An Artificial Enzyme for Asymmetric Nitrocyclopropanation of α,ß-Unsaturated Aldehydes-Design and Evolution.

The introduction of an abiological catalytic group into the binding pocket of a protein host allows for the expansion of enzyme chemistries. Here, we report the generation of an artificial enzyme by genetic encoding of a non-canonical amino acid that contains a secondary amine side chain. The non-canonical amino acid and the binding pocket function synergistically to catalyze the asymmetric nitrocyclopropanation of α,ß-unsaturated aldehydes by the iminium activation mechanism. The designer enzyme was evolved to an optimal variant that catalyzes the reaction at high conversions with high diastereo- and enantioselectivity. This work demonstrates the application of genetic code expansion in enzyme design and expands the scope of enzyme-catalyzed abiological reactions.

Extraction and characterization of anti-virus anthraquinones from Nicotiana tabacum-derived Aspergillus oryzae YNCA1220.

In this study, seven novel anthraquinones (1-7) and four described anthraquinones (8-11) were purified from Nicotiana tabacum-derived Aspergillus oryzae YNCA1220. It is worth noting that only analogs of 4 and 5 have been reported as natural products to date, while the nuclei of compounds 1-3, 6 and 7 were isolated for the first time in nature. Among them, compounds 1-3 bear an unusual anthra[2,3-b]furan-9,10-dione nucleus, 4 and 5 possess a rare 3-methyl-1H-pyrrol-2-yl substituent, and 6 and 7 are new framework anthraquinones bearing a 6-methyl-1,7-dihydro-2H-azepin-2-one ring. Interestingly, the in vivo assays indicated that 1, 4 and 5 had inactivation effects against tobacco mosaic virus (TMV) with inhibition rates of 41.6%, 55.4% and 38.6%, respectively, at a concentration of 50 µg/mL, which were better than that of the positive control agent, ningnanmycin (33.8%). Compounds 1, 4 and 5 also had protective effects with inhibition rates of 48.7%, 60.2% and 43.5% at the same concentration, while 4 had a better curative effect than ningnanmycin at a concentration of 100 µg/mL. In addition, mechanistic studies also revealed that a potent direct effect on TMV, the induction of SAR in tobacco plants, and the effective regulation of defense enzymes, defense genes, and defense hormones may be the reasons for the significant effects of 4 against TMV. At the same time, downregulation of the expression of total NtHsp70 protein by inhibiting the related Hsp70 genes may also be involved in tobacco resistance to TMV. To evaluate whether compounds have broader antiviral activities, the antirotavirus activities of new isolates were also evaluated and found to be highly effective with a therapeutic index (TI) value ranging from 11.6 to 17.7. This study suggests that the above anthraquinone compounds, particularly 4, have broad spectrum antiviral activities. The successful isolation and structure identification of the above anthraquinones provide new materials for the screening of anti-TMV agents and contribute to the improved utilization of N. tabacum-derived fungi.

The anti-TMV potency of the tobacco-derived fungus Aspergillus versicolor and its active alkaloids, as anti-TMV activity inhibitors.

Nicotiana tabacum (tobacco) has attracted interest as one of the most economically important industrial crops widely cultivated in China, whose dried leaves are popularly consumed medicinally and recreationally by human societies. In this study, five undescribed alkaloids derivatives, isoaspergillines A-E, together with eight known alkaloids, notoamide D, (1R,4S)-4-benzyl-1-isopropyl-2,4-dihydro-1H-pyrazino-[2,1-b]quinazoline-3,6-dione, protuboxepin K, notoamide C, notoamide M, deoxybrevianamide E, cyclo (D-Pro-L-Trp), and versicolamide B, were obtained from the culture of the Nicotiana tabacum-derived fungus Aspergillus versicolor. Their structures were mainly elucidated through comprehensive analyses of spectroscopic data. Bioactivity evaluation of all isolated compounds revealed that isoaspergilline A and notoamide M exhibited anti-TMV activities with IC50 values of 20.0 and 22.8 µM, respectively. Molecular docking suggested that isoaspergilline A and notoamide M were well located into the active site of anti-TMV by interacting with SER138, SER143, and ASN73 residues. This study enlightens the therapeutic potential of the endophytic fungus A. versicolor and it is helpful to find undescribed anti-TMV activity inhibitors, as well as searching for new anti-TMV candidates from natural sources.

