diffusion barrier and thus allow more Li+ to participate in deeper phase transitions. Meanwhile, the surface and grain boundaries of NRLOs are found to be modified by generating a dense and uniform LiWxOy phase, which further extends its cycle life by reducing side reactions with electrolytes. This work enables a comprehensive understanding of the capacity-increased mechanism and endows the remarkable potential of microstructural engineering for capacity- and lifespan-increased NRLOs.

Recent research progress based on ferroptosis-related signaling pathways and the tumor microenvironment on it effects.

The existing therapies for cancer are not remote satisfactory due to drug-resistance in tumors that are malignant. There is a pressing necessity to take a step forward to develop innovative therapies that can complement current ones. Multiple investigations have demonstrated that ferroptosis therapy, a non-apoptotic modality of programmed cell death, has tremendous potential in face of multiple crucial events, such as drug resistance and toxicity in aggressive malignancies. Recently, ferroptosis at the crosswalk of chemotherapy, materials science, immunotherapy, tumor microenvironment, and bionanotechnology has been presented to elucidate its therapeutic feasibility. Given the burgeoning progression of ferroptosis-based nanomedicine, the newest advancements in this field at the confluence of ferroptosis-inducers, nanotherapeutics, along with tumor microenvironment are given an overview. Here, the signaling pathways of ferroptosis-related were first talked about briefly. The emphasis discussion was placed on the pharmacological mechanisms and the nanodrugs design of ferroptosis inducing agents based on multiple distinct metabolism pathways. Additionally, a comprehensive overview of the action mechanisms by which the tumor microenvironment influences ferroptosis was elaborately descripted. Finally, some limitations of current researches and future research directions were also deliberately discussed to provide details about therapeutic avenues for ferroptosis-related diseases along with the design of anti-drugs.

Synergistic anti-tumor effect of dual drug co-assembled nanoparticles based on ursolic acid and sorafenib.

Both ursolic acid (UA) and sorafenib (Sora) have been generally utilized in cancer treatment, and the combination of the two has also shown a good anti-tumor effect. However, single-agent therapy for Hepatocellular carcinoma (HCC) has the disadvantages of multi-drug resistance, poor water solubility and low bioavailability, and the application of traditional nanocarrier materials is limited due to their low drug loading and low carrier-related toxicity. Therefore, we prepared US NPs with different proportions of UA and Sora by solvent exchange method for achieving synergistic HCC therapy. US NPs had suitable particle size, good dispersibility and storage stability, which synergistically inhibited the proliferation of HepG2 cells, SMMC7721 cells and H22 cells. In addition, we also proved that US NPs were able to suppress the migration of HepG2 cells and SMMC7721 cells and reduce the adhesion ability and colony formation ability of these cells. According to the results, US NPs could degrade the membrane potential of mitochondrial, participate in cell apoptosis, and synergistically induce autophagy. Collectively, the carrier-free US NPs provide new strategies for HCC treatment and new ideas for the development of novel nano-drug delivery systems containing UA and Sora.

Calcium carbonate synthesis from Kambara reactor desulphurization slag via indirect carbonation for CO2 capture and utilization.

In this work, industrial Kambara reactor desulphurization slag (KR slag) was indirectly carbonated. The effects of leaching time, leaching temperature, leaching agent types, and leaching agent concentration on the leaching ratio of calcium from KR slag were investigated. Subsequently, precipitated calcium carbonate (PCC) was synthesized by bubbling CO2 gas (flow rate of 15 mL/min) into 400 mL leaching solutions at 40 °C for 120 min with magnetic stirring at 300 rpm. It is found that calcium in KR slag can be selectively extracted using a diluted solution of ammonium acetate (CH3COONH4) or ammonium chloride (NH4Cl), while ammonium sulfate ((NH4)2SO4) solution is not suitable as leaching agent due to the formation of slightly soluble calcium sulfate (CaSO4). The leaching ratio of calcium is improved by extending the leaching time or increasing the leaching solvent concentration. However, leaching temperature has little effect on calcium extraction. After carbonating the NH4Cl- and CH3COONH4-leachate for 120 min, calcite and vaterite type PCC with a purity of 99% is synthesized. Each gram of KR slag can produce 0.794 g and 0.803 g PCC using NH4Cl and CH3COONH4 leaching agents respectively. Calculations show that 349.6 kg CO2 is captured by per ton of KR slag. The CO2 capture capacity of KR slag is significantly higher compared with previously studied materials.

