Pazopanib stimulates senescence of renal carcinoma cells through targeting nuclear factor E2-related factor 2 (Nrf2).

Renal cell carcinoma (RCC) is the most common kidney cancer with high mortality rate. Pazopanib has been approved for the treatment of RCC. However, the underlying mechanism is not clear. Here, we report a novel finding by showing that treatment with Pazopanib could promote cellular senescence of the human RCC cell line ACHN. Cells were stimulated with 5, 10, and 20 µM Pazopanib, respectively. Cellular senescence was measured using senescence-associated ß-galactosidase (SA-ß-Gal) staining. Western blot analysis and real-time polymerase chain reaction were used to measure the mRNA and protein expression of nuclear factor E2-related factor 2 (Nrf2), γH2AX, human telomerase reverse transcriptase (hTERT), telomeric repeat binding factor 2 (TERF2), p53 and plasminogen activator inhibitor (PAI). First, we found that exposure to Pazopanib reduced the cell viability of ACHN cells. Additionally, Pazopanib induced oxidative stress  by increasing the production of reactive oxygen species, reducing the levels of glutathione peroxidase, and promoting nuclear translocation of Nrf2. Interestingly, Pazopanib exposure resulted in DNA damage by increasing the expression of γH2AX. Importantly, Pazopanib increased cellular senescence and reduced telomerase activity. Pazopanib also reduced the gene expression of hTERT but increased the gene expression of TERF2. Correspondingly, we found that Pazopanib increased the expression of p53 and PAI at both the mRNA and protein levels. To elucidate the underlying mechanism, the expression of Nrf2 was knocked down by transduction with Ad- Nrf2 shRNA. Results indicate that silencing of Nrf2 in ACHN cells abolished the effects of Pazopanib in stimulating cellular senescence and reducing telomerase activity. Consistently, knockdown of Nrf2 restored the expression of p53 and PAI in ACHN cells. Based on these results, we explored a novel mechanism whereby which Pazopanib displays a cytotoxicity effect in RCC cells through promoting cellular senescence mediated by Nrf2.

T6SS translocates a micropeptide to suppress STING-mediated innate immunity by sequestering manganese.

Cellular ionic concentrations are a central factor orchestrating host innate immunity, but no pathogenic mechanism that perturbs host innate immunity by directly targeting metal ions has yet been described. Here, we report a unique virulence strategy of Yersinia pseudotuberculosis (Yptb) involving modulation of the availability of Mn2+, an immunostimulatory metal ion in host cells. We showed that the Yptb type VI secretion system (T6SS) delivered a micropeptide, TssS, into host cells to enhance its virulence. The mutant strain lacking TssS (ΔtssS) showed substantially reduced virulence but induced a significantly stronger host innate immune response, indicating an antagonistic role of this effector in host antimicrobial immunity. Subsequent studies revealed that TssS is a Mn2+-chelating protein and that its Mn2+-chelating ability is essential for the disruption of host innate immunity. Moreover, we showed that Mn2+ enhances the host innate immune response to Yptb infection by activating the stimulator of interferon genes (STING)-mediated immune response. Furthermore, we demonstrated that TssS counteracted the cytoplasmic Mn2+ increase to inhibit the STING-mediated innate immune response by sequestering Mn2+ Finally, TssS-mediated STING inhibition sabotaged bacterial clearance in vivo. These results reveal a previously unrecognized bacterial immune evasion strategy involving modulation of the bioavailability of intracellular metal ions and provide a perspective on the role of the T6SS in pathogenesis.

22R- but not 22S-hydroxycholesterol is recruited for diosgenin biosynthesis.

Diosgenin is an important compound in the pharmaceutical industry and it is biosynthesized in several eudicot and monocot species, herein represented by fenugreek (a eudicot), and Dioscorea zingiberensis (a monocot). Formation of diosgenin can be achieved by the early C22,16-oxidations of cholesterol followed by a late C26-oxidation. This study reveals that, in both fenugreek and D. zingiberensis, the early C22,16-oxygenase(s) shows strict 22R-stereospecificity for hydroxylation of the substrates. Evidence against the recently proposed intermediacy of 16S,22S-dihydroxycholesterol in diosgenin biosynthesis was also found. Moreover, in contrast to the eudicot fenugreek, which utilizes a single multifunctional cytochrome P450 (TfCYP90B50) to perform the early C22,16-oxidations, the monocot D. zingiberensis has evolved two separate cytochrome P450 enzymes, with DzCYP90B71 being specific for the 22R-oxidation and DzCYP90G6 for the C16-oxidation. We suggest that the DzCYP90B71/DzCYP90G6 pair represent more broadly conserved catalysts for diosgenin biosynthesis in monocots.