Isoindolin-1-ones from the stems of Nicotiana tabacum and their antiviral activities.

In previous studies, several isoindolin-1-one analogs that exhibited significant anti-tobacco mosaic virus (anti-TMV) activities were isolated from Nicotiana tabacum. Since gene-editing mutants provide a new sample for the discovery of active metabolites, we focused on the stems of YN-18-23 (a mutant N. tabacum for gene editing with the alkaloid metabolic pathway cultivated by Yunnan Tobacco Company), which led to the isolation of four new (1-4) and four known (5-8) isoindolin-1-ones. To the best of our knowledge, nicindole C (3) is the first subclass of isoindolin-1-one bearing a pentacyclic ketone, while nicindole D (4) is the first example of isoindolin-1-one bearing a methyl-pyridin-2-(1H)-one moiety. Compounds 1-4 were tested for their anti-TMV activities, and the results revealed that compounds 1, 3, and 4 exhibited high anti-TMV activities at concentrations of 20 µM with inhibition rates of 48.6, 42.8, and 71.5%, respectively. These rates are higher than the inhibition rate of the positive control (33.2%). The mechanistic study of compound 4, which had the highest anti-TMV activity revealed that increased potentiation of defense-related enzyme activities and downregulation of expression of the NtHsp70 protein may induce resistance in tobacco against the viral pathogen TMV. Molecular docking studies also revealed that the isoindolin-1-one substructure is fundamental for anti-TMV activity. The methyl-pyridin-2-(1H)-one moiety in compound 4 and the 2-oxopropyl groups in compounds 1 and 3 at the N-2 position may increase inhibitory activities. This study of the structure-activity relationship is helpful for finding new anti-TMV activity inhibitors. To study whether the isoindolin-1-ones have broader antiviral activities, compounds 1-4 were also tested for their anti-rotavirus activities. Compound 4 exhibited high anti-rotavirus activity with a therapeutic index (TI) value of 20.7. This TI value is close to that of the positive control (20.2).

Cyclopiazonic acid type indole alkaloids from Nicotiana tabacum-derived fungus Aspergillus versicolor and their anti-tobacco mosaic virus activities.

Indole alkaloids have attracted widespread attention of chemists and biologists. Therefore, the aim of this study is to screen more bioactivities indole alkaloids from the microorganisms. In this study, five undescribed CPA-type indole alkaloids, aspergillines F-J, and three known CPA-type indole alkaloids, aspergilline A, aspergilline C, and cyclopiamide E, were obtained from the Nicotiana tabacum-derived fungus Aspergillus versicolor. Notably, aspergillines F and G represent the first examples of indole alkaloids with a benzo[cd]indol-2(1H)-one skeleton, and aspergilline J is also the firstly obtained indole alkaloids bearing a N-1-(2-(1H-imidazole-5-yl)ethyl) moiety. Aspergillines F-J and cyclopiamide E were tested for their anti-TMV activities, and the results revealed that aspergillines G and J exhibited obvious anti-TMV activities with inhibition rates of 41.2 and 56.8% at the concentration of 20 µM, respectively. These rates are high than that of positive control (with inhibition rate of 32.5%). In addition, the molecular docking studies for the isolated CPA-type indole alkaloids may also reveal that the benzo[cd]indol-2(1H)-one substructure is the fundamental for anti-TMV activity and the oxygen-containing substituent groups at C-19 also increases the inhibitory activity. This study of structure-activity relationship is helpful to find new anti-TMV activity inhibitors.

ATF2 accelerates the invasion and metastasis of hepatocellular carcinoma through targeting the miR-548p/TUFT1 axis.

