[Cloning and expression analysis of kaurenoic acid oxidase gene in Tripterygium wilfordii].

Kaurenoic acid oxidase involved in biosynthesis pathway of gibberellin. According to the transcriptome database, the specific primers were designed and used in cloning the full-length cDNA of TwKAO, the bioinformatic analysis of the sequence was performed. The qRT-PCR were used to detect the expression level of TwKAO after MeJA treatment.The full-length cDNA of the TwKAO was 1 874 bp encoding a polypeptide of 487 amino acids.The calculate molecular weight was about 56.02 kDa,and the theoretical isoelectric point (pI) was 8.89. The relative expression level of TwKAO was deduced by MeJA and reached the highest at 12 h after the treatment.Plant tissue expression analysis indicated that, TwKAO expressed the highest in leaves,while lowest in roots.For the first time, we cloned and analyzed the expression characteristics of TwKAO, which laid a foundation for deep analysis of growing development and terpenoid secondary metabolites in T. wilfordii.

OsWOX3A is involved in negative feedback regulation of the gibberellic acid biosynthetic pathway in rice (Oryza sativa).

The plant-specific WUSCHEL-related homeobox (WOX) nuclear proteins have important roles in the transcriptional regulation of many developmental processes. Among the rice (Oryza sativa) WOX proteins, a loss of OsWOX3A function in narrow leaf2 (nal2) nal3 double mutants (termed nal2/3) causes pleiotropic effects, such as narrow and curly leaves, opened spikelets, narrow grains, more tillers, and fewer lateral roots, but almost normal plant height. To examine OsWOX3A function in more detail, transgenic rice overexpressing OsWOX3A (OsWOX3A-OX) were generated; unexpectedly, all of them consistently exhibited severe dwarfism with very short and wide leaves, a phenotype that resembles that of gibberellic acid (GA)-deficient or GA-insensitive mutants. Exogenous GA3 treatment fully rescued the developmental defects of OsWOX3A-OX plants, suggesting that constitutive overexpression of OsWOX3A downregulates GA biosynthesis. Quantitative analysis of GA intermediates revealed significantly reduced levels of GA20 and bioactive GA1 in OsWOX3A-OX, possibly due to downregulation of the expression of KAO, which encodes ent-kaurenoic acid oxidase, a GA biosynthetic enzyme. Yeast one-hybrid and electrophoretic mobility shift assays revealed that OsWOX3A directly interacts with the KAO promoter. OsWOX3A expression is drastically and temporarily upregulated by GA3 and downregulated by paclobutrazol, a blocker of GA biosynthesis. These data indicate that OsWOX3A is a GA-responsive gene and functions in the negative feedback regulation of the GA biosynthetic pathway for GA homeostasis to maintain the threshold levels of endogenous GA intermediates throughout development.

Separation and purification of glycosides from medicinal plants based on strong polar separation medium with online closed-loop mode.

Natural glycosides widely distributed in medicinal plants are valuable sources of therapeutic agents, showing various pharmacological effects. The separation and purification of natural glycosides are meaningful for their pharmacological research, which face with great challenges due to the complex of medicinal plants samples. In this work, two kinds of functional monolithic separation mediums A and S were fabricated and fully applied in the online extraction, separation and purification of active glycoside components from medicinal plants with a simple-procedure closed-loop mode. Chrysophanol glucoside and physcion glucoside were detected and separated from Rhei Radix et Rhizoma using separation medium A as a solid-phase extraction adsorbent. Rhapontin was isolated and purified from Rheum hotaoense C. Y. Cheng et Kao using separation medium S as the stationary phase of high-performance liquid chromatography. Compared to the reported literatures, high yield of 5.68, 1.20 and 4.76 mg g-1 of these three products were obtained with high purity. These two online closed-loop mode methods were carried out using high-performance liquid chromatography system, in which the sample injection, isolation and purification procedures are all online mode, and reduced loss compared to offline extraction and purification procedures, thus achieving high recovery and high purity.

A novel tumor-specific broad-spectral monoclonal antibody to PL2L60 is highly effective for the treatment of various types of cancers from human and mouse.