Cloning and functional characterization of FtWRKY29, a low phosphorus stress-inducible transcription factor in Tartary buckwheat.

The regulation of the expression of genes related to abiotic stress in plants is significantly influenced by the binding of the transcription factor (TF) WRKY to the W-box elements in their promoters. The findings of this study have confirmed that the ability of Tartary buckwheat (Fagopyrum tataricum Gaertn.) to tolerate phosphorus (P) deficiency is regulated by FtWRKY29, which is classified as a member of group II of the WRKY family. The roots predominantly exhibited an enhanced expression of FtWRKY29, which was significantly upregulated in response to low-P-induced stress. The W-box motif was bound to by FtWRKY29 which enhanced the transcription of genes and was localized to the nucleus. The overexpression of FtWRKY29 in Arabidopsis thaliana produced transgenic lines that exhibited phenotypes typical of diminished sensitivity to low-P-induced stress by promoting root growth, increasing P-uptake, and regulating the accumulation of anthocyanin. The low-P-responsive genes, PHT1;1, PHT1;4, and PHO1 were significantly up-regulated in these lines. In addition, the overexpression of FtWRKY29 restored the P-absorption ability of the wrky75 mutant to a certain extent. Moreover, the binding of FtWRKY29 to the promoter of PHT1;1 activated its expression in tobacco. It was also observed that FtWRKY29 interacts with AtMPK3, AtMPK6, FtMPK3, and FtMPK7. This study provides preliminary evidence that FtWRKY29 improved the tolerance of transgenic A. thaliana plants to low-P-induced stress and deepened the understanding of the regulatory mechanism behind the same in Tartary buckwheat.

CRISPR/Cas9-based application for cancer therapy: Challenges and solutions for non-viral delivery.

CRISPR/Cas9 genome editing is a promising therapeutic technique, which makes precise and rapid gene editing technology possible on account of its high sensitivity and efficiency. CRISPR/Cas9 system has been proved to able to effectively disrupt and modify genes, which shows great potential for cancer treatment. Current researches proves that virus vectors are capable of effectively delivering the CRISPR/Cas9 system, but immunogenicity and carcinogenicity caused by virus transmission still trigger serious consequences. Therefore, the greatest challenge of CRISPR/Cas9 for cancer therapy lies on how to deliver it to the target tumor site safely and effectively. Non-viral delivery systems with specific targeting, high loading capacity, and low immune toxicity are more suitable than viral vectors, which limited by uncontrollable side effects. Their medical advances and applications have been widely concerned. Herein, we present the molecule mechanism and different construction strategies of CRISPR/Cas9 system for editing genes at the beginning of this research. Subsequently, several common CRISPR/Cas9 non-viral deliveries for cancer treatment are introduced. Lastly, based on the main factors limiting the delivery efficiency of non-viral vectors proposed in the existing researches and literature, we summarize and discuss the main methods to solve these limitations in the existing tumor treatment system, aiming to introduce further optimization and innovation of the CRISPR/Cas9 non-viral delivery system suitable for cancer treatment.

Transcriptomic and Metabonomic Profiling Reveals the Antihyperlipidemic Effects of Tartary Buckwheat Sprouts in High-Fat-Diet-Fed Mice.

