Characterization and Engineering of Two Highly Paralogous Sesquiterpene Synthases Reveal a Regioselective Reprotonation Switch.

Sesquiterpene synthases (STPSs) catalyze carbocation-driven cyclization reactions that can generate structurally diverse hydrocarbons. The deprotonation-reprotonation process is widely used in STPSs to promote structural diversity, largely attributable to the distinct regio/stereoselective reprotonations. However, the molecular basis for reprotonation regioselectivity remains largely understudied. Herein, we analyzed two highly paralogous STPSs, Artabotrys hexapetalus (-)-cyperene synthase (AhCS) and ishwarane synthase (AhIS), which catalyze reactions that are distinct from the regioselective protonation of germacrene A (GA), resulting in distinct skeletons of 5/5/6 tricyclic (-)-cyperene and 6/6/5/3 tetracyclic ishwarane, respectively. Isotopic labeling experiments demonstrated that these protonations occur at C3 and C6 of GA in AhCS and AhIS, respectively. The cryo-electron microscopy-derived AhCS complex structure provided the structural basis for identifying different key active site residues that may govern their functional disparity. The structure-guided mutagenesis of these residues resulted in successful functional interconversion between AhCS and AhIS, thus targeting the three active site residues [L311-S419-C458]/[M311-V419-A458] that may act as a C3/C6 reprotonation switch for GA. These findings facilitate the rational design or directed evolution of STPSs with structurally diverse skeletons.

Nitrate leaching characteristics of red soils from different parent materials in subtropical China.

Globally, nitrate (NO3-) leaching from agroecosystems has been of major concern. There is evidence that NO3- leaching exhibits intense seasonal variation in subtropical regions. However, influencing factors to the seasonal dynamics remain unclear. In this study, a two-year field lysimeters experiment was conducted with three red soils derived from different parent materials (Quaternary red clay (QR), red sandstone (RS), and basalt (BA)). An N fertilizer (15N-enriched urea, 10 atom% excess) of 200 kg N ha-1 yr-1 was applied for maize. The effect of parent material on NO3- leaching characteristics was examined in surface (0-20 cm) and subsoil (20-100 cm) layers. The results showed due to the weakening of abundant drainage, there was no significant effect of parent materials on NO3- leaching characteristics in surface layers. Environmental factors (precipitation and temperature) and fertilization together led to obvious seasonal characteristics, i.e. abundant NO3- leaching during both crop growth and fallow periods. In subsoil layers, NO3- leaching characteristics were completely different among three soils. The concentrations and δ15N of NO3- in QR and RS soils showed a continuous increase after first year's fertilization, while those in BA soil remained relatively stable after reaching peak levels around harvest in first year. Meanwhile, the NO3- leaching amount in BA soil was significantly lower than in the other two soils. These might be explained by different NO3- adsorption capacities caused by the differences in mineral composition and free iron and aluminium contents. These elucidated in subsoil layers, NO3- leaching characteristics highly depended on parent materials. Meanwhile, adsorption capacity was limited and cannot slow NO3- leaching in the long run. Our results suggest that seasonal variation of NO3- leaching in surface layers and temporary retardant effect from NO3- adsorption capacity in subsoil layers should receive much attention when calculating and predicting NO3- leaching in subtropical regions.

Two new bibenzyl methylglucosides as SIRT3 activators obtained through microbial transformation.

Microbial transformation of dihydroresveratrol (DHRSV) using Beauveria bassiana has produced two new methylglucosylated derivatives of DHRSV (1 and 2), whose structures were characterized as 4'-O-(4″-O-methyl-ß-D-glucopyranosyl)-dihydroresveratrol (4'-O-MG DHRSV, 1) and 3-O-(4″-O-methyl-ß-D-glucopyranosyl)-dihydroresveratrol (3-O-MG DHRSV, 2) on the basis of spectroscopic methods. They showed moderate SIRT3 agonistic activity, and compound 2 exhibited the best deacetylation of 406.63% at 10 µM. The activity of 2 increased by 3.12-fold compared with that of DHRSV, since 2 performed better in molecular docking assay (GScore -8.445).

Identification of volatile compounds and their bioactivities from unpolar fraction of Alpinia oxyphylla Miq. and mining key genes of nootkatone biosynthesis.