Genome-wide identification and expression pattern analysis of R2R3-MYB transcription factor gene family involved in puerarin biosynthesis and response to hormone in Pueraria lobata var. thomsonii.

BACKGROUND: R2R3-MYB transcription factors regulate secondary metabolism, stress responses and development in various plants. Puerarin is a bioactive ingredient and most abundant secondary metabolite isolated from Pueraria lobata. The biosynthesis of puerarin proceeds via the phenylpropanoid pathway and isoflavonoids pathway, in which 9 key enzymes are involved. The expression of these structural genes is under control of specific PtR2R3-MYB genes in different plant tissues. However, how PtR2R3-MYB genes regulates structural genes in puerarin biosynthesis remains elusive. This study mined the PtR2R3-MYB genes involved in puerarin biosynthesis and response to hormone in Pueraria lobata var. thomsonii. RESULTS: A total of 209 PtR2R3-MYB proteins were identified, in which classified into 34 subgroups based on the phylogenetic topology and the classification of the R2R3-MYB superfamily in Arabidopsis thaliana. Furtherly physical and chemical characteristics, gene structure, and conserved motif analysis were also used to further analyze PtR2R3-MYBs. Combining puerarin content and RNA-seq data, speculated on the regulated puerarin biosynthesis of PtR2R3-MYB genes and structural genes, thus 21 PtR2R3-MYB genes and 25 structural genes were selected for validation gene expression and further explore its response to MeJA and GSH treatment by using qRT-PCR analysis technique. Correlation analysis and cis-acting element analysis revealed that 6 PtR2R3-MYB genes (PtMYB039, PtMYB057, PtMYB080, PtMYB109, PtMYB115 and PtMYB138) and 7 structural genes (PtHID2, PtHID9, PtIFS3, PtUGT069, PtUGT188, PtUGT286 and PtUGT297) were directly or indirectly regulation of puerarin biosynthesis in ZG11. It is worth noting that after MeJA and GSH treatment for 12-24 h, the expression changes of most candidate genes were consistent with the correlation of puerarin biosynthesis, which also shows that MeJA and GSH have the potential to mediate puerarin biosynthesis by regulating gene expression in ZG11. CONCLUSIONS: Overall, this study provides a comprehensive understanding of the PtR2R3-MYB and will paves the way to reveal the transcriptional regulation of puerarin biosynthesis and response to phytohormone of PtR2R3-MYB genes in Pueraria lobata var. thomsonii.

A Yersinia T6SS Effector YezP Engages the Hemin Uptake Receptor HmuR and ZnuABC for Zn2+ Acquisition.

Metal ions are essential nutrients for all life forms, and restriction of metal ion availability is an effective host defense against bacterial infection. Meanwhile, bacterial pathogens have developed equally effective means to secure their metal ion supply. The enteric pathogen Yersinia pseudotuberculosis was found to uptake zinc using the T6SS4 effector YezP, which is essential for Zn2+ acquisition and bacterial survival under oxidative stress. However, the mechanism of this zinc uptake pathway has not been fully elucidated. Here, we identified the hemin uptake receptor HmuR for YezP, which can mediate import of Zn2+ into the periplasm by the YezP-Zn2+ complex and demonstrated that YezP functions extracellularly. This study also confirmed that the ZnuCB transporter is the inner membrane transporter for Zn2+ from the periplasm to cytoplasm. Overall, our results reveal the complete T6SS/YezP/HmuR/ZnuABC pathway, wherein multiple systems are coupled to support zinc uptake by Y. pseudotuberculosis under oxidative stress. IMPORTANCE Identifying the transporters involved in import of metal ions under normal physiological growth conditions in bacterial pathogens will clarify its pathogenic mechanism. Y. pseudotuberculosis YPIII, a common foodborne pathogen that infects animals and humans, uptake zinc via the T6SS4 effector YezP. However, the outer and inner transports involved in Zn2+ acquisition remain unknown. The important outcomes of this study are the identification of the hemin uptake receptor HmuR and inner membrane transporter ZnuCB that import Zn2+ into the cytoplasm via the YezP-Zn2+ complex, and elucidation of the complete Zn2+ acquisition pathway consisting of T6SS, HmuRSTUV, and ZnuABC, thereby providing a comprehensive view of T6SS-mediated ion transport and its functions.