Flavonoids are known for potent antioxidant activity and antihyperlipidemia. As a result of the few antinutritional factors and high bioactive substances, such as flavonoids, sprouts of tartary buckwheat (Fagopyrum tataricum, STB) have become healthy food. This study aims to unravel the antihyperlipidemic effects of STB in vivo and its potential mechanism through transcriptomic and metabonomic analysis. The physiological parameters of mice administered the high-fat diet with or without 2.5 and 5% of STB for 10 weeks were recorded. Liquid chromatography-tandem mass spectrometry and RNA sequencing were applied to obtain the serum lipid metabolomic and hepatic transcriptomic profiling, respectively. Results revealed that STB could significantly alleviate the increase of body weight, liver, and abdominal adipose while ameliorating the lipid content in serum and insulin resistance of mice fed with a high-fat diet. Notably, the metabonomic analysis identified the core differential metabolites mainly enriched in the pathways, such as fat digestion and absorption, insulin resistance, and other processes. Transcriptomic results revealed that STB significantly altered the expression levels of PIK3R1, LRP5, SLC10A2, and FBXO21. These genes are involved in the PI3K-AKT signaling pathway, digestion and absorption of carbohydrates, and type II diabetes mellitus pathways. In this study, STB exhibited remarkable influence on the metabolism of lipids and glucose, exerting antihyperlipidemic effects. STB have the potential for the development and application of a lipid-lowering health food.

Tartary buckwheat FtF3'H1 as a metabolic branch switch to increase anthocyanin content in transgenic plant.

Tartary buckwheat (TB) is a pseudocereal rich in flavonoids, mainly including flavonols and anthocyanins. The flavonoid 3'-hydroxylase (F3'H) is a key enzyme in flavonoid biosynthesis and is encoded by two copies in TB genome. However, its biological function and effects on flavonol and anthocyanin synthesis in TB have not been well validated yet. In this study, we cloned the full-length FtF3'H1 gene highly expressed in all tissues (compared with FtF3'H2) according to TB flowering transcriptome data. The corresponding FtF3'H1 protein contains 534 amino acids with the molecular properties of the typical plant F3'H and belongs to the CYP75B family. During the flowering stage, the FtF3'H1 expression was highest in flowers, and its expression pattern showed a significant and positive correlation with the total flavonoids (R 2 > 0.95). The overexpression of FtF3'H1 in Arabidopsis thaliana, Nicotiana tabacum and TB hairy roots resulted in a significant increase in anthocyanin contents (p < 0.05) but a decrease in rutin (p < 0.05). The average anthocyanin contents were 2.94 mg/g (fresh weight, FW) in A. thaliana (about 135% increase), 1.18 mg/g (FW) in tobacco (about 17% increase), and 1.56 mg/g (FW) TB hairy roots (about 44% increase), and the rutin contents were dropped to about 53.85, 14.99, 46.31%, respectively. However, the expression of genes involved in anthocyanin (DFRs and ANSs) and flavonol (FLSs) synthesis pathways were significantly upregulated (p < 0.05). In particular, the expression level of DFR, a key enzyme that enters the anthocyanin branch, was upregulated thousand-fold in A. thaliana and in N. tabacum. These results might be attributed to FtF3'H1 protein with a higher substrate preference for anthocyanin synthesis substrates. Altogether, we identified the basic biochemical activity of FtF3'H1 in vivo and investigated its involvement in anthocyanin and flavonol metabolism in plant.

Tartary Buckwheat R2R3-MYB Gene FtMYB3 Negatively Regulates Anthocyanin and Proanthocyanin Biosynthesis.

Anthocyanins and proanthocyanidins (PAs) are vital secondary metabolites in Tartary buckwheat because of their antioxidant capacities and radical scavenging functions. It has been demonstrated that R2R3-MYB transcription factors (TFs) are essential regulators of anthocyanin and PA biosynthesis in many plants. However, their regulatory mechanisms in Tartary buckwheat remain to be clarified. Here, we confirmed the role of FtMYB3 in anthocyanin and PA biosynthesis. FtMYB3, which belongs to the subgroup 4 R2R3 family was predominantly expressed in roots. The transcriptional expression of FtMYB3 increased significantly under hormone treatment with SA and MeJA and abiotic stresses including drought, salt, and cold at the seedling stage. Functional analyses showed that FtMYB3 negatively regulated anthocyanin and PA biosynthesis, primarily via downregulating the expression of the DFR, ANS, BAN, and TT13 in transgenic Arabidopsis thaliana, which may depend on the interaction between FtMYB3 and FtbHLH/FtWD40. Altogether, this study reveals that FtMYB3 is a negative regulatory transcription factor for anthocyanin and PA biosynthesis in Tartary buckwheat.