There are numerous antibodies used for cancer therapy in clinic, but they are essentially less efficacy than expected. None of them has tumor-specific and broad-spectral properties. PIWIL2-like (PL2L) protein 60 (PL2L60) is a product of alienated activation of PIWIL2 gene, and has been found to be specifically and widely expressed in various types of cancers, including hematopoietic and solid ones. Current study aims to investigate whether a monoclonal antibody (mAb) to PL2L60 has both tumor-specific and broad-spectral properties, which can be used universally to treat various types of cancers. The expression of PL2L60 protein in the cell surface and cytoplasm were determined in a panel of human and mouse tumor cell lines by flow cytometry, immunofluorescent microscopy and Western Blotting. The apoptosis and the cell cycle arrest of the tumor cells treated with mAb KAO3 were evaluated by flow cytometry. The tumorigenesis of the mAb KAO3-pretreated tumor cells was determined by tumor incidence and tumor size, and the efficacy of mAb KAO3 treatment on tumor growth in tumors-bearing mice were kinetically evaluated. Complement-dependent cytotoxicity (CDC) assay was used to determine the capacity of mAb KAO3 to kill tumor cells. Treatment of human or mouse tumor cells from hematopoietic or solid tumors with mAb KAO3 at the time of inoculation efficiently inhibited tumorigenesis in the severe combined immunodeficient (SCID) mice. Moreover, injection of mAb KAO3 into established tumors significantly inhibited their growth, and prolonged survival of the tumor-bearing mice, including lymphoma, breast cancer, lung cancer and cervical cancer. The efficacy of mAb KAO3 treatment is likely associated with its binding to PL2L60 expressed on tumor cell surface, which may lead to cancer cell death through blocking cell cycling and/or activation of complement. In conclusion, we have identified a tumor-specific mAb to PL2L60 (KAO3), which may be used potentially to treat all the types of human cancers including from both hematopoietic and solid ones.

Investigating the nexus between hydroelectricity energy, renewable energy, nonrenewable energy consumption on output: evidence from E7 countries.

The transition of most economies especially heavily industrialized nations like China, Turkey, Russia, India, Indonesia, Brazil, and Mexico fondly known as E7 are fast emerging economies with its impact on economic growth and ecosystem. On the above highlight, the present study explores the dynamic interaction between hydroelectricity energy, renewable energy consumption, nonrenewable energy consumption on economic growth over annual time frequency data from 1990 to 2018. To this end, Kao co-integration technique is adopted in conjunction with panel ordinary least squares, dynamic ordinary least squares, and fully modified ordinary least square estimators over the identified blocs while the heterogeneous causality test of Dumitrescu and Hurlin is employed to detect the direction of causality among the variables. Empirical result shows long-run analysis reveals long-run equilibrium relationship between the examined variables. Furthermore, a one-way causality relationship is observed between economic growth and nonrenewable energy, economic growth and renewable energy, and economic growth and pollutant emission. The present study identifies a U-shaped pattern among nonrenewable energy consumption and economic growth in the long-run. These findings suggest that as economic growth increases, there is less strengthening of energy from the nonrenewable energy consumption hence, portentous deterioration in nonrenewable usage while authenticating the proficiency of nonrenewable energy consumption in the E7 countries. Further policy prescriptions are rendered in the concluding section.

[A review of Hirata Atsutane's Yi zong zhong jing kao (Studies on Zhongjing, the Medical Master)].

In the book named Yi zong zhong jing kao (Studies on Zhongjing, the Medical Master), through a series of examinations, the author, Hirata Atsutane of the late Edo period of Japan, argued that the author of the book Shang han lun (Treatise on Febrile Diseases) is Ge Xuan, brother of Ge Hong's grandfather, whereas the names of Dai Ba, Zhang Ji, Zhong Jing, etc., in ancient books are but his other names or his alias. With Ge Xuan as a basis, Hirata Atsutane explored its former and later history and eventually pointed out that medical prescriptions of traditional Chinese medicine were originally formulated by the gods, and fundamentally rooted from the Japanese gods, or called Shinto in ancient Japan, which is the foundation of Traditional Chinese Medical art. Prospectively, Hirata Atsutane's such conclusion is incredible. Actually, Hirata Atsutane used a series of textual investigation to interpret the doctrine of supernatural medicine, or the idea of "medical Shinto" he advocated. This thought also corresponds to the thinking of recede-to-ancient Shinto, reflecting his view of superiority of Japan over China. In a broader sense, such viewpoint on China can also be seen as a strong nationalist view in Japan, emphasized by the intelligentsia represented by classical scholars and even by Japan as a whole in the mid-late Edo period.

Development of a new field-test procedure for cocaine.

The Scott test, widely used as the field test for cocaine, is performed in three steps. If a sample contains cocaine, blue precipitates appear in step 1, the precipitates are dissolved and the solution turns pink in step 2, and the lower layer turns blue in step 3. However, some pyrrolidine-type cathinones produce cocaine-like results when tested, necessitating modification of the test procedure. Filtration of the second-step mixture weakened the blue color in step 3; however, the blue color did not completely disappear. Adding the Chen-Kao reagent to the test procedure enhanced the differentiation: when the reagent was added to cocaine, the solution was initially turbid, but then became clear over time; its addition to cathinones resulted in turquoise or light sky-blue precipitation. These results indicated that the Chen-Kao test was useful for exclusion of cathinones. A combination of the modified Scott test and the Chen-Kao test was successfully applied to the forensic samples containing cocaine or pyrrolidine-type cathinones. In conclusion, a combination of these tests will be the useful field-test procedure for cocaine.

[Effectiveness of three biological larvicides and of an insect growth regulator against Anopheles arabiensis in Senegal]. / Efficacité de trois larvicides d'origine biologique et d'un régulateur de croissance contre Anopheles arabiensis au Sénégal.

Urban malaria is a major public health problem in Africa. In Senegal, the environmental changes seem to favor the persistence of malaria transmission in Dakar suburbs by creating, throughout the year, potential breeding sites of malaria vectors. In such a situation and in a context of a growing threat of insecticide resistance in anopheline vectors, the larval control making use of products from biological origin or growth regulators could represent an additional tool to the current strategies developed against anophelines. In this study conducted in 2012, the efficiency and residual effect of three biological larvicides (VectoBac® WG, Vecto-Max® CG, and VectoBac® GR) and an insect growth regulator (MetaLarv™) were evaluated on Anopheles gambiae s.l. larvae in seminatural conditions (experimental station) and natural breeding sites in the suburbs of Dakar. The formulations were tested according to the manufacturer recommendations, namely 0.03 g/m2 for VectoBac® WG, 0.5 g/m2 for VectoBac® GR, 0.75 g/m2 for VectoMax® CG, and 0.5 g/m2 for MetaLarv™. In experimental station, the treatment with larvicides was effective over a period of 14 days with a mortality ranging between 92% and 100%. The insect growth regulator remained effective up to 55 days with a single emergence recorded in the 27th day after treatment. In natural conditions, a total effectiveness (100% mortality) of larvicides was obtained 48 hours after treatment, then a gradual recolonization of breeding sites was noted. However, the insect growth regulator has reduced adult emergence higher than 80% until the end of follow-up (J28). This study showed a good efficiency of the larvicides and of the growth regulator tested. These works provide current data on potential candidates for the implementation of larval control interventions in addition to that of chemical adulticide for control of urban malaria.

Hormonal diterpenoids derived from ent-kaurenoic acid are involved in the blue-light avoidance response of Physcomitrella patens.

Gibberellins (GAs) are diterpenoid hormones that regulate growth and development in flowering plants. The moss Physcomitrella patens has part of the GA biosynthetic pathway from geranylgeranyl diphosphate to ent-kaurenoic acid via ent-kaurene, but it does not produce GA. Disruption of the ent-kaurene synthase gene in P. patens suppressed caulonemal differentiation. Application of ent-kaurene or ent-kaurenoic acid restored differentiation, suggesting that derivative(s) of ent-kaurenoic acid, but not GAs, are endogenous regulator(s) of caulonemal cell differentiation. The protonemal growth of P. patens shows an avoidance response under unilateral blue light. Physiological studies using gene mutants involved in ent-kaurene biosynthesis confirmed that diterpenoid(s) regulate the blue-light response. Here, we discuss the implications of these findings, and provide data for the ent-kaurene oxidase gene-disrupted mutant.

Analysis of ent-kaurenoic acid by ultra-performance liquid chromatography-tandem mass spectrometry.

ent-Kaurenoic acid (KA) is a key intermediate connected to a phytohormone gibberellin. To date, the general procedure for quantifying KA is by using traditional gas chromatography-mass spectrometry (GC-MS). In contrast, gibberellins, which are more hydrophilic than KA, can be easily quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this study, we have established a new method to quantify KA by LC-MS/MS by taking advantage of a key feature of KA, namely the lack of fragmentation that occurs in MS/MS when electrospray ionization (ESI) is in the negative mode. Q1 and Q3 were adopted as identical channels for the multiple reaction monitoring of KA. The method was validated by comparing with the results obtained by selected ion monitoring in GC-MS. This new method could be applicable for the quantification of other hydrophobic compounds.

Gelatinomyces siamensis gen. sp. nov. (Ascomycota, Leotiomycetes, incertae sedis) on bamboo in Thailand.

Gelatinomyces siamensis gen. sp. nov., incertae sedis within Leotiomycetes, the Siamese jelly-ball, is described. The fungus was collected from bamboo culms and branches in Nam Nao National Park, Phetchabun, Thailand. It presents as a ping-pong ball-sized and golf ball-like gelatinous ascostroma. The asci have numerous ascospores, are thick-walled, and arise on discoid apothecia which are aggregated and clustered to form the spherical gelatinous structures. An hyphomycete asexual morph is morphologically somewhat phialophora-like, and produces red pigments. On the basis of phylogenetic analysis based on rRNA, SSU, and LSU gene sequences, the lineage is closest to Collophora rubra. However, ITS sequences place the fungus on a well-separated branch from that fungus, and the morphological and ecological differences exclude it from Collophora.

Molecular evolution of the ent-kaurenoic acid oxidase gene in Oryzeae.

We surveyed the substitution patterns in the ent-kaurenoic acid oxidase (KAO) gene in 11 species of Oryzeae with an outgroup in the Ehrhartoidaea. The synonymous and non-synonymous substitution rates showed a high positive correlation with each other, but were negatively correlated with codon usage bias and GC content at third codon positions. The substitution rate was heterogenous among lineages. Likelihood-ratio tests showed that the non-synonymous/synonymous rate ratio changed significantly among lineages. Site-specific models provided no evidence for positive selection of particular amino acid sites in any codon of the KAO gene. This finding suggested that the significant rate heterogeneity among some lineages may have been caused by variability in the relaxation of the selective constraint among lineages or by neutral processes.