Computed tomography radiomic features and clinical factors predicting the response to first transarterial chemoembolization in intermediate-stage hepatocellular carcinoma.

BACKGROUND: According to clinical practice guidelines, transarterial chemoembolization (TACE) is the standard treatment modality for patients with intermediate-stage hepatocellular carcinoma (HCC). Early prediction of treatment response can help patients choose a reasonable treatment plan. This study aimed to investigate the value of the radiomic-clinical model in predicting the efficacy of the first TACE treatment for HCC to prolong patient survival. METHODS: A total of 164 patients with HCC who underwent the first TACE from January 2017 to September 2021 were analyzed. The tumor response was assessed by modified response evaluation criteria in solid tumors (mRECIST), and the response of the first TACE to each session and its correlation with overall survival were evaluated. The radiomic signatures associated with the treatment response were identified by the least absolute shrinkage and selection operator (LASSO), and four machine learning models were built with different types of regions of interest (ROIs) (tumor and corresponding tissues) and the model with the best performance was selected. The predictive performance was assessed with receiver operating characteristic (ROC) curves and calibration curves. RESULTS: Of all the models, the random forest (RF) model with peritumor (+10 mm) radiomic signatures had the best performance [area under ROC curve (AUC) = 0.964 in the training cohort, AUC = 0.949 in the validation cohort]. The RF model was used to calculate the radiomic score (Rad-score), and the optimal cutoff value (0.34) was calculated according to the Youden's index. Patients were then divided into a high-risk group (Rad-score > 0.34) and a low-risk group (Rad-score ≤ 0.34), and a nomogram model was successfully established to predict treatment response. The predicted treatment response also allowed for significant discrimination of Kaplan-Meier curves. Multivariate Cox regression identified six independent prognostic factors for overall survival, including male [hazard ratio (HR) = 0.500, 95% confidence interval (CI): 0.260-0.962, P = 0.038], alpha-fetoprotein (HR = 1.003, 95% CI: 1.002-1.004, P < 0.001), alanine aminotransferase (HR = 1.003, 95% CI: 1.001-1.005, P = 0.025), performance status (HR = 2.400, 95% CI: 1.200-4.800, P = 0.013), the number of TACE sessions (HR = 0.870, 95% CI: 0.780-0.970, P = 0.012) and Rad-score (HR = 3.480, 95% CI: 1.416-8.552, P = 0.007). CONCLUSIONS: The radiomic signatures and clinical factors can be well-used to predict the response of HCC patients to the first TACE and may help identify the patients most likely to benefit from TACE.

Identification of P450 Candidates Associated with the Biosynthesis of Physalin-Class Compounds in Physalis angulata.

The Physalis genus has long been used as traditional medicine in the treatment of various diseases. Physalins, the characteristic class of compounds in this genus, are major bioactive constituents. To date, the biogenesis of physalins remains largely unknown, except for the recently established knowledge that 24-methyldesmosterol is a precursor of physalin. To identify the genes encoding P450s that are putatively involved in converting 24-methyldesmosterol to physalins, a total of 306 P450-encoding unigenes were retrieved from our recently constructed P. angulata transcriptome. Extensive phylogenetic analysis proposed 21 P450s that might participate in physalin biosynthesis. To validate the candidates, we developed a virus-induced gene silencing (VIGS) system for P. angulata, and four P450 candidates were selected for the VIGS experiments. The reduction in the transcripts of the four P450 candidates by VIGS all led to decreased levels of physalin-class compounds in the P. angulata leaves. Thus, this study provides a number of P450 candidates that are likely associated with the biosynthesis of physalin-class compounds, forming a strong basis to reveal the unknown physalin biosynthetic pathway in the future.