AIM: Due to high invasion and metastasis, hepatocellular carcinoma (HCC) is known as one of the most fatal carcinomas. We aim to further investigate regulatory mechanisms of invasion and metastasis to elucidate HCC pathogenesis and develop novel medications. METHODS: Patient specimens were collected for assessing gene expression and correlation between gene expressions. The expression of Ki67 and E-cadherin in subcutaneous xenograft tumor were examined by immunohistochemistry staining. The expression of activating transcription factor 2 (ATF2), miR-548p and TUFT1 were determined using Real-time quantitative reverse transcription polymerase chain reaction. Epithelial-mesenchymal transition and PI3K/AKT signaling-associated markers were examined with western blot. The proliferation, migration and invasion were assessed by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide, colony formation and transwell assays, respectively. Cell apoptosis was assessed via Annexin V and propidium iodide staining. Gene interaction was confirmed using chromatin immunoprecipitation and luciferase activity assays. Subcutaneous and intravenous xenograft mouse models were established for analyzing HCC growth and metastasis in vivo. RESULTS: ATF2 was up-regulated in HCC patients and cells. ATF2 promoted HCC cell proliferation, migration and invasion and inhibited cell apoptosis through directly targeting miR-548p and controlling its expression. miR-548p suppressed HCC cell proliferation, migration and invasion and enhanced cell apoptosis. miR-548p directly bound to the 3'UTR of TUFT1 to restrain its expression and subsequently suppress the PI3K/AKT signaling. ATF2 knock-down significantly suppressed the growth and metastasis of HCC. CONCLUSION: ATF2 accelerates HCC progression by promoting cell proliferation, migration, invasion and metastasis, which is dependent on regulating the miR-548p/TUFT1 axis.

Pyrrolizidine alkaloids from the seeds of Scleropyrum wallichianum.

Two new pyrrolizidine alkaloids, sclerwalins A and B (1 and 2), and one known 9-O-E-hydroxysenecioylretronecine (3) were first isolated from the seeds of Scleropyrum wallichianum. Their chemical structures were elucidated by extensive 1 D NMR and 2 D NMR (HSQC, HMBC, COSY, and ROESY), MS and IR spectra. Cytotoxicities of all isolates were evaluated against five human tumor cell lines (HL-60, A-549, SMMC-7721, MCF-7 and SW480).[Formula: see text].

[Spatial and temporal variability of climatic potential productivity in Yunnan Province, China.] / 云南省气候生产潜力的时空变化.

We used the mean annual temperature and mean annual precipitation data during 1961 and 2017 of 101 national meteorological stations in Yunnan Province to calculate three climate-induced potential productivity in Yunnan Province by Miami model and the Thornthwaite Memorial model. The abrupt test was carried out by Mann-Kendell method. The spatial and temporal distribution characteristics and future trends of the three climate-induced potential productivities were analyzed. Results showed that the average values of the temperature potential productivity (Yt), precipitation potential productivity (Yr) and evapotranspiration potential productivity (Ye) during the study period was 1968, 1477 and 1434 g·m-2·a-1, respectively. The value of Yt was rising in Yunnan Province. For the value of Yr /Yt, there was a large difference in water-heat ratio among regions, as well as the binding conditions. There was an abrupt change in climate-induced potential productivity, with Yt began to abrupt change significantly in 2001. There was no abrupt change in Yr, but Ye had abrupt change in 2002-2004. The spatial distribution of climate production potential and climate tendency were uneven. The annual average value of Yt, Yr and Ye was 1030-2465, 927-2341 and 832-1995 g·m-2·a-1, respectively. The climate-induced potential productivity was the lowest in the northwestern and northeastern Yunnan and the highest in the southwestern and southern Yunnan. Most of the climatic propensity rates of Yt, Yr and Ye showed increase, decrease and increase trends respectively. Eight schemes simulating future climate change (i.e., temperature increased by 1 ℃, precipitation increased by 10%, temperature decreased by 1 ℃, precipitation decreased by 10%, temperature increased by 1 ℃ and precipitation decreased by 10%, temperature increased by 1 ℃ and precipitation increased by 10%, the temperature decrease by 1 ℃ and the precipitation increased by 10%, the temperature decrease by 1 ℃ and precipitation decreased by 10%) would lead to Ye changes of 6-45, 13-77.2, 15-67, -87 to -17, -74-46, 58-96, -54-57, -101 to -59 g·m-2·a-1, respectively. On the whole, if the climate tends to be "warm and wet" in the future, it will be beneficial for crop production. However, if it tends to be "cold and dry", it will be unfavorable to crop production in the study area.

