Synergistic anticancer effects of ginsenoside CK and gefitinib against gefitinib-resistant NSCLC by regulating the balance of angiogenic factors through HIF-1α/VEGF.

Drug resistance is a serious problem for gefitinib in the treatment of lung cancer. Ginsenoside CK, a metabolite of diol ginsenosides, have many excellent pharmacological activities, but whether ginsenoside CK can overcome gefitinib resistance remains unclear. In our study, the sensitizing activity of ginsenoside CK on gefitinib-resistant non-small cell lung cancer (NSCLC) in vitro and in vivo was investigated. Ginsenoside CK was confirmed to enhance the anti-proliferation, pro-apoptotic and anti-migration effects of gefitinib in primary and acquired resistant NSCLC. Furthermore, the combined administration of CK and gefitinib effectively promoted the sensitivity of lung cancer xenograft to gefitinib in vivo, and the tumor inhibition rate reached 70.97% (vs. gefitinib monotherapy 32.65%). Subsequently, tubule formation experiment and western blot results showed that co-treatment of ginsenoside CK inhibited the angiogenesis ability of HUVEC cells, and inhibited the expression of HIF-1α, VEGF, FGF and MMP2/9. More interestingly, ginsenoside CK co-treatment enhanced the expression of anti-angiogenic factor PF4, increased pericellular envelope, and promoted the normalization of vascular structure. In conclusion, ginsenoside CK improved the resistance of gefitinib by regulating the balance of angiogenic factors through down-regulating the HIF-1α/VEGF signaling pathway, providing a theoretical basis for improving the clinical efficacy of gefitinib and applying combined strategies to overcome drug resistance.

The Incidence and Effect of Different Organ Metastasis on the Prognosis of NSCLC.

OBJECTIVE: The aim of this study was to explore the effect of different organ metastasis on the prognosis of non-small cell lung cancer (NSCLC). METHODS: Patients with distant metastatic NSCLC were selected from Surveillance, Epidemiology, and End Results database during 2016 to 2019. The incidence of different organ metastasis and their association with clinicopathological factors were explored. Overall survival (OS) and lung cancer-specific survival (LCSS) for metastatic NSCLC were calculated, and multivariate Cox regression analysis was performed with a nomogram for OS being constructed based on Cox regression. RESULTS: Total 26,210 patients with distant metastatic NSCLC were included in this study. Around 48.9% of the metastatic NSCLC were multiple-organ metastasis and bone was the most commonly involved organ (44.4%). For patients with single-organ metastasis, the prognosis for lung or distant lymph nodes (LNs) metastasis was better than others (with median OS of 15 and 16 months for lung and distant LNs metastasis, respectively), and liver metastasis resulted in the worst prognosis with median OS of 8 months. A nomogram was constructed to visualize Cox regression model, along with the receiver operating characteristic (ROC) curve demonstrated good discrimination for the predictive model with 1- and 2-year area under the curve of ROC of 0.687 and 0.702, respectively. CONCLUSION: The prognosis of NSCLC patients with distant metastasis was poor. Liver metastasis results in the worst prognosis among the single-organ metastasis. The nomogram developed based on the Cox regression model has provided a useful tool to estimate the probability of OS of the metastatic NSCLC.

The role of HPV status in patients with overlapping grey zone cancer in oral cavity and oropharynx.