Initial experience with surrounding en bloc transurethral resection of bladder tumor and simultaneous intravesical treating for non-muscle invasive bladder cancer.

PURPOSE: The high recurrence rate after traditional transurethral resection of bladder tumor (TURBT) remains a challenge for management of non-muscle invasive bladder tumor (NMIBC). The aim of this study was to evaluate feasibility, efficacy and safety of surrounding en bloc resection using a general wire bipolar loop electrode and simultaneous intravesical chemotherapy. METHODS: We retrospectively analyzed data of 111 consecutive patients with NMIBC treated from June 2018 to December 2021. These patients underwent conventional TURBT and immediate intravesical chemotherapy (n = 45) or surrounding en bloc TURBT and simultaneous intravesical chemotherapy in the Urology Department of Harbin Medical University Cancer Hospital, The former and latter were defined as the conventional TURBT group and the surrounding en bloc TURBT group, respectively. All patients were followed up from 6 to 40 months, with an average of 24 months. Demographic characteristics, location and number of tumors, perioperative and postoperative data, pathological results and recurrence were documented. RESULTS: There were no significant differences in clinicopathological data between the conventional TURBT group (n = 45) and the surrounding en bloc TURBT group (n = 66). Operative time and complications associated with TURBT were comparable in the two groups. Recurrent tumors were found during follow-up in 2 (3.0%) of 66 patients in the surrounding en bloc group and 9 (20%) of 45 patients in the conventional group (p < 0.05). Lower urinary tract symptoms developed in 2 (3.0%) of 66 patients after surrounding en bloc TURBT and in 11(24.4%) of 45 patients after conventional TURBT (p < 0.05). CONCLUSION: Surrounding en bloc TURBT and simultaneous intravesical chemotherapy might significantly decrease the recurrence rate of NMIBC, and showed favorable safety and tolerability profiles. The general bipolar loop electrode was appropriate to complete the procedure.

LncRNA HOTAIR induces sunitinib resistance in renal cancer by acting as a competing endogenous RNA to regulate autophagy of renal cells.

BACKGROUND: Cell autophagy has been proposed to be involved in drug resistance therapy. However, how the long non-coding RNA (lncRNA) reduces risks of drug resistance in renal cancer (RC) cells needs a thorough inquiry. This study was assigned to probe the effect and mechanism of HOTAIR on sunitinib resistance of RC. METHODS: Clinical RC tissues and para-carcinoma tissues were obtained to detect the expressions of miR-17-5p, HOTAIR and Beclin1. Sunitinib-resistant cells (786-O-R and ACHN-R) were constructed using parental RC cells (786-O and ACHN). The resistance of 786-O-R and ACHN-R cells to sunitinib was examined. Western blot and qRT-PCR were assayed to obtain the expressions of miR-17-5p, HOTAIR and Beclin1. The effects of HOTAIR knockdown or miR-17-5p overexpression/knockdown on cell autophagy and sunitinib resistance were measured by MDC staining, immunofluorescence and Western blot. The sensitivity of RC cells to sunitinib and change in cell clone formation after sunitinib treatment were assessed by CCK-8 assay and colony formation assay, respectively. The relationships among HOTAIR, miR-17-5p and Beclin1 were verified by dual-luciferase reporter gene and RIP assay. The role of HOTAIR knockdown in sunitinib resistance was verified in nude mice. RESULTS: HOTAIR expression in sunitinib-resistant cells is higher than that in parental cells. Knockdown of HOTAIR in sunitinib-resistant cells lead to refrained sunitinib resistance and cell autophagy both in vivo and in vitro. Activation of autophagy could raise resistance to sunitinib in RC cells, while inhibition of autophagy could improve the sensitivity of sunitinib-resistant cells to sunitinib. HOTAIR could compete with miR-17-5p to regulate Beclin1 expression. Knockdown of miR-17-5p in parental cells increases cell resistant to sunitinib, and overexpression of miR-17-5p in sunitinib-resistant cells increases cell sensitive to sunitinib. CONCLUSION: HOTAIR negatively targets miR-17-5p to activate Beclin1-mediated cell autophagy, thereby enhancing sunitinib resistance in RC cells.