[Effect of Bushen Yanggu Decoction on Multidrug Resistance of Multiple Myeloma Cell Line KM3/BTZ].

OBJECTIVE: To explore the effect of Bushen Yanggu Decoction (BYD) on drug resistance and proliferation of human multiple myeloma-resistant KM3/BTZ cells. METHODS: Human multidrug-resistant KM3/BTZ cells were established by Bortezomib (BTZ) gradient induction. The effects of commonly used chemotherapeutic drugs and serum containing Bushen Yanggu Decoction (BYD) on the proliferation of KM3 cells and KM3/BTZ cells were detected by MTT assay. RT-qPCR and Western blot were used to detect the expression of Par-4, HSP27 and P-gp genes. Flow cytometry was used to detect cell apoptosis. RESULTS: The established KM3/BTZ cells could produce varying degree of resistance to commonly used chemotherapeutic drugs. Among them, the highest resistance index (RI) to BTZ was 20.269. MTT assay showed that the proliferation of KM3/BTZ cells treated with serum containing Bushen Yanggu Decoction was inhibited, and the inhibitory effect increased with the serum concentration incranse of Bushen Yanggu Decoction. The serum containing Bushen Yanggu Decoction could inhibit the proliferation of KM3/BTZ cells, and induce apoptosis, significantly reduce the drug-resistance of KM3/BTZ cells, up-regulate the expression of Par-4, down-regulate the expression of HSP27 and P-gp. CONCLUSION: Bushen Yanggu Decoction can effectively inhibit the proliferation of KM3/BTZ cells and induce apoptosis. Bushen Yanggu Decoction can effectively reverse the multidrug-resistance of KM3/BTZ cells. The mechanism may be related with the decrease of expression of HSP27 and P-gp and the increase of expression of Par-4.

Simultaneous determination of alkaloids dicentrine and sinomenine in Stephania epigeae by 1H NMR spectroscopy.

Stephania epigaea Lo is an important herbal medicine used as antiphlogistic and analgesic drugs. Its major components are dicentrine (1) and sinomenine (2). In the present study, a rapid, accurate, and precise method for simultaneous quantitation of dicentrine (1) and sinomenine (2) in S. epigeae using 1H NMR spectra was developed. The deuterated solvent of DMSO-d6 enabled satisfactory separation of the signals to be integrated in 1H NMR spectrum and dimethyl terephthalate was selected as an internal standard. The feature signals of δ 7.57 and 5.70 were selected for quantifying the dicentrine (1) and sinomenine (2), respectively. Validation of the quantitative method was performed in terms of specificity, accuracy, precision, and stability. This work implied that quantitative 1H NMR represents a feasible alternative to high-performance liquid chromatography-based methods for quantitation of dicentrine (1) and sinomenine (2) in S. epigeae and is suitable for the quality control of S. epigeae.

Continuously adjustable period optical grating based on flexoelectric effect of a bent-core nematic liquid crystal in planar cells.

The structures of flexodomains, which are similar to optical gratings and can be controlled by the amplitude of applied voltage and temperature, were verified through polarizing microscopy and light diffraction techniques. The properties of the optical grating induced by a bent-core nematic liquid crystal in planar cells with varied cell gaps and pretilt angles were studied. The period of optical grating decreases with the increase in the amplitude of the applied voltage and pretilt angle. In addition, the period increases with the increase in cell gap and temperature. The period of optical grating has a linear relationship with temperature. The continuously adjustable period has the potential to become an important and extended application of optical grating.

Isopentylated diphenyl ether derivatives from the fermentation products of an endophytic fungus Phomopsis fukushii.

Three new isopentylated diphenyl ethers, (1-3), together with two known isopentylated diphenyl ethers derivatives (4 and 5) were isolated from the fermentation products of an endophytic fungus Phomopsis fukushii. Their structures were elucidated by spectroscopic methods, including extensive 1D- and 2D NMR techniques. Compounds 1-3 were evaluated for their anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) activity. The results showed that compounds 1-3 showed strong activity with diameter of inhibition zone (IZD) of 21.8 ± 2.4 mm, 16.8 ± 2.2 mm, and 15.6 ± 2.0 mm, respectively.