PURPOSE: We aimed to explore the clinicodemographic characteristics and prognosis of grey zone squamous cell cancer (GZSCC) located in the overlapping or ambiguous area of oral cavity and oropharynx and to identify valuable factors that would improve its differential diagnosis and prognosis. METHODS: Information of GZSCC patients in the Surveillance, Epidemiology, and End Results (SEER) database were compared to patients with oral cavity (OCSCC) and oropharyngeal (OPSCC) squamous cell carcinomas with corresponding HPV status, respectively. Kaplan-Meier method with log-rank test and multivariate Cox regression analysis were applied to assess associations between clinical characteristics and overall survival (OS). A predictive model integrating age, gender, marital status, HPV status and staging variables was conducted to classify GZSCC patients into three risk groups and verified internally by tenfold cross validation. RESULTS: A total of 3318 GZSCC, 10792 OPSCC and 6656 OCSCC patients were identified. HPV-positive GZSCC patients had the best 5-year OS as HPV-positive OPSCC (81% vs. 82%). However, the 5-year OS of HPV-negative/unknown GZSCC (43%/42%) were the worst among all groups, indicating that HPV status and the overlapping nature of tumors were valuable prognostic predictors in GZSCC patients. Compared with the strategy of dividing GZSCC into two groups by HPV status, the predictive model integrating more variables could additionally identify a unique high-risk GZSCC group with the lowest OS rate. CONCLUSIONS: GZSCC patients had distinct clinical characteristics and prognosis compared with OPSCC and OCSCC, integrating HPV status and other clinical factors could help distinguish GZSCC and predict their prognosis.

Four Undescribed Pyranones from the Scutellaria formosana-Derived Endophytic Fungi Ascomycota sp. FAE17.

Four undescribed pyranone derivatives, named ascomycopyrones A-D (1-4), as well as one known analogue simplicilopyrone (5) (this is the first study to report the absolute configuration), were isolated from the endophytic fungus Ascomycota sp. FAE17 derived from the flowers of Scutellaria formosa. The structures of these pyranones were identified by comprehensive spectroscopic and MS analyses, and the absolute configurations were determined by their experimental and quantum chemical electronic circular dichroism (ECD) calculations. All isolated compounds were tested for various bioactivities, including antibacterial, cytotoxic activity, and NO inhibitory activity. Unfortunately, none of the compounds showed significant bioactivities.

Construction, expression, purification, characterization, and structural analysis of microbial transglutaminase variants.

Microbial transglutaminase (MTG, EC 2.3.2.13) derived from Streptomyces mobaraensis is widely used in the food and pharmaceutical industry because of its ability to synthesize isopeptide bonds between the proteinogenic side chains of glutamine and lysine. The half-life (t1/2 ) of the activated wild-type enzyme at 60°C is 2 min. To improve the activity and thermostability of MTG for higher temperature application, three variants (Mut1, Mut2, and Mut3) were obtained by combining key amino acid mutations on the basis of previous research results. The best variant Mut2 with a specific combination of five of seven substitutions (S2P-S23V-Y24N-R215A-H289Y) shows a 10-fold increased half-life at 60°C (t1/2  = 27.6 min), and a 2.4-fold increased specific enzyme activity (39.3 U/mg). As measured by circular dichroism, the curve of Mut2 was basically the same as that of MTG-WT. The structural simulation of Mut2 shows that the overall structure is discoid with a crack, but the crack openings are wider than that of MTG-WT. Furthermore, structural analysis of Mut2 showed that there were seven hydrogen bonds and one π-anion interaction between Mut2 and its adjacent amino acids, and the number of hydrogen bonds was one more than that of MTG-WT (six hydrogen bonds).

Rs6757 in microRNA-3976 binding site of CD147 confers risk of hepatocellular carcinoma in South Chinese population.