In this study, analysis of the chemical constituents and bioactivities of the unpolar fractions [petroleum ether (PE) and chloroform (C)] of fruits and leaves of Alpinia oxyphylla Miq. were carried out, as well as the bioactivities of the main compounds nootkatone and valencene. From PE and C fractions of the fruits, and PE fraction of the leaves, 95.80%, 59.30%, and 82.11% of the chemical constituents respectively were identified by GC-MS. Among these identified compounds, nootkatone was the main compound in all of three fractions, while valencene was the second main compound in the PE fractions of the fruits and leaves. The bioactivities results showed that all of the fractions and the major compound nootkatone showed tyrosinase inhibitory, as well as inhibitory effect on NO production in LPS-stimulated RAW264.7 cells. While valencene only presented inhibitory activity on NO production in RAW264.7 cells. The critical genes involved in nootkatone biosynthesis in A. oxyphylla were identified from the public transcriptome datasets, and protein sequences were preliminarily analyzed. Our studies develop the usage of the unpolar fractions of A. oxyphylla, especially its leaves as the waste during its production, and meanwhile provide the gene resources for nootkatone biosynthesis.

[Clinicopathological Features and Prognosis of Patients Newly Diagnosed With Lung Adenocarcinoma With Both EGFR Mutation and C-MET Amplification].

Objective To explore the clinicopathological features and prognosis of the patients newly diagnosed with lung adenocarcinoma with both EGFR mutation and C-MET amplification.Methods The pathological sections were reviewed.EGFR mutation was detected by amplification refractory mutation system-quantitative real-time polymerase chain reaction,and C-MET amplification by fluorescence in situ hybridization.The clinicopathological features and survival data of the patients newly diagnosed with lung adenocarcinoma with both EGFR mutation and C-MET amplification were analyzed retrospectively.Results In 11 cases of EGFR mutation combined with C-MET amplification,complex glands and solid high-grade components were observed under a microscope in 10 cases except for one case with a cell block,the tissue structure of which was difficult to be evaluated.The incidence of lung adenocarcinoma in the patients with EGFR mutation combined with C-MET amplification at clinical stage Ⅳ was higher than that in the EGFR mutation or C-MET amplification group (all P<0.001),whereas the difference was not statistically significant between the EGFR mutation group and C-MET amplification group at each clinical stage (all P>0.05).There was no significant difference in the trend of survival rate between EGFR gene group and C-MET amplification group (χ2=0.042,P=0.838),while the survival of the patients with EGFR mutation combined with C-MET amplification was worse than that of the patients with EGFR mutation (χ2=246.72,P<0.001) or C-MET amplification (χ2=236.41,P<0.001).Conclusions The patients newly diagnosed with lung adenocarcinoma with EGFR mutation plus C-MET amplification demonstrate poor histological differentiation,rapid progress,and poor prognosis.The patients are often in the advanced stage when being diagnosed with cancer.Attention should be paid to this concurrent adverse driving molecular event in clinical work.With increasing availability,the inhibitors targeting C-MET may serve as an option to benefit these patients in the near future.

Soil acidification in a tailing area of ionic rare earth in Southeast China.

Ionic rare earth ores are now commonly mined using the ammonium sulfate in situ leaching method, causing soil acidification in tailings. To evaluate the degree of soil acidification in tailings and the influence of mining activities on acidification, we selected an ionic rare earth tailing and a nearby unmined area in Southeast China. This tailing had been closed for 12 years. We sampled the soil from the surface to the bedrock in layers and determined soil properties related to soil acidification. The results showed that the average soil pH was 5.0 in the unmined area and 4.5 in the mined area (tailing area). Rare earth mining led to a decrease in soil pH of 0.47 units per 10 years, which was 2-5 times higher than that of other land uses. The shallow soil acidification in the mined area is not affected by mining. Deep soils were significantly acidified and the H+ concentration in the soil solution was approximately nine times that of the unmined area soil. Deep soil acidification is influenced very little by natural factors. The average soil ammonium­nitrogen (NH4+-N) and nitrate­nitrogen contents in the mined area were 58.34 mg kg-1 and 8.19 mg kg-1, respectively, 84 times and 21 times that of the unmined area. There were large amounts of NH4+, NO3-, and H+ in the soil of the mined area, indicating that soil acidification is closely related to exogenous NH4+-N input and nitrogen transformation. Nitrification is the most important driver of soil acidification in mining areas. Continued nitrification of excess NH4+-N will continue to produce H+ and migrate with water, which will cause long-term harm to the soil and surrounding environment in the mining area. Therefore, it is necessary to remove the enriched NH4+-N in tailings soil to avoid further soil acidification.