Irinotecan/scFv co-loaded liposomes coaction on tumor cells and CAFs for enhanced colorectal cancer therapy.

BACKGROUND: Cancer-associated fibroblasts (CAFs), as an important component of stroma, not only supply the "soils" to promote tumor invasion and metastasis, but also form a physical barrier to hinder the penetration of therapeutic agents. Based on this, the combinational strategy that action on both tumor cells and CAFs simultaneously would be a promising approach for improving the antitumor effect. RESULTS: In this study, the novel multifunctional liposomes (IRI-RGD/R9-sLip) were designed, which integrated the advantages including IRI and scFv co-loading, different targets, RGD mediated active targeting, R9 promoting cell efficient permeation and lysosomal escape. As expected, IRI-RGD/R9-sLip showed enhanced cytotoxicity in different cell models, effectively increased the accumulation in tumor sites, as well as exhibited deep permeation ability both in vitro and in vivo. Notably, IRI-RGD/R9-sLip not only exhibited superior in vivo anti-tumor effect in both CAFs-free and CAFs-abundant bearing mice models, but also presented excellent anti-metastasis efficiency in lung metastasis model. CONCLUSION: In a word, the novel combinational strategy by coaction on both "seeds" and "soils" of the tumor provides a new approach for cancer therapy, and the prepared liposomes could efficiently improve the antitumor effect with promising clinical application prospects.

Combined anti-hepatocellular carcinoma therapy inhibit drug-resistance and metastasis via targeting "substance P-hepatic stellate cells-hepatocellular carcinoma" axis.

Peripheral nerves have emerged as the important components in tumor microenvironment (TME), which could activate hepatic stellate cells (HSCs) by secreting substance P (SP), leading to hepatocellular carcinoma (HCC) invasion and metastasis. Herein, we proposed a novel anti-HCC concept of blocking "SP-HSCs-HCC" axis for omnidirectional inhibition of HCC development. To pursue this aim, the novel CAP/GA-sHA-DOX NPs were developed for targeted co-delivery of capsaicin (CAP) and doxorubicin (DOX) using glycyrrhetinic acid (GA) modified sulfated-HA (sHA) as nanocarriers. Among that, CAP could inhibit the activation of HSCs as an inhibitor of SP. Notably, to real mimic "SP-HSCs-HCC" axis for in vitro and in vivo evaluation, both "SP + LX-2+BEL-7402" co-cultured cell model and "SP + m-HSC + H22" co-implantation mice model were attempted for the first time. Furthermore, in vivo anti-HCC effects were performed in three different tumor-bearing models: subcutaneous implantation of H22 or "SP + m-HSC + H22", intravenous injection of H22 for lung metastasis, and orthotopic implantation of H22 for primary HCC. Our results showed that CAP/GA-sHA-DOX NPs could be efficiently taken up by tumor cells and activated HSCs (aHSCs) simultaneously, and effectively inhibit tumor drug-resistance and migration by blocking SP-induced HSCs activation. In addition, CAP/GA-sHA-DOX NPs exhibited low ECM deposition, less tumor angiogenesis, and superior in vivo anti-HCC effects. The anti-HCC mechanisms revealed that CAP/GA-sHA-DOX NPs could down-regulate the expression level of Vimentin and P-gp, reverse epithelial-mesenchymal transition (EMT) of tumor cells. In brief, the nano-sized combination therapy based on GA-sHA-DOX polymers could effectively inhibit drug-resistance and metastasis of HCC by blocking "SP-HSCs-HCC" axis, which provides a promising approach for cancer therapy.

Dual-Ligand-Modified Liposomes Co-Loaded with Anti-Angiogenic and Chemotherapeutic Drugs for Inhibiting Tumor Angiogenesis and Metastasis.