BACKGROUND: Cluster of differentiation 147 (CD147) overexpression plays a key role in the proliferation, differentiation, invasion, metastasis, and prognosis of hepatocellular carcinoma (HCC). The aim of this study was to explore the relationship between rs6757 and the HCC risk in the South Chinese population, and the functional significance of rs6757 by affecting the efficacy of microRNA-3976 (miR-3976) binding to the CD147 3'-UTR. METHODS: We performed a retrospective case-control study to analyze the association between rs6757 and the risk of HCC. We chose candidate microRNAs with the potential of interacting with rs6757 through a series of silico analyses. A luciferase reporter gene assay was implemented to detect the binding extent of microRNAs to each polymorphic allele of rs6757. RESULTS: An obvious association between rs6757 and the risk of HCC was detected in C vs. T (OR = 1.826, 95% CI [1.263-2.642]), CC vs. TT (OR = 4.513, 95% CI [1.510-13.489]), dominant genetic model (OR = 1.824, 95% CI [1.120-2.965]), and recessive genetic model (OR = 3.765, 95% CI [1.286-11.020]). Bioinformatics analysis indicated that miR-3976 binding sites containing the rs6757-T allele had lower free energies than those with the C allele, the lower free energies, the higher affinities. Luciferase activity was remarkably decreased by miR-3976 binding to the CD147 3'-UTR bearing rs6757 T allele, which could be reversed by miR-3976 inhibitors. Furthermore, miR-3976 reduced the luciferase expression in a manner of dose-dependent when cotransfected with constructs with the CD147-TT-pSICHECK2. CONCLUSIONS: The research we have done suggests that rs6757 confers the CD147 allele-specific translational suppression by miR-3976, which provides a theoretical basis for antineoplastic therapy targeting CD147.

Ophiopogon Polysaccharide Liposome Regulated the Immune Activity of Kupffer Cell through miR-4796.

The purpose of this article is to study the effects and mechanism of miR-4796 in the process of ophiopogon polysaccharide liposome (OPL) regulation of the immune activity of Kupffer cells (KCs). In this study, KCs were used as cell models, and were treated with OPL in different concentrations after being transfected with miR-4796 mimic or miR-4796 inhibitor. Firstly, the secretion of NO and iNOS, phagocytic activity, the expression of surface molecules CD14 and MHC II, apoptosis and ROS secretion were measured by Griess, flow cytometry, fluorescence staining and ELISA. Then, real-time PCR and Western blot were used to measure the expression of TLR4, IKKß, MyD88 and NF-κB in the TLR4-NF-κB signaling pathway. The results showed that after transfection with miR-4796 mimic, the secretion of NO and iNOS, cell migration, cell phagocytosis and expression levels of CD14 and MHC II in the OPL group were significantly higher than those in the miR-4796 mimic control group (p < 0.05; p < 0.01). In addition, the mRNA and protein expression levels of TLR4, MyD88 and NF-κB were significantly higher than those in miR-4796 mimic control group (p < 0.05; p < 0.01). After transfection with miR-4796 inhibitor, the secretion of NO and iNOS, cell migration, cell phagocytosis, expression of CD14 and MHCII in OPL group were significantly higher than those in the miR-4796 inhibitor control group (p < 0.05; p < 0.01). These results indicated that OPL could regulate the immune activity of KCs by regulating miR-4796 and activating the TLR4-NF-κB signaling pathway.

FGFR leads to sustained activation of STAT3 to mediate resistance to EGFR-TKIs treatment.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have led to great advances in the treatment of non-small cell lung cancer (NSCLC), but the emergence of drug resistance severely limits their clinical use. Thus, elucidation of the mechanism underlying resistance to EGFR-TKIs is of great importance. In our study, sustained activation of STAT3 was confirmed to be involved in resistance to EGFR-TKIs, and this resistance occurred regardless of exposure time, EGFR-TKIs type, and even cancer cell type. Mechanistically, the sustained activation of STAT3 was not related to gp130/JAK signalling pathway or HER2/EGFR heterodimer formation, while related to the expression and activation levels of STAT3. Furthermore, FGFR was shown to bind more strongly to STAT3 after gefitinib treatment, and the inhibition of FGFR reduced the phosphorylation of STAT3, thereby counteracting the effects of EGFR-TKIs and resulting in the synergistic inhibition of cancer cell proliferation. Taken together, the FGFR/STAT3 axis mediates the sustained activation of STAT3 upon EGFR-TKI treatment. This finding elucidates a new mechanism underlying drug resistance to EGFR-TKIs that the FGFR/STAT3 axis mediates the sustained activation of STAT3, providing theoretical support for considering the combination of TKIs and FGFR inhibitors in clinical cancer treatment.