[Icaritin Reverses Multidrug Resistance of Multiple Myeloma Cell Line KM3/BTZ].

OBJECTIVE: To investigate the reversing effect of icaritin on multidrug resistance of multiple myeloma cell lines KM3/BTZ and its underlying mechanism. METHODS: KM3/BTZ cells were established by a gradually ascending gradient induction of bortezomib (BTZ). The sensitivities of KM3 and KM3/BTZ cells to 7 chemotherapeutic drugs, the inhibition and reversal effects of icaritin on proliferation and drug-resistance of KM3/BTZ cells were analyzed by MTT. The apoptosis was analyzed by flow cytometry, and the expression of Par-4, HSP27 and P-gp were detected by Western blot. RESULTS: KM3/BTZ cells were not only resistant to BTZ, but also to other 6 chemotherapeutic drugs. The resistance index (RI) to BTZ was 17.84, and higher than that of other chemotherapeutic drugs. Icaritin inhibited the proliferation and induced the apoptosis of KM3/BTZ cells. The IC50 value of BTZ decreased from 0.345 µg/ml to 0.149 µg/ml, and the reversal index was 2.38 (P<0.05). The expression of Par-4 protein increased in a concentration-dependent manner, while the expression of HSP27 and P-gp were down-regulated. CONCLUSION: Icaritin can inhibit cell proliferation and induce apoptosis of KM3/BTZ cells, moreover, can effectively reverse the multidrug resistance of KM3/BTZ cells. The mechanism may be related with down-regulation of HSP27 and P-gp expression, and up-regulation of Par-4 expression.

Diet-induced obesity accelerates blood lactate accumulation of rats in response to incremental exercise to maximum.

Blood lactate increases during incremental exercise at high-intensity workloads, and limited exercise capacity is a characteristic of obese animals. This study examined whether blood lactate changes in response to incremental exercise is disrupted in obese animals. Muscular and hepatic proteins that are critical in lactate metabolism were also investigated. Rats were randomized to either standard chow (control) or high-fat diet (HFD) groups. All animals underwent an incremental treadmill test after 14 wk of diet intervention. Blood lactate levels were measured before and after the treadmill test. Activities of mitochondrial oxidative phosphorylation and glycolysis were examined in muscle tissues. Proteins in the liver and skeletal muscles that participate in the turnover of blood lactate were determined by Western blot. Running time in the incremental treadmill test decreased in the HFD group, and blood lactate accumulated faster in these animals than in the control group. Animals with HFD had a decreased level of hepatic monocarboxylate transporter 2, the protein responsible for blood lactate uptake in the liver. Skeletal muscles of animals with HFD showed greater glycolytic activity and decreased content of lactate dehydrogenase B, which converts lactate to pyruvate. We conclude that blood lactate accumulated faster during incremental exercise in obese animals and was associated with their decreased exercise performance. Changes in the metabolic pattern of muscles and changes of liver and muscle proteins associated with lactate utilization likely contribute to the abnormal response of blood lactate to incremental exercise in obese animals.

Implementation of ultrafast X-ray diffraction at the 1W2B wiggler beamline of Beijing Synchrotron Radiation Facility.

The implementation of a laser pump/X-ray probe scheme for performing picosecond-resolution X-ray diffraction at the 1W2B wiggler beamline at Beijing Synchrotron Radiation Facility is reported. With the hybrid fill pattern in top-up mode, a pixel array X-ray detector was optimized to gate out the signal from the singlet bunch with interval 85 ns from the bunch train. The singlet pulse intensity is ∼2.5 × 10(6) photons pulse(-1) at 10 keV. The laser pulse is synchronized to this singlet bunch at a 1 kHz repetition rate. A polycapillary X-ray lens was used for secondary focusing to obtain a 72 µm (FWHM) X-ray spot. Transient photo-induced strain in BiFeO3 film was observed at a ∼150 ps time resolution for demonstration.

A new phenolic compound with antioxidant activity from the branches and leaves of Pyrus pashia.