BACKGROUND: Tumor angiogenesis has been proven to potentiate tumor growth and metastasis; therefore, the strategies targeting tumor-related angiogenesis have great potentials in antitumor therapy. METHODS: Here, the GA&Gal dual-ligand-modified liposomes co-loaded with curcumin and combretastatin A-4 phosphate (CUCA/GA&Gal-Lip) were prepared and characterized. A novel "BEL-7402+HUVEC" co-cultured cell model was established to mimic tumor microenvironment. The cytotoxicity and migration assays were performed against the novel co-cultured model. Angiogenesis ability was evaluated by tube formation test, and in vivo metastatic ability was evaluated by lung metastasis test. RESULTS: The result demonstrated that dual-ligand-modified liposomes showed greater inhibition of tumor angiogenesis and metastasis in comparison with other combined groups. Significantly, the mechanism analysis revealed that curcumin and combretastatin A-4 phosphate could inhibit tumor angiogenesis and metastasis via down-regulation of VEGF and VEGFR2 expression, respectively, and that GA&Gal-Lip could improve antitumor effect by GA/Gal-mediated active-targeting delivery. CONCLUSION: CUCA/GA&Gal-Lip hold great potentials in hepatoma-targeting delivery of antitumor drugs and can achieve anti-angiogenic and anti-metastatic effects by simultaneously blocking VEGF/VEGFR2 signal pathway, therefore exhibiting superior anti-hepatoma efficacy.

Acidic tumor microenvironment-sensitive liposomes enhance colorectal cancer therapy by acting on both tumor cells and cancer-associated fibroblasts.

Cancer-associated fibroblasts (CAFs) play a crucial role in facilitating tumor invasion and metastasis, which act as the "soil" in the tumor microenvironment (TME). Accordingly, it would be a promising strategy to enhance the antitumor effect by killing both tumor cells and CAFs simultaneously. Herein, novel TME acid-responsive liposomes for co-delivery of IRI and 398 (IRI&398-s-LPs) were developed, in which the rapid release of both drugs could be triggered under acidic conditions. Notably, a CT-26/3T3 cell co-culture system was used to mimic the real TME both in vitro and in vivo. Cellular immunofluorescence revealed that IRI&398-s-LPs could efficiently decrease the activation of CAFs. In vitro cytotoxicity evaluation demonstrated that IRI&398-s-LPs exhibited higher cytotoxicity than the other liposomal formulations in the CT-26 and CT-26/3T3 cell co-culture system. In vivo NIRF imaging showed that the IRI&398-s-LPs could increase drug accumulation in the tumor sites. Furthermore, IRI&398-s-LPs not only presented superior in vivo anti-tumor activity in CT-26 bearing BALB/c mice, but also enhanced the effect in CT-26/3T3 cell bearing mice with decreased collagen and CAF biomarker expression. Furthermore, IRI&398-s-LPs also presented superior anti-metastatic efficiency in a lung metastasis model. These results indicated that this combinational strategy for eliminating both tumor cells and CAFs provides a new approach for cancer therapy, and the prepared TME-responsive liposomes for co-delivery of drugs hold promising clinical application prospects.

Co-Delivery of Curcumin and Capsaicin by Dual-Targeting Liposomes for Inhibition of aHSC-Induced Drug Resistance and Metastasis.

Recent research studies have shown that the low survival rate of liver cancer is due to drug resistance and metastasis. In the tumor microenvironment (TME), activated hepatic stellate cells (aHSCs) have been proven to favor the development of liver cancer. Hence, the combination therapy dual-targeting aHSCs and tumor cells might be an effective strategy for treatment of liver cancer. In this study, the novel multifunctional liposomes (CAPS-CUR/GA&Gal-Lip) were prepared for co-delivery of curcumin (CUR) and capsaicin (CAPS), in which glycyrrhetinic acid (GA) and galactose (Gal) were chosen as targeting ligands to modify the liposomes (Lip) for dual-targeting liver cancer. To mimic TME, a novel HSCs+HepG2 (human hepatoma cell line) cocultured model was established for the antitumor effect in vitro. The results showed that, compared to HepG2 cells alone, the cocultured model promoted drug resistance and migration by upregulating the expression of P-glycoprotein (P-gp) and Vimentin, which were effectively inhibited by CAPS-CUR/GA&Gal-Lip. The efficacy of the in vivo antitumor was evaluated by three mice models: subcutaneous H22 (mouse hepatoma cell line) tumor-bearing mice, H22+m-HSC (mouse hepatic stellate cell) tumor-bearing mice, and orthotopic H22 cells-bearing mice. The results showed that CAPS-CUR/GA&Gal-Lip exhibited lesser extracellular matrix (ECM) deposition, lesser tumor angiogenesis, and superior antitumor effect compared with the no- and/or Gal-modified Lip, which was attributed to the simultaneous blocking of the activation of HSCs and inhibition of the metastasis of tumor cells. The dual-targeting method using Lip is thus a potential strategy for liver cancer treatment.