Giant exchange bias induced via tuning interfacial spins in polycrystalline Fe3O4/CoO bilayers.

A giant exchange bias (EB) of 9600 Oe was observed in polycrystalline Fe3O4/CoO layers at 10 K after 20 kOe field cooling, and was attributed to the strong exchange coupling formed by the interfacial spins between the polycrystalline Fe3O4 and the CoO layer. It was found that at 10 K, the magnetic-moment difference (ΔM) between the zero field cooling curves and field cooling curves first increases and then decreases with the change of the field, and it reaches the maximum value at a field of 20 kOe, which suggests that the interfacial spins can be tuned by the cooling field. Furthermore, other magnetic properties, including field dependence, temperature dependence, and training effects, were investigated, which further confirmed that the interfacial spins play an important role in the EB effect. This work provides a method to tune the magnitude of the EB effect and reveals the mechanism of the dependency of EB on interfacial spins, which could guide the design of giant-EB-effect materials.

Spongy p-Toluenesulfonic Acid-doped Polypyrrole with Extraordinary Rate Performance as Durable Anodes of Sodium-Ion Batteries at Different Temperatures.

Sodium-ion batteries (SIBs) have potential as an energy storage system because they have similar electrochemical properties as lithium-ion batteries, abundant resource reserves, and extremely high safety performance. Compared with traditional graphite materials, conductive polymers are more suitable as an anode electrode material for SIBs. In this study, a simple and scalable approach has been used to synthesize p-toluenesulfonic acid-doped polypyrrole (p-TSA-PPy). The as-obtained material showed remarkable rate capacities and cyclability. At room temperature (25 °C), its discharge capacities could reach 185, 162, and 135 mAh g-1 under 10, 30, and 50 C rates after 250 cycles, respectively. More importantly, the capacity of the p-TSA-PPy could still be maintained at 120.5 mAh g-1 even at the 2000th cycle at 10 C. In addition, it achieves attractive electrochemical performance at different temperatures (0 and 50 °C).

VLRs expression were significantly affected by complement C3 knockdown morphants in Lampetra morii.

The complement component 3 of the lamprey, a jawless vertebrate, functions as an opsonin during the phagocytosis of rabbit red cells. Furthermore, lamprey C3 may be activated and cleaved into C3b, which is attached to the surface of target cells in the cytolytic process. However, the mechanism mediating the biological function of C3 in the lamprey is unknown. To our knowledge, this study is the first to show that variable lymphocyte receptors (VLRs) expression were significantly affected by complement C3 knockdown morphants in Lampetra morii. We identified the C3 gene in the lamprey genome based on its orthologs, conserved synteny, functional domains, phylogenetic tree, and conserved motifs. Additionally, we determined the optimal infection concentration of Aeromonas hydrophila to perform immune stimulation experiments in the lamprey larvae. The quantitative real-time polymerase chain reaction and immunofluorescence analyses revealed that the expression of Lampetra morii C3 (lmC3) was significantly upregulated in the larvae infected with 107 CFU/mL of A. hydrophila. The lmC3 morphants (lmC3 MO) of lamprey larvae were generated by morpholino-mediated knockdown. The lmC3 MO larvae were highly susceptible to A. hydrophila infection, which indicated that lmC3 is critical in lamprey immune response. The expression of a selected panel of orthologous genes was comparatively analyzed in the infected wild type, infected lmC3 MO, infected control MO, uninfected wild type and uninfected lmC3 MO one-month-old ammocoete larvae. The knockdown of lmC3 strongly affected the expression of VLRA+/VLRB+/VLRC+-associated genes, which was also confirmed by immunohistochemical analysis. Thus, VLR expression were significantly affected by complement C3 knockdown morphants in Lampetra morii.

Lamprey VLRB participates in pathogen detection, VLRB/L-BLNK/L-NF-κB (B-like cells) signal transduction, and development.