Microbial biotransformation to obtain stilbene methylglucoside with GPR119 agonistic activity.

Introduction: Limitation of pharmaceutical application of resveratrol (RSV) and piceatannol (PIC) continue to exist, there is a need to obtain the superior analogs of two stilbenes with promoted activity, stability, and bioavailability. Microbial transformation has been suggested as a common and efficient strategy to solve the above problems. Methods: In this study, Beauveria bassiana was selected to transform RSV and PIC. LC-MS and NMR spectroscopies were used to analyze the transformed products and identify their structures. The biological activities of these metabolites were evaluated in vitro with GPR119 agonist and insulin secretion assays. Single factor tests were employed to optimize the biotransformation condition. Results: Three new methylglucosylated derivatives of PIC (1-3) and two known RSV methylglucosides (4 and 5) were isolated and characterized from the fermentation broth. Among them, 1 not only showed moderate GPR119 agonistic activity with 65.9%, but also promoted insulin secretion level significantly (12.94 ng/mg protein/hour) at 1 µM. After optimization of fermentation conditions, the yield of 1 reached 45.53%, which was increased by 4.2-fold compared with the control. Discussion: Our work presents that 3-O-MG PIC (1), obtained by microbial transformation, is an effective and safer ligand targeting GPR119, which lays a foundation for the anti-diabetic drug design in the future.

Mapping high resolution National Soil Information Grids of China.

Soil spatial information has traditionally been presented as polygon maps at coarse scales. Solving global and local issues, including food security, water regulation, land degradation, and climate change requires higher quality, more consistent and detailed soil information. Accurate prediction of soil variation over large and complex areas with limited samples remains a challenge, which is especially significant for China due to its vast land area which contains the most diverse soil landscapes in the world. Here, we integrated predictive soil mapping paradigm with adaptive depth function fitting, state-of-the-art ensemble machine learning and high-resolution soil-forming environment characterization in a high-performance parallel computing environment to generate 90-m resolution national gridded maps of nine soil properties (pH, organic carbon, nitrogen, phosphorus, potassium, cation exchange capacity, bulk density, coarse fragments, and thickness) at multiple depths across China. This was based on approximately 5000 representative soil profiles collected in a recent national soil survey and a suite of detailed covariates to characterize soil-forming environments. The predictive accuracy ranged from very good to moderate (Model Efficiency Coefficients from 0.71 to 0.36) at 0-5 cm. The predictive accuracy for most soil properties declined with depth. Compared with previous soil maps, we achieved significantly more detailed and accurate predictions which could well represent soil variations across the territory and are a significant contribution to the GlobalSoilMap.net project. The relative importance of soil-forming factors in the predictions varied by specific soil property and depth, suggesting the complexity and non-stationarity of comprehensive multi-factor interactions in the process of soil development.

Chromosome-scale genome assembly of Rhododendron molle provides insights into its evolution and terpenoid biosynthesis.

BACKGROUND: Rhododendron molle (Ericaceae) is a traditional Chinese medicine, which has been used to treat rheumatism and relieve pain since ancient times. The characteristic grayanoids of this plant have been demonstrated to be the chemical basis for the analgesic activity. Moreover, unlike morphine, these diterpenoids are non-addictive. Grayanoids mainly distribute in the leaves, flowers, roots, and fruits of R. molle, with low content. Currently the research on the biosynthesis of grayanoids is hindered, partially due to lack of the genomic information. RESULTS: In the present study, a total of 744 Mb sequences were generated and assembled into 13 chromosomes. An ancient whole-genome duplication event (Ad-ß) was discovered that occurred around 70 million years ago. Tandem and segmental gene duplications led to specific gene expansions in the terpene synthase and cytochrome P450 (CYP450) gene families. Two diterpene synthases were demonstrated to be responsible for the biosynthesis of 16α-hydroxy-ent-kaurane, the key precursor for grayanoids. Phylogenetic analysis revealed a species-specific bloom of the CYP71AU subfamily, which may involve the candidate CYP450s responsible for the biosynthesis of grayanoids. Additionally, three putative terpene biosynthetic gene clusters were found. CONCLUSIONS: We reported the first genome assembly of R. molle and investigated the molecular basis underpinning terpenoids biosynthesis. Our work provides a foundation for elucidating the complete biosynthetic pathway of grayanoids and studying the terpenoids diversity in R. molle.