FtMYB6, a Light-Induced SG7 R2R3-MYB Transcription Factor, Promotes Flavonol Biosynthesis in Tartary Buckwheat (Fagopyrum tataricum).

Tartary buckwheat (Fagopyrum tataricum) is rich in flavonols, which are thought to be highly beneficial for human health. However, little is known about the regulatory mechanism of flavonol biosynthesis in Tartary buckwheat. In this study, we identified and characterized a novel SG7 R2R3-MYB transcription factor in Tartary buckwheat, FtMYB6. We showed that FtMYB6 is located in the nucleus and acts as a transcriptional activator. The FtMYB6 promoter showed strong spatiotemporal specificity and was induced by light. The expression of FtMYB6 showed a significant correlation with rutin accumulation in the roots, stems, leaves, and flowers. Overexpression of FtMYB6 in transgenic Tartary buckwheat hairy roots and tobacco (Nicotiana tabacum) plants significantly increased the accumulation of flavonols. In transient luciferase (LUC) activity assay, FtMYB6 promoted the activity of FtF3H and FtFLS1 promoters and inhibited the activity of the Ft4CL promoter. Collectively, our results suggest that FtMYB6 promotes flavonol biosynthesis by activating FtF3H and FtFLS1 expression.

Improving decolorization of dyes by laccase from Bacillus licheniformis by random and site-directed mutagenesis.

BACKGROUND: Dye wastewater increases cancer risk in humans. For the treatment of dyestuffs, biodegradation has the advantages of economy, high efficiency, and environmental protection compared with traditional physical and chemical methods. Laccase is the best candidate for dye degradation because of its multiple substrates and pollution-free products. METHODS: Here, we modified the laccase gene of Bacillus licheniformis by error-prone PCR and site-directed mutagenesis and expressed in E. coli. The protein was purified by His-tagged protein purification kit. We tested the enzymatic properties of wild type and mutant laccase by single factor test, and further evaluated the decolorization ability of laccase to acid violet, alphazurine A, and methyl orange by spectrophotometry. RESULTS: Mutant laccase Lacep69and D500G were superior to wild type laccase in enzyme activity, stability, and decolorization ability. Moreover, the laccase D500G obtained by site-directed mutagenesis had higher enzyme activity in both, and the specific activity of the purified enzyme was as high as 426.13 U/mg. Also, D500G has a higher optimum temperature of 70 °C and temperature stability, while it has a more neutral pH 4.5 and pH stability. D500G had the maximum enzyme activity at a copper ion concentration of 12 mM. The results of decolorization experiments showed that D500G had a strong overall decolorization ability, with a lower decolorization rate of 18% for methyl orange and a higher decolorization rate of 78% for acid violet. CONCLUSION: Compared with the wild type laccase, the enzyme activity of D500G was significantly increased. At the same time, it has obvious advantages in the decolorization effect of different dyes. Also, the advantages of temperature and pH stability increase its tolerance to the environment of dye wastewater.

Ferrous iron-induced increases in capitate glandular trichome density and upregulation of CbHO-1 contributes to increases in blinin content in Conyza blinii.