In jawed vertebrates, B cell receptors (BCR) are primary pathogen detectors that activate downstream signaling pathways to express adaptive immune effectors. In jawless vertebrates, the variable lymphocyte receptors (VLR) B positive lymphocytes can express and secrete specific VLRB molecules in an analogous manner to that of immunoglobulins by B cells in jawed vertebrates. Our study is the first to demonstrate the possibility of incubation of fertilized eggs and artificial breeding of Lampetra morii larvae throughout their life cycle under laboratory condition. We also found that VLRB, lamprey B-cell linker (L-BLNK), and lamprey nuclear factor-kappa B (L-NF-κB) play key roles in early larval development. Aeromonas hydrophila was found to be a lethal pathogen of L. morii larvae causing rapid infection at a concentration of 107 cfu/mL qRT-PCR results revealed that gene expression levels of VLRB, L-BLNK, and L-NF-κB were up-regulated significantly. Ten-day infection trials showed that VLRB, L-BLNK, and L-NF-κB are crucial for lamprey immune response. Furthermore, the expression levels of L-BLNK and L-NF-κB were down-regulated drastically both at mRNA and protein levels after bacterial infection than in the naive group of VLRB morphants. A similar expression pattern of VLRB and L-BLNK was found in L-NF-κB morphants post bacterial infection. The results were strikingly different in the other two morphants. The VLRB and L-NF-κB expression levels were found to be down-regulated at mRNA and protein levels by less than 30% and 45%, respectively, in L-BLNK morphants compared to those in the naive group. These results indicate that L-BLNK and L-NF-κB might participate in VLRB-mediated immune response. Additionally, in VLRB morphants, the mRNA expression levels of some genes, especially the ones expressed in VLRB+ lymphocytes but not in VLRA+ lymphocytes, were found to be affected. Therefore, these findings of B-like lymphocytes in lamprey offer key evidence with regard to the evolution of adaptive immunity.

Determination of water content of crude oil by azeotropic distillation Karl Fischer coulometric titration.

The determination of water content in crude oil is important for oil transportation, refining, and trade. However, the sensitivity and accuracy of the conventional azeotropic distillation (AD) method are inadequate. Karl Fischer titration methods may give false high results because of the reducing sulfur compounds in crude oil. The present study developed an azeotropic distillation Karl Fischer coulometric titration (AD-KFCT) method which required a modified instrument and a new calibration procedure. The method was modified to decrease the interference caused by reducing sulfur compounds. A certified reference material for water content in liquid was used to determine the recovery of water mass achieved using the AD-KFCT method. The effect of the sample polarity on the method accuracy was assessed. The relative error and relative standard deviations of the water content in three crude oils containing known amounts of water were -7.5% to 2.9% and 0.4% to 6.0%, respectively. The interference by reducing sulfur compounds was studied. The crude oil containing 1-propanethiol was measured using a sulfur dioxide-free anolyte, and the amount of iodine consumed by the distilled 1-propanethiol was determined. The contribution of 1-propanethiol was then subtracted from the water content measured using the normal Karl Fischer reagent. Finally, the relative error of the modified water content in the crude oil samples containing 1-propanethiol was -4.4% to 0.7%. Therefore, the modified AD-KFCT method is accurate and convenient for crude oil.

Superior electrochemical performance of a novel LiFePO4/C/CNTs composite for aqueous rechargeable lithium-ion batteries.