[Clinicopathological Characteristics of Lymphadenitis Caused by Talaromyces marneffei Diagnosed by Core Needle Biopsy].

Objective To analyze the clinicopathological characteristics of lymphadenitis caused by Talaromyces marneffei (TM).Method s The clinical data,pathological features,pathogen examination,and treatment of 15 cases of TM-caused lymphadenitis were analyzed retrospectively.Results The 15 cases included 14 males and 1 females,who were aged 26-67 years,with an average age of (49.1±11.87) years.The 15 cases,including 13 cases of acquired immunodeficiency syndrome and 2 cases of diabetes mellitus,were accompanied by superficial lymph node enlargement in the neck and supraclavicular,axillary,and inguinal regions.The structure of cord-like lymph node tissue punctured by thick needle was completely or partially replaced by inflammatory lesions. Under microscope,8 cases showed mainly diffuse infiltration of phagocytes with pathogens;5 cases presented mainly extensive coagulation necrosis with a small amount of pathogens and nuclear debris;2 cases were characterized by small nodular hyperplasia of fibroblasts,formation of granulomatous structure,and scattered distribution of a few multinucleated giant cells.The pathogens were relatively consistent in size and shape,which were round,oval or sausage-shaped and clustered like mulberry.Diastase periodic acid-Schiff staining and hexamine silver staining highlighted the bacterial structure with transverse septum.TM growth was detected in the blood,alveolar lavage fluid,sputum or lymph node extract fungal culture of the 15 patients.Owing to the adequate antifungal treatment in time,these 15 patients were discharged after their conditions were improved.Conclusion Lymphadenitis is one of the major manifestations of the systemic invasion of TM at the late stage,which is tended to be misdiagnosed.Through core needle biopsy of lymph node,it can be diagnosed as soon as possible to avoid delayed treatment and improve the cure rate.

Unusual (2R,6R)-bicyclo[3.1.1]heptane ring construction in fungal α-trans-bergamotene biosynthesis.

Bergamotenes are bicyclo[3.1.1]heptane sesquiterpenes found abundantly in plants and fungi. Known bergamotene derivatives all possess (2S,6S)-bergamotene backbone. In this study, two (+)-α-trans-bergamotene derivatives (1 and 2) with unusual (2R,6R) configuration were isolated and elucidated from marine fungus Nectria sp. HLS206. The first (+)-α-trans-bergamotene synthase NsBERS was characterized using genome mining and heterologous expression-based strategies. Based on homology search, we characterized another (+)-α-trans-bergamotene synthase LsBERS from Lachnellula suecica and an (+)-α-bisabolol synthase BcBOS from Botrytis cinerea. We proposed that the cyclization mechanism of (+)-α-trans-bergamotene involved endo-anti cyclization of left-handed helix farnesyl pyrophosphate by (6R)-bisabolyl cation, which was supported by molecular docking. The biosynthesis-based volatiles (3-6) produced by heterologous fungal expression systems elicited significant electroantennographic responses of Helicoverpa armigera and Spodoptera frugiperda, respectively, suggesting their potential in biocontrol of these pests. This work enriches diversity of sesquiterpenoids and fungal sesquiterpene synthases, providing insight into the enzymatic mechanism of formation of enantiomeric sesquiterpenes.

Soil acidification and loss of base cations in a subtropical agricultural watershed.

Soil acidification along with base cations loss degrades soil quality and is a major environmental problem, especially in agroecosystems with extensive nitrogen (N) fertilization. So far, the rates of proton (H+) production and real soil acidification (loss of base cations) remain unclear in subtropical agricultural watersheds. To assess the current status and future risk of soil acidification in subtropical red soil region of China, a two-year monitoring was conducted in a typical agricultural watershed with upland, paddy fields, and orchards where high N fertilizers are applied (320 kg N ha-1 yr-1). H+ production, neutralization and base cations losses were quantified based on the inputs (rainwater, inflow of water, and fertilizer) and outputs (outflow of water, groundwater drainage, and plant uptake) of major elements (K+, Ca2+, Na+, Mg2+, Al3+, NH4+, NO3-, SO42-, Cl-, and H+). The result showed that total H+ production in the watershed was 5152 molc ha-1 yr-1. N transformation was the most important H+ source (68%), followed by excess plant uptake of cations (25%) and H+ deposition (7%). Base cations exchange and weathering of minerals (3842 molc ha-1 yr-1) dominated H+ neutralization, followed by SO42- adsorption (1081 molc ha-1 yr-1), while H+ and Al3+ leaching amounted to 431 molc ha-1 yr-1, only. These results state clearly that despite significant soil acidification, the acidification of surface waters is minor, implying that soils have buffered substantially the net H+ addition. As a result of soil buffering, there was abundant loss of base cations, whose rate is significantly higher than the previously reported weathering rate of minerals in red soils (3842 vs 230-1080 molc ha-1 yr-1). This suggests that the pool of exchangeable base cations is being depleted in the watershed, increasing the vulnerability of the watershed, and posing a serious threat to future recovery of soils from acidification.

Significant loss of soil inorganic carbon at the continental scale.

Widespread soil acidification due to atmospheric acid deposition and agricultural fertilization may greatly accelerate soil carbonate dissolution and CO2 release. However, to date, few studies have addressed these processes. Here, we use meta-analysis and nationwide-survey datasets to investigate changes in soil inorganic carbon (SIC) stocks in China. We observe an overall decrease in SIC stocks in topsoil (0-30 cm) (11.33 g C m-2 yr-1) from the 1980s to the 2010s. Total SIC stocks have decreased by ∼8.99 ± 2.24% (1.37 ± 0.37 Pg C). The average SIC losses across China (0.046 Pg C yr-1) and in cropland (0.016 Pg C yr-1) account for ∼17.6%-24.0% of the terrestrial C sink and 57.1% of the soil organic carbon sink in cropland, respectively. Nitrogen deposition and climate change have profound influences on SIC cycling. We estimate that ∼19.12%-19.47% of SIC stocks will be further lost by 2100. The consumption of SIC may offset a large portion of global efforts aimed at ecosystem carbon sequestration, which emphasizes the importance of achieving a better understanding of the indirect coupling mechanisms of nitrogen and carbon cycling and of effective countermeasures to minimize SIC loss.

Nitrate runoff loss and source apportionment in a typical subtropical agricultural watershed.

Nitrate (NO3-) loss and enrichment in water bodies caused by fertilization are a major environmental problem in agricultural areas. However, the quantitative contribution of different NO3- sources, especially chemical fertilizers (CF) and soil organic nitrogen (SON), to NO3- runoff loss remains unclear. In this study, a systematic investigation of NO3- runoff and its sources was conducted in a subtropical agricultural watershed located in Yujiang County, Jiangxi Province, China. A semi-monthly sampling was performed at the inlet and outlet from March 2018 to February 2019. Hydrochemical and dual NO3- isotope (15 N and 18O) approaches were combined to estimate the NO3- runoff loss and quantify the contribution of different sources with a Bayesian isotope mixing model. Source apportionment by Stable Isotope Analysis in R (SIAR) suggested that NO3- in runoff was mainly derived from nitrification of ammonium (NH4+) mineralized from SON (37-52%) and manure/sewage (M&S) (25-47%), while the contribution of CF was relatively small (14-25%). The contribution of various sources showed seasonal variations, with a greater contribution of CF in the wet growing season (March to August). Compared with the inlet which contributed 37-40% to runoff NO3-, SON contributed more at the outlet (49-52%). Denitrification in the runoff was small and appeared to be confined to the dry season (September to February), with an estimated NO3- loss of 2.73 kg N ha-1. The net NO3- runoff loss of the watershed was 34.5 kg N ha-1 yr-1, accounting for 15% of the annual fertilization rate (229 kg N ha-1 yr-1). Besides M&S (22%), fertilization and remineralization of SON (CF + SON) were the main sources for the NO3- runoff loss (78%), suggesting accelerated nitrification of NH4+ from CF (24%) and SON mineralization (54%). Our study indicates that NO3- runoff loss in subtropical agricultural watersheds is dominated by nonpoint source pollution from fertilization. SON played a more important role than CF. Besides, the contribution of sewage should not be neglected. Our data suggest that a combination of more rational fertilizer N application (CF), better management of SON, and better treatment of domestic sewage could alleviate NO3- pollution in subtropical China.

New monoterpenoids from the stigmas of Crocus sativus.

One new compound, crocusatin M (1), and three new glycosidic compounds, crocusatins N-P (2-4), along with nine known compounds were isolated from the dried stigmas of Crocus sativus. The structures of new compounds were elucidated on the basis of spectroscopic analysis, and the absolute configurations of 1, 2, and 3 were unambiguously assigned by the comparison of experimental and calculated ECD data. This is the first report of the isolation of 4 with the HMG moiety from the genus Crocus. Compounds 1 and 4 exhibited weak anti-inflammatory activities on inhibiting lipopolysaccharide (LPS)-induced NO production.

Seasonal dynamics of soil pH and N transformation as affected by N fertilization in subtropical China: An in situ 15N labeling study.

Nitrogen (N)-induced soil acidification has received much attention worldwide. Nitrification and soil N mineralization are two key N cycle processes that affect soil acidification. However, the seasonal dynamics of soil pH under their combined influence is unclear. We studied the effect of N fertilization on soil pH and N transformations using 15N tracing in field lysimeters with soils developed from different parent materials (Quaternary red clay, sandstone, and basalt). Maize was planted with 200 kg N ha-1 yr-115N-labeled urea addition. During 7-45 days after fertilization, proton (H+) production due to nitrification of fertilizer N, nitrate (NO3-) leaching, and plant uptake exceeded H+ consumption by base cations mobilization and leaching, resulting in a significant soil pH decline. When nitrification activity decreased (after 45 days), due to exhausted ammonium (NH4+) availability, soil pH rose again. During the fallow period, acid neutralization due to base cation mobilization, and ammonification of soil organic N (SON) offset H+ production caused by nitrification of mineralized SON, leading to a sustained rise in soil pH. After the one-year experiment, no significant soil pH decrease was observed in any of the soils. Parent material had little effect on the seasonal dynamics of soil acidification, which appeared to be controlled by fertilization, environmental factors (temperature and moisture), and plant uptake. In subtropical regions, monitoring of soil pH on an annual basis may mask the effect of N fertilization on soil acidification.

Construction of acetyl-CoA and DBAT hybrid metabolic pathway for acetylation of 10-deacetylbaccatin III to baccatin III.

10-Deacetylbaccatin III (10-DAB) C10 acetylation is an indispensable procedure for Taxol semi-synthesis, which often requires harsh conditions. 10-Deacetylbaccatin III-10-ß-O-acetyltransferase (DBAT) catalyzes the acetylation but acetyl-CoA supply remains a key limiting factor. Here we refactored the innate biosynthetic pathway of acetyl-CoA in Escherichia coli and obtained a chassis with acetyl-CoA productivity over three times higher than that of the host cell. Then, we constructed a microbial cell factory by introducing DBAT gene into this chassis for efficiently converting 10-DAB into baccatin III. We found that baccatin III could be efficiently deacetylated into 10-DAB by DBAT with CoASH and K+ under alkaline condition. Thus, we fed acetic acid to the engineered strain both for serving as a substrate of acetyl-CoA biosynthesis and for alleviating the deacetylation of baccatin III. The fermentation conditions were optimized and the baccatin III titers reached 2, 3 and 4.6 g/L, respectively, in a 3-L bioreactor culture when 2, 3 and 6 g/L of 10-DAB were supplied. Our study provides an environment-friendly approach for the large scale 10-DAB acetylation without addition of acetyl-CoA in the industrial Taxol semi-synthesis. The finding of DBAT deacetylase activity may broaden its application in the structural modification of pharmaceutically important lead compounds.

Distribution of the Alpha-Synuclein in the Brain and the Primary Organs of the Rhesus Monkey.

Previous studies have shown that abnormal aggregation of alpha-synuclein (α-syn) protein is a major trigger of neurodegenerative diseases. The expression level of α-syn in different brain regions and the disease-susceptible regions varies with the development of the disease. The expression pattern of the α-syn protein in mouse brain has been precisely described in the literature. Some studies have also reported the ubiquitous expression of the α-syn protein in the central and peripheral in nonhuman primates (NHPs). However, little is known about the expression pattern of α-syn in the brain or in the primary organs of NHPs. Here, we investigated the expression profile of α-syn in different brain regions and the primary organs of NHPs. The α-syn protein was mainly distributed in layers III and V of the cerebral cortex and the hippocampus. In addition, strong immunofluorescent signals were detected in the striatum and the substantia nigra, especially in the globus pallidus and the substantia nigra pars compacta, where the expression was significantly and particularly strong, compared with that in the cerebellum or the cortex. In the cerebellum, intense α-syn signal was observed in the molecular layer, where it was significantly higher than in the nucleus or the medulla. In the brain, the α-syn was always detected both in the cytoplasm and the synapses. Additionally, the α-syn was widely expressed in primary organs. The α-syn signal was higher in the liver and small intestine than in the spleen. Thus, the regions displaying the highest α-syn expression are also those affected during the progression of neurodegenerative diseases. These results may provide basic reference data for the study of multi-systemic mechanism of neurodegenerative diseases.

The Characteristic Fragrant Sesquiterpenes and 2-(2-Phenylethyl)chromones in Wild and Cultivated "Qi-Nan" Agarwood.

Recently, cultivated "Qi-Nan" (CQN) agarwood has emerged as a new high-quality agarwood in the agarwood market owing to its similar characteristics, such as high content of resin and richness in two 2-(2-phenylethyl)chromone derivatives, 2-(2-phenylethyl)chromone (59) and 2-[2-(4-methoxyphenyl)ethyl]chromone (60), to the wild harvested "Qi-Nan" (WQN) agarwood. In this study, we compared the chemical constituents and fragrant components of two types of WQN agarwood from A. agallocha Roxb. and A. sinensis, respectively, with CQN agarwood and ordinary agarwood varieties. Additionally, we analyzed different samples of WQN agarwood and CQN agarwood by GC-MS, which revealed several noteworthy differences between WQN and CQN agarwood. The chemical diversity of WQN was greater than that of CQN agarwood. The content of (59) and (60) was higher in CQN agarwood than in WQN agarwood. For the sesquiterpenes, the richness and diversity of sesquiterpenes in WQN agarwood, particularly guaiane and agarofuran sesquiterpenes, were higher than those in CQN. Moreover, guaiane-furans sesquiterpenes were only detected by GC-MS in WQN agarwood of A. sinensis and could be a chemical marker for the WQN agarwood of A. sinensis. In addition, we summarized the odor descriptions of the constituents and established the correlation of scents and chemical constituents in the agarwood.

High-level soluble expression of human Cu,Zn superoxide dismutase with high activity in Escherichia coli.

As the most important member of antioxidant defense system, human Cu,Zn superoxide dismutase (hCu,Zn-SOD) protects cells against the free radicals produced by aerobic metabolism. hCu,Zn-SOD has been widely used in food, cosmetic and medicine industry due to its health benefits and therapeutic potentials. However, a more extensive application of hCu,Zn-SOD is limited by the challenge of expensive and low production of high-activity hCu,Zn-SOD in large scale. In this study, the codon-optimized hCu,Zn-SOD gene was synthesized, cloned into pET-28a( +) and transformed into Escherichia coli BL21(DE3). After induction with IPTG or lactose, hCu,Zn-SOD was highly expressed as soluble form in LB medium with 800 µM Cu2+ and 20 µM Zn2+ at 25 °C. The recombinant hCu,Zn-SOD was efficiently purified by nickel affinity chromatography. Through optimization of fed-batch fermentation conditions, 342 mg purified hCu,Zn-SOD was obtained from 1 L cultures fermented in a 3-L bioreactor. Furthermore, the recombinant hCu,Zn-SOD retained the enzymatic specific activity of 46,541 U/mg. This study has opened up an effective avenue for industrial production of hCu,Zn-SOD through microbial fermentation in the future.