MAIN CONCLUSION: Ferrous iron can promote the development of glandular trichomes and increase the content of blinin, which depends on CbHO-1 expression. Conyza blinii (C. blinii) is a unique Chinese herbal medicine that grows in Sichuan Province, China. Because the habitat of C. blinii is an iron ore mining area with abundant iron content, this species can be used as one of the best materials to study the mechanism of plant tolerance to iron. In this study, C. blinii was treated with ferrous-EDTA solutions at different concentrations, and it was found that the tolerance value of C. blinii to iron was 200 µM. Under this concentration, the plant height, root length, biomass, and iron content of C. blinii increased to the maximum values, and the effect was dependent on the upregulated expression of CbHO-1. At the same time, under ferrous iron, the photosynthetic capacity and capitate glandular trichome density of C. blinii also significantly increased, providing precursors and sites for the synthesis of blinin, thus significantly increasing the content of blinin. These processes were also dependent on the high expression of CbHO-1. Correlation analysis showed that there were strong positive correlations between iron content, capitate glandular trichome density, CbHO-1 gene expression, and blinin content. This study explored the effects of ferrous iron on the physiology and biochemistry of C. blinii, greatly improving our understanding of the mechanism of iron tolerance in C. blinii.

FtMYB18 acts as a negative regulator of anthocyanin/proanthocyanidin biosynthesis in Tartary buckwheat.

KEY MESSAGE: FtMYB18 plays a role in the repression of anthocyanins and proanthocyanidins accumulation by strongly down-regulating the CHS and DFR genes in Tartary buckwheat, and the C5 motif plays an important role in this process. Anthocyanins and proanthocyanidins (PAs) are important flavonoids in Tartary buckwheat (Fagopyrum tataricum Gaertn.), which provides various vibrant color and stronge abiotic stress resistance. Their synthesis is generally regulated by MYB transcription factors at transcription level. However, the negative regulations of MYB and their effects on flavonol metabolism are poorly understood. A SG4-like MYB subfamily TF, FtMYB18, containing C5 motif was identified from Tartary buckwheat. The expression of FtMYB18 was not only showed a negative correlation with anthocyanins and PAs content but also strongly respond to MeJA and ABA. As far as the transgenic lines with FtMYB18 overexpression, anthocyanins and PAs accumulations were decreased through down-regulating expression levels of NtCHS and NtDFR in tobacco, AtDFR and AtTT12 in Arabidopsis, FtCHS, FtDFR and FtANS in Tartary buckwheat hairy roots, respectively. However, FtMYB18 showed no effect on the FLS gene expression and the metabolites content in flavonol synthesis branch. The further molecular interaction analysis indicated FtMYB18 could mediate the inhibition of anthocyanins and PAs synthesis by forming MBW transcriptional complex with FtTT8 and FtTTG1, or MYB-JAZ complex with FtJAZ1/-3/-4/-7. Importantly, in FtMYB18 mutant lines with C5 motif deletion (FtMYB18-C), both of anthocyanins and PAs accumulations had recovered to the similar level as that in wild type, which was attributed to the weakened MBW complex activity or the deficient molecular interaction between FtMYB18ΔC5 with FtJAZ3/-4. The results showed that FtMYB18 could suppress anthocyanins and PAs synthesis at transcription level through the specific interaction of C5 motif with other proteins in Tartary buckwheat.

Study on different particle sizes of DOX-loaded mixed micelles for cancer therapy.

Nano-based drug delivery systems have been widely applied in cancer therapy, among that, particle sizes may affect the delivery efficiency of nanocarriers. The purpose of this study was to evaluate the potential impacts of particle size on tumor therapy, in consideration of this, lipid/glycocholic acid mixed micelles (LGs) were designed as the model nanocarriers. Doxorubicin (DOX) loaded LGs with two different particle sizes at around 10 nm and 100 nm, respectively, were successfully prepared by controlling the ratio of EPC to GAH. In vitro release study showed that the release behaviors of DOX in mixed micelles with two different particle sizes was basically consistent and showed sustained release. DOX-LGs at 10 nm exhibited higher cellular uptake capacity, compared with DOX-LGs at 100 nm. Besides, in vivo NIFR imaging also demonstrated that DOX-LGs at 10 nm had more accumulation in tumor site. Furthermore, DOX-LGs at 10 nm presented both higher in vitro cytotoxicity and superior in vivo antitumor activity than that of 100 nm. In vivo safety evaluations showed that the mixed micelles had lower toxicities than free DOX solution formulations. These results indicated that the nanoparticles with smaller particle size could improve the profiles in cellular uptake, tumor accumulation as well as anti-tumor efficacy, which would provide a theoretical principle for the design of nanoparticles.