The branches and leaves of Pyrus pashia are used to cure abdominal pain and diarrhoea in Chinese folk medicine. A new phenilic compound, 4-O-ß-d-glucopyranosylbenzyl-benzoate ester (1), along with 21 known ones (2-22) were isolated from the branches and leaves of this plant. Compounds 2 and 3 displayed remarkable antioxidant activities against 1,1-diphenyl-2-picrylhydrazyl radical (IC50 = 13.26 ± 0.04 µM, 13.28 ± 0.11 µM, respectively), which were at the same grade as positive control rutin. The caffeoyl group in compounds 2 and 3 was supposed to play an important role in the antioxidant activities.

Proaporphine and aporphine alkaloids with acetylcholinesterase inhibitory activity from Stephania epigaea.

An unusual proaporphine alkaloid bearing an isopropanenitrile group at isoquinoline nitrogen, named epiganine A (1) and a new aporphine alkaloid, epiganine B (2), together with eight known alkaloids, pronuciferine (3), dehydrodicentrine (4), romerine (5), romeline (6), N-methylcalycinine (7), phanostenine (8), dicentrine (9), and N-methyllaurotetanine (10), were isolated from the roots of Stephania epigaea. The absolute configuration of 1 was determined by calculating electronic circular dichroism (ECD) and comparing with experimental data. Compounds 2 and 4 showed strong acetylcholinesterase (AChE) inhibitory effects with the IC50 values of 4.36 and 2.98µM, respectively. Compounds 5-9 also exhibited potent AChE inhibitory activities.

Late-onset caloric restriction alters skeletal muscle metabolism by modulating pyruvate metabolism.

Caloric restriction (CR) attenuates age-related muscle loss. However, the underlying mechanism responsible for this attenuation is not fully understood. This study evaluated the role of energy metabolism in the CR-induced attenuation of muscle loss. The aims of this study were twofold: 1) to evaluate the effect of CR on energy metabolism and determine its relationship with muscle mass, and 2) to determine whether the effects of CR are age dependent. Young and middle-aged rats were randomized into either 40% CR or ad libitum (AL) diet groups for 14 wk. Major energy-producing pathways in muscles, i.e., glycolysis and mitochondrial oxidative phosphorylation (OXPHOS), were examined. We found that the effects of CR were age dependent. CR improved muscle metabolism and normalized muscle mass in middle-aged animals but not young animals. CR decreased glycolysis and increased the cellular dependency for OXPHOS vs. glycolysis in muscles of middle-aged rats, which was associated with the improvement of normalized muscle mass. The metabolic reprogramming induced by CR was related to modulation of pyruvate metabolism and increased mitochondrial biogenesis. Compared with animals fed AL, middle-aged animals with CR had lower lactate dehydrogenase A content and greater mitochondrial pyruvate carrier content. Markers of mitochondrial biogenesis, including AMPK activation levels and SIRT1 and COX-IV content, also showed increased levels. In conclusion, 14 wk of CR improved muscle metabolism and preserved muscle mass in middle-aged animals but not in young developing animals. CR-attenuated age-related muscle loss is associated with reprogramming of the metabolic pathway from glycolysis to OXPHOS.

Diterpenoid alkaloids from Aconitum vilmorinianum.

Diterpenoid alkaloids, named vilmorines A-D, in addition to fifteen known alkaloids, were isolated from roots of Aconitum vilmorinianum. Their structures were established on the basis of extensive spectroscopic analyses. Antibacterial and antioxidant studies on isolated compounds were also carried out.

[Treatment of hypertension by acupuncture method of "activating blood and dispersing wind, harmonizing Gan-Pi": an analysis of its principle of the circular motion of ancient Chinese medicine].

Hypertension is one of main risk factors for the occurrence and death of stroke and coronary heart disease. Its prevalence rate is rising year by year. It severely threatens the health of the human beings. The acupuncture method of "activating blood and dispersing wind, harmonizing Gan-Pi" for treating hypertension launched by Academician SHI Xue-min has aroused great attention due to good cur- ative effect and less adverse reactions. In this paper principles of the circular motion covered by the acupuncture method of "activating blood and dispersing wind, harmonizing Gan-Pi" were clarified.