Olivine LiFePO4 covered flocculent carbon layers wrapped with carbon nanotubes (CNTs) prepared by sol-gel method and calcination is used as the cathode material for aqueous rechargeable lithium-ion batteries (ARLBs). The phase structures and morphologies of the composite material are characterized by X-ray diffraction (XRD), selected area electron diffraction (SAED), and transmission electron microscopy (TEM). The mechanism and method through which CNTs and flocculent carbon improve the electrochemical performance are investigated in an aqueous lithium-ion battery by setting up a comparative experiment. The ARLB system is assembled using a LiFePO4/C/CNTs cathode and a zinc anode in 1 mol L-1 ZnSO4·7H2O and saturated LiNO3 aqueous solution (pH = 6), which can deliver a capacity of 158 mA h g-1 at a rate of 1C. Even at a rate of 50C, it still has a capacity of 110 mA h g-1 after 250 cycles with fantastic capacity retention (95.7%). The lithium-ion diffusion coefficient increases by an order of magnitude due to the addition of CNTs together with flocculent carbon. Four LEDs are successfully powered by the ARLBs for more than one minute to demonstrate the practical application. The excellent rate capabilities and thrilling discharge capacity at a high rate indicate that this cathode material possesses excellent electrochemical performance, and this ARLB system exhibits excellent potential as a power source for environmental applications.

Three new methylated Δ8-pregnene steroids from the Polyalthia laui-derived fungus Stemphylium sp. AZGP4-2.

Three new methylated Δ8-pregnene steroids, stemphylisteroids A-C (1-3) were isolated from the medicinal plant Polyalthia laui-derived fungus Stemphylium sp. AZGP4-2. Their structures were elucidated by the detailed analysis of comprehensive spectroscopic data. The absolute configuration of 1 was determined by X-ray crystallographic analysis. Compound 1 show antibacterial activity against Escherichia coli with the MIC value of 6.25 µg/mL, and 2 exhibited a broad spectrum of antibacterial activities against six pathogenic bacteria with the MIC values ranging from 12.5 to 50 µg/mL. The discovery of three methylated Δ8-pregnene steroids 1-3 are a further addition to diverse and complex array of methylated steroids.

Four New Chromone Derivatives from Colletotrichum gloeosporioides.

Four previously unreported chromones, 5-hydroxy-2-(hydroxymethyl)-8-methoxy-4H-chromen-4-one (1), (5R,7S)-5,7-dihydroxy-2-propyl-5,6,7,8-tetrahydro-4H-chromen-4-one (2), (5R,7S)-5,7-dihydroxy-2-methyl-5,6,7,8-tetrahydro-4H-chromen-4-one (3), and (5R,7S)-5,7-dihydroxy-2-[(E)-prop-1-en-1-yl]-5,6,7,8-tetrahydro-4H-chromen-4-one (4), as well as one known analogue 5-hydroxy-2-methyl-4H-chromen-4-one (5) were isolated from the fermentation broth of the endophytic fungus Colletotrichum gloeosporioides derived from the mangrove Ceriops tagal. Their structures were elucidated based on extensive spectroscopic analyses. The absolute configurations of 2-4 were determined by comparison the experimental and calculated electronic circular dichroism (ECD) spectra. Compound 2 showed cytotoxic activity against A549 cell line with the IC50 value of 0.094 mm.

Nurses' Attitudes Toward Internet-Based Home Care: A Survey Study.

Internet-based home care has emerged as a way to relieve the burden of hospitals and meet patients' need for home care. This study aims to explore nurses' attitudes toward Internet-based home care. A cross-sectional online survey was conducted in Ningbo City in China. A self-designed Internet-based home care attitudes questionnaire for nurses (23 items) was used. There were 2039 nurses from 13 hospitals who participated in this online survey. Results reveal that, 1369 nurses (67.1%) were willing to provide Internet-based home care. However, there were significant differences in the attitudes of nurses with different ages (H = 11.86, P = .001), years of work experience (H = 24.257, P = .000), positions (H = 8.850, P = .031), and types of phones (H = 13.096, P = .001). More than 80% of nurses were willing to provide hypodermic and intramuscular injection. But there was a significant difference in the attitudes toward hypodermic injection, intramuscular injection, and pressure ulcer care in nurses with different ages (H = 13.039, P = .005; H = 9.178, P = .027; H = 10.997, P = .012) and a significant difference in the attitudes toward pressure ulcer care in nurses with different years of work experience (H = 15.259, P = .002). Results also indicated that most nurses were worried about their own safety and personal rights protection during Internet-based home care.

