Functional verification of the JmLFY gene associated with the flowering of Juglans mandshurica Maxim.

In this study, a pBI121-JmLFY plant expression vector was constructed on the basis of obtaining the full-length sequence of the JmLFY gene from Juglans mandshurica, which was then used for genetic transformation via Agrobacterium inflorescence infection using wild-type Arabidopsis thaliana and lfy mutants as transgenic receptors. Seeds of positive A. thaliana plants with high expression of JmLFY were collected and sowed till the homozygous T3 regeneration plants were obtained. Then the expression of flowering-related genes (AtAP1, AtSOC1, AtFT and AtPI) in T3 generation plants were analyzed and the results showed that JmLFY gene overexpression promoted the expression of flowering-related genes and resulted in earlier flowering in A. thaliana. The A. thaliana plants of JmLFY-transformed and JmLFY-transformed lfy mutants appeared shorter leaves, longer fruit pods, and fewer cauline leaves than those of wild-type and the lfy mutants plants, respectively. In addition, some secondary branches in the transgenic plants converted into inflorescences, which indicated that the overexpression of JmLFY promoted the transition from vegetative growth to reproductive growth, and compensate the phenotypic defects of lfy mutant partially. The results provides a scientific reference for formulating reasonable genetic improvement strategies such as shortening childhood, improving yield and quality, and breeding desirable varieties, which have important guiding significance in production.

Hydrogen Generation by Hydrolysis of MgH2-LiH Composite.

As a most promising material for hydrogen generation by hydrolysis, magnesium hydride (MgH2) is also trapped by its yielded byproduct Mg(OH)2 whose dense passivated layers prevent the further contact of intimal MgH2 with water. In this work, LiH, as a destroyer, has been added to promote the hydrogen properties of MgH2. The results demonstrate that even 3 wt % LiH was added into MgH2-G, the hydrogen generation yield can increase about 72% compared to the hydrogen generation yield of MgH2-G. The possible mechanism is that Mg2+ from the hydrolysis of MgH2 preferentially bound with OH- ions from the hydrolysis of LiH to form Mg(OH)2 precipitation, which is dispersed in water rather than coated on the surface of MgH2. Moreover, adding MgCl2 into hydrolysis solution, using ball milling technology, and increasing the hydrolysis temperature can make the hydrolysis rate higher and reaction process more complete. It is noted that a too high weight ratio of LiH with too high of a hydrolysis temperature will make the reaction too violent to be safe in the experiment. We determinate the best experimental condition is that the LiH ratio added into MgH2 is 3 wt %, the hydrolysis temperature is 60 °C, and the concentration of MgCl2 hydrating solution is 1 M. MgH2-LiH composite hydrogen generation technology can meet the needs of various types of hydrogen supply and has broad application prospects.

Transcriptome-based analysis of the hormone regulation mechanism of gender differentiation in Juglans mandshurica Maxim.

Juglans mandshurica Maxim is a hermaphroditic plant belonging to the genus Juglans in the family Juglandaceae. The pollination period of female flowers is different from the loose powder period of male flowers on the same tree. In several trees, female flowers bloom first, whereas in others, male flowers bloom first. In this study, male and female flower buds of J. mandshurica at the physiological differentiation stage were used. Illumina-based transcriptome sequencing was performed, and the quality of the sequencing results was evaluated and analyzed. A total of 138,138 unigenes with an average length of 788 bp were obtained. There were 8,116 differentially expressed genes (DEGs); 2,840 genes were upregulated, and 5,276 genes were downregulated. The DEGs were classified by Gene Ontology and analyzed by Kyoto Encyclopedia of Genes and Genomes. The signal transduction factors involved in phytohormone synthesis were selected. The results displayed that ARF and SAUR were expressed differently in the auxin signaling pathway. Additionally, DELLA protein (a negative regulator of gibberellin), the cytokinin synthesis pathway, and A-ARR were downregulated. On April 2nd, the contents of IAA, GA, CTK, ETH and SA in male and female flower buds of two types of J. mandshurica were opposite, and there were obvious genes regulating gender differentiation. Overall, we found that the sex differentiation of J. mandshurica was related to various hormone signal transduction pathways, and hormone signal transduction plays a leading role in regulation.

Flower Development of Heterodichogamous Juglans mandshurica (Juglandaceae).

Juglans mandshurica is a monoecious heterodichogamous species with protogynous and protandrous mating strategies that occur at a 1:1 ratio and are randomly distributed in the population. The inconsistent male and female flowering periods of the same mating type result in an imbalance of the ratio of male and female flowers, contributing to the low yield of this species. However, little more is known about its floral development. Following three consecutive years of observations, histological analysis, and scanning electron microscopy, we found that the morphological and anatomical development of the male and female flowers were synchronous. The male floral morphological development of J. mandshurica was divided into seven phases, while that of the female flower was nine. Four stages were shared between the male and female flower's anatomical development. Our findings indicate that there was minimal overlap between sexual functions within the same mating type, guaranteeing synchronization, mutual non-interference, outcrossing, and avoidance of self-fertilization. These results provide a theoretical basis for the improvement of fruit yield and quality through the reasonable allocation of protogynous and protandrous individuals in a population, and for artificial pollination control. Further, these findings lay a foundation for further research on the genetic mechanisms and environmental effects on flower development of heterodichogamous J. mandshurica.

Study on follicular fluid metabolomics components at different ages based on lipid metabolism.

BACKGROUND: Follicular fluid is an important external environment for the growth and development of oocytes. A thorough identification of specific components in follicular fluid can