Discovery of a non-stereoselective cytochrome P450 catalyzing either 8α- or 8ß-hydroxylation of germacrene A acid from the Chinese medicinal plant, Inula hupehensis.

Sesquiterpene lactones (STLs) are C15 terpenoid natural products with α-methylene γ-lactone moiety. A large proportion of STLs in Asteraceae species is derived from the central precursor germacrene A acid (GAA). Formation of the lactone rings depends on the regio-(C6 or C8) and stereoselective (α- or ß-)hydroxylations of GAA, producing STLs with four distinct stereo-configurations (12,6α-, 12,6ß-, 12,8α-, and 12,8ß-olide derivatives of GAA) in nature. Curiously, two configurations of STLs (C12,8α and C12,8ß) are simultaneously present in the Chinese medicinal plant, Inula hupehensis. However, how these related yet distinct STL stereo-isomers are co-synthesized in I. hupehensis remains unknown. Here, we describe the functional identification of the I. hupehensis cytochrome P450 (CYP71BL6) that can catalyze the hydroxylation of GAA in either 8α- or 8ß-configuration, resulting in the synthesis of both 8α- and 8ß-hydroxyl GAAs. Of these two products, only 8α-hydroxyl GAA spontaneously lactonizes to the C12,8α-STL while the 8ß-hydroxyl GAA remains stable without lactonization. Chemical structures of the C12,8α-STL, named inunolide, and 8ß-hydroxyl GAA were fully elucidated by nuclear magnetic resonance analysis and mass spectrometry. The CYP71BL6 displays 63-66% amino acid identity to the previously reported CYP71BL1/2 catalyzing GAA 6α- or 8ß-hydroxylation, indicating CYP71BL6 shares the same evolutionary lineage with other stereoselective cytochrome P450s, but catalyzes hydroxylation in a non-stereoselective manner. We observed that the CYP71BL6 transcript abundance correlates closely to the accumulation of C12,8-STLs in I. hupehensis. The identification of CYP71BL6 provides an insight into the biosynthesis of STLs in Asteraceae.

Siderophore-Mediated Iron Acquisition Enhances Resistance to Oxidative and Aromatic Compound Stress in Cupriavidus necator JMP134.

Many bacteria secrete siderophores to enhance iron uptake under iron-restricted conditions. In this study, we found that Cupriavidus necator JMP134, a well-known aromatic pollutant-degrading bacterium, produces an unknown carboxylate-type siderophore named cupriabactin to overcome iron limitation. Using genome mining, targeted mutagenesis, and biochemical analysis, we discovered an operon containing six open reading frames (cubA-F) in the C. necator JMP134 genome that encodes proteins required for the biosynthesis and uptake of cupriabactin. As the dominant siderophore of C. necator JMP134, cupriabactin promotes the growth of C. necator JMP134 under iron-limited conditions via enhanced ferric iron uptake. Furthermore, we demonstrated that the iron concentration-dependent expression of the cub operon is mediated by the ferric uptake regulator (Fur). Physiological analyses revealed that the cupriabactin-mediated iron acquisition system influences swimming motility, biofilm formation, and resistance to oxidative and aromatic compound stress in C. necator JMP134. In conclusion, we identified a carboxylate-type siderophore named cupriabactin, which plays important roles in iron scavenging, bacterial motility, biofilm formation, and stress resistance.IMPORTANCE Since siderophores have been widely exploited for agricultural, environmental, and medical applications, the identification and characterization of new siderophores from different habitats and organisms will have great beneficial applications. Here, we identified a novel siderophore-producing gene cluster in C. necator JMP134. This gene cluster produces a previously unknown carboxylate siderophore, cupriabactin. Physiological analyses revealed that the cupriabactin-mediated iron acquisition system influences swimming motility, biofilm formation, and oxidative stress resistance. Most notably, this system also plays important roles in increasing the resistance of C. necator JMP134 to stress caused by aromatic compounds, which provide a promising strategy to engineer more efficient approaches to degrade aromatic pollutants.

Comparative Transcriptome Analysis Identifies Genes Involved in Diosgenin Biosynthesis in Trigonella foenum-graecum L.

Trigonella foenum-graecum L. (fenugreek) is a valuable resource of producing diosgenin which serves as a substrate for synthesizing more than two hundred kinds of steroidal drugs. Phytochemical analysis indicated that methyl jasmonate (MeJA) efficiently induced diosgenin biosynthesis in fenugreek seedlings. Though early steps up to cholesterol have recently been elucidated in plants, cytochrome P450 (CYP)- and glycosyltransferase (GT)-encoding genes involved in the late steps from cholesterol to diosgenin remain unknown. This study established comparative fenugreek transcriptome datasets from the MeJA-treated seedlings and the corresponding control lines. Differential gene expression analysis identified a number of MeJA-induced CYP and GT candidate genes. Further gene expression pattern analysis across a different MeJA-treating time points, together with a phylogenetic analysis, suggested specific family members of CYPs and GTs that may participate in the late steps during diosgenin biosynthesis. MeJA-induced transcription factors (TFs) that may play regulatory roles in diosgenin biosynthesis were also discussed. This study provided a valuable genetic resource to functionally characterize the genes involved in diosgenin biosynthesis, which will push forward the production of diosgenin in microbial organisms using a promising synthetic biology strategy.

Molecular characterization of the C-glucosylation for puerarin biosynthesis in Pueraria lobata.

C-glycosyltransferases (CGTs) are important enzymes that are responsible for the synthesis of the C-glycosides of flavonoids and isoflavonoids. Flavonoid CGTs have been molecularly characterized from several plant species; however, to date, no gene encoding an isoflavonoid CGT has been reported from any plant species. A significant example of an isoflavonoid C-glycoside is puerarin, a compound that contributes to the major medicinal effects of Pueraria lobata. Little is known about the C-glucosylation that occurs during puerarin biosynthesis. One possible route for puerarin synthesis is via the C-glucosylation of daidzein. This study describes the molecular cloning and functional characterization of a novel glucosyltransferase (PlUGT43) from P. lobata. Biochemical analyses revealed that PlUGT43 possesses an activity for the C-glucosylation of daidzein to puerarin; it shows activity with the isoflavones daidzein and genistein, but displays no activity towards other potential acceptors, including flavonoids. To validate the in vivo function of PlUGT43, the PlUGT43 gene was over-expressed in soybean hairy roots that naturally synthesize daidzein but that do not produce puerarin. The expression of PlUGT43 led to the production of puerarin in the transgenic soybean hairy roots, confirming a role for PlUGT43 in puerarin biosynthesis.

Supermacrocyclic Assemblies by Hydrogen-Bond Codes of C7-Phenol Pyrazolo and Pyrrolo Derivatives of Adenine.

Hydrogen bond (HB) mediated base pair motifs are versatile scaffolds of diverse supramolecular constructs. Here, we report that two new four- and six-membered supermacrocyclic assemblies with intriguing geometries could self-assemble from two new adenine derivatives, APN (1) and APC (2). The conversion of a conventional HB acceptor, N8 of 1, to a non-conventional HB donor, C8-H of 2, had a pronounced impact on the overall intricate HB network and self-assembly patterns, epitomizing the subtleties in design and exploitation of such base-pair motifs as promising tectons for building supramolecular architectures.

Comparative Transcriptome Analysis Identifies Putative Genes Involved in Dioscin Biosynthesis in Dioscorea zingiberensis.

Dioscorea zingiberensis is a perennial herb native to China. The rhizome of D. zingiberensis has long been used as a traditional Chinese medicine to treat rheumatic arthritis. Dioscin is the major bioactive ingredient conferring the medicinal property described in Chinese pharmacopoeia. Several previous studies have suggested cholesterol as the intermediate to the biosynthesis of dioscin, however, the biosynthetic steps to dioscin after cholesterol remain unknown. In this study, a comprehensive D. zingiberensis transcriptome derived from its leaf and rhizome was constructed. Based on the annotation using various public databases, all possible enzymes in the biosynthetic steps to cholesterol were identified. In the late steps beyond cholesterol, cholesterol undergoes site-specific oxidation by cytochrome P450s (CYPs) and glycosylation by UDP-glycosyltransferases (UGTs) to yield dioscin. From the D. zingiberensis transcriptome, a total of 485 unigenes were annotated as CYPs and 195 unigenes with a sequence length above 1000 bp were annotated as UGTs. Transcriptomic comparison revealed 165 CYP annotated unigenes correlating to dioscin biosynthesis in the plant. Further phylogenetic analysis suggested that among those CYP candidates four of them would be the most likely candidates involved in the biosynthetic steps from cholesterol to dioscin. Additionally, from the UGT annotated unigenes, six of them were annotated as 3-O-UGTs and two of them were annotated as rhamnosyltransferases, which consisted of potential UGT candidates involved in dioscin biosynthesis. To further explore the function of the UGT candidates, two 3-O-UGT candidates, named Dz3GT1 and Dz3GT2, were cloned and functionally characterized. Both Dz3GT1 and Dz3GT2 were able to catalyze a C3-glucosylation activity on diosgenin. In conclusion, this study will facilitate our understanding of dioscin biosynthesis pathway and provides a basis for further mining the genes involved in dioscin biosynthesis.

Effects of sulforaphane on neural stem cell proliferation and differentiation.

Sulforaphane (SFN) is a natural organosulfur compound with anti-oxidant and anti-inflammation properties. The objective of this study is to investigate the effect of SFN on the proliferation and differentiation of neural stem cells (NSC). NSCs were exposed to SFN at the concentrations ranging from 0.25 to 10 µM. Cell viability was evaluated with MTT assay and lactate dehydogenase (LDH) release assay. The proliferation of NSCs was evaluated with neurosphere formation assay and Ki-67 staining. The level of Tuj-1 was evaluated with immunostaining and Western blot to assess NSC neuronal differentiation. The expression of key proteins in the Wnt signaling pathway, including ß-catenin and cyclin D1, in response to SFN treatment or the Wnt inhibitor, DKK-1, was determined by Western blotting. No significant cytotoxicity was seen for SFN on NSCs with SFN at concentrations of less than 10 µM. On the contrary, SFN of low concentrations stimulated cell proliferation and prominently increased neurosphere formation and NSC differentiation to neurons. SFN treatment upregulated Wnt signaling in the NSCs, whereas DKK-1 attenuated the effects of SFN. SFN is a drug to promote NSC proliferation and neuronal differentiation when used at low concentrations. These protective effects are mediated by Wnt signaling pathway.

"W-shaped" injection current dependence of electroluminescence linewidth in green InGaN/GaN-based LED grown on silicon substrate.

Injection current, and temperature, dependences of the electroluminescence (EL) spectrum from green InGaN/GaN multiple quantum well (MQW)-based light-emitting diodes (LED) grown on a Si substrate, are investigated over a wide range of injection currents (0.5 µA-350 mA) and temperatures (6-350 K). The results show that an increasing temperature can result in the change of injection current-dependent behavior of the EL spectrum in initial current range. That is, with increasing the injection current in the low current range, the emission process of the MQWs is dominated by filling effect of low-energetic localized states at the low temperature range of around 6 K, and by Coulomb screening of the quantum confinement Stark effect followed by a filling effect of the higher levels of the low-energetic localized states at the intermediate temperature range of around 160 K. However, when the temperature is further raised to the higher temperature range of around 350 K, the emission process of the MQWs in the low current range is dominated by carrier-scattering effect followed by non-radiative recombination process. The aforementioned current-dependent behaviors of the EL spectrum are mainly attributed to the strong localized effect of the green LED, as confirmed by the anomalous temperature dependence of the EL spectrum measured at the low injection current of 5 µA. In addition, the injection current dependence of external quantum efficiency at different temperatures shows that, with increasing temperature from 6 to 350 K, in addition to the enhanced non-radiative recombination, electron overflow becomes more significant, especially in the higher temperature range above 300 K.

Identification and Functional Characterization of Sesquiterpene Synthases from Xanthium strumarium.

Xanthium strumarium synthesizes various pharmacologically active sesquiterpenes. The molecular characterization of sesquiterpene biosynthesis in X. strumarium has not been reported so far. In this study, the cDNAs coding for three sesquiterpene synthases (designated as XsTPS1, XsTPS2 and XsTPS3) were isolated using the X. strumarium transcriptome that we recently constructed. XsTPS1, XsTPS2 and XsTPS3 were revealed to have primary activities forming germacrene D, guaia-4,6-diene and germacrene A, respectively, by either ectopic expression in yeast cells or purified recombinant protein-based in vitro assays. Quantitative real-time PCRs and metabolite analysis for the different plant parts showed that the transcript abundance of XsTPS1-XsTPS3 is consistent with the accumulation pattern of their enzymatic products, supporting their biochemical functions in vivo. In particular, we discovered that none of the XsTPS2 product, guaia-4,6-diene, can be detected in one of the X. strumarium cultivars used in this study (it was named the Hubei-cultivar), in which a natural deletion of two A bases in the XsTPS2 cDNA disrupts its activity, which further confirmed the proposed biochemical role of XsTPS2 in X. strumarium in vivo.

Improvement of betulinic acid biosynthesis in yeast employing multiple strategies.

BACKGROUND: Betulinic acid (BA) is a lupane-type triterpene which has been considered as a promising agent to cure melanoma with no side effects. Considering that BA is naturally produced in small quantities in plants, we previously reported the success in engineering its production in yeast. In the present study, we attempted to improve BA biosynthesis in yeast by the use of different strategies. RESULTS: We first isolated a gene encoding a lupeol C-28 oxidase (LO) from Betula platyphylla (designated as BPLO). BPLO showed a higher activity in BA biosynthesis compared to the previously reported LOs. In addition, two yeast platforms were compared for engineering the production of BA, which demonstrated that the WAT11 strain was better to host BA pathway than the CEN.PK strain. Based on the WAT11-chassiss, the Gal80p mutant was further constructed. The mutant produced 0.16 mg/L/OD600 of BA, which was 2.2 fold of that produced by the wild type strain (0.07 mg/L/OD600). CONCLUSIONS: This study reported our efforts to improve BA production in yeast employing multiple strategies, which included the identification of a novel LO enzyme with a higher activity in BA biosynthesis, the evaluation of two yeast strains for hosting the BA pathway, and the up-regulation of the expression of the BA pathway genes by managing yeast GAL gene regulon circuit.

Identification of biological targets of therapeutic intervention for clear cell renal cell carcinoma based on bioinformatics approach.

BACKGROUND: We aimed to discover the potential microRNA (miRNA) targets and to explore the underlying molecular mechanisms of clear cell renal cell carcinoma (ccRCC). METHODS: Microarray data of GSE16441 was downloaded from Gene Expression Omnibus database. Differentially expressed genes (DEGs) and differentially expressed miRNAs between ccRCC tumors and matched non-tumor samples were analyzed. Target genes of differentially expressed miRNAs were screened. Besides, functional enrichment analysis of DEGs was performed, followed by protein-protein interaction (PPI) network construction and sub-module analysis. Finally, the integrated miRNA-DEGs network was constructed. RESULTS: A total of 1758 up- and 2465 down-regulated DEGs were identified. Moreover, 15 up- and 12 down-regulated differentially expressed miRNAs were screened. The up-regulated DEGs were significantly enriched in pathways such as cell adhesion molecules and focal adhesion. Besides, the down-regulated DEGs were enriched in oxidative phosphorylation, and citrate cycle (TCA cycle). Moreover, eight sub-modules of PPI network were obtained. Totally, eight down-regulated miRNAs were identified to significantly regulate the DEGs and miRNA-200c that could regulate collagen, type V, alpha 2 (COL5A2) as well as COL5A3 was found to be the most significant. Additionally, 10 up-regulated miRNAs were identified to be significantly associated with the DEGs. Thereinto, miRNA-15a that could regulate ATPase, H(+) transporting, lysosomal 21 kDa, V0 subunit b (ATP6V0B) and miRNA-155 were found to be the most significant. CONCLUSIONS: miRNA-200c that could regulate COL5A2 and COL5A3, miRNA-15a that could regulate ATP6V0B and miRNA-155 may play key roles in ccRCC progression. These miRNAs may be potential targets for ccRCC treatment.