Nine hub genes as the potential indicator for the clinical outcome of diabetic nephropathy.

Diabetic nephropathy (DN) is one of the most serious and dangerous chronic complications caused by diabetes mellitus, and the identification and development of novel biomarkers could be beneficial for the diagnosis and prognosis of DN patients. This study focused on researching the differently expressed pattern of the DN samples from glomeruli and tubulointerstitium. Significance analysis of microarrays (SAM) was used to identify differentially regulated genes in 44 microdissected human kidney samples. Functional enrichment analysis was used to analyze the functions these genes are mostly enriched in. Besides, protein-protein interaction (PPI) network was used to select the hub genes that were associated with DN. The gene expression pattern of DN samples from glomeruli and tubulointerstitium was found to be quite different. It showed that the recurrence rate of DN originating from glomeruli and control samples was lower than that from tubulointerstitium and control samples. A total of 332 differentially expressed genes were identified between glomeruli tissues and tubulointerstitium tissues. PPI network analysis was performed on these 332 genes. Finally, nine hub genes were selected as the most potential biomarkers in the occurrence of DN. In conclusion, a total of 332 genes were found to be related to DN, and these genes were found to be mostly enriched in pathways such as immunity, inflammatory, and vascular pathways. Three genes VAV1, LCK, and Plk had the potential to serve as indicators for the occurrence and development of DN in clinical management.

Improved the expression level of active transglutaminase by directional increasing copy of mtg gene in Pichia pastoris.

BACKGROUND: The microbial transglutaminase (MTG) is inactive when only the mature sequence is expressed in Pichia pastoris. Although co-expression of MTG and its N-terminal pro-peptide can obtain the active MTG, the enzyme activity was still low. One of the basic steps for strain improvement is to ensure a sufficient level of transcription of the heterologous gene, based on promoter strength and gene copy number. To date, high-copy-number recombinants of P. pastoris are achievable only by cloning of gene concatemers, so methods for rapid and reliable multicopy strains are therefore desirable. RESULTS: The coexpression strains harboring different copies mtg were obtained successfully by stepwise increasing Zeocin concentration based on the rDNA sequence of P. pastoris. The genome of coexpression strains with the highest enzyme activity was analyzed by real-time fluorescence quantitative PCR, and three copies of mtg gene (mtg-3c) was calculated according to the standard curve of gap and mtg genes (gap is regarded as the single-copy reference gene). The maximum enzyme activity of mtg-3c was up to 1.41 U/mL after being inducted for 72 h in 1 L flask under optimal culture conditions, and two protein bands were observed at the expected molecular weights (40 kDa and 5 kDa) by Western blot. Furthermore, among the strains detected, compared with mtg-2c, mtg-6c or mtg-8c, mtg-3c is the highest expression level and enzyme activity, implying that mtg-3c is the most suitable for co-expression pro-peptide and MTG. CONCLUSIONS: This study provides an effective strategy for improving the expression level of active MTG by directional increasing of mtg copies in P. pastoris.

3,4- seco-Norclerodane Diterpenoids from the Roots of Polyalthia laui.

Ten new clerodane diterpenoids, polylauioids A-J (1-10), and five known analogues (11-15) were isolated from the roots of Polyalthia laui. Among the new compounds, 3 and 8 are artifacts. The structures were elucidated using spectroscopic methods and by comparison with published NMR spectroscopic data. The absolute configurations of 4, 5, and 7 were defined based on single-crystal X-ray diffraction and electronic circular dichroism data. Compounds 1 and 2 represent the first examples of rearranged 3,4- seco-norclerodane diterpenoids, and a putative biosynthesis pathway for these compounds is proposed. Compounds 1, 4, 6, 7, 9, and 10 showed anti-HIV activities with EC50 values ranging from 12.2 to 35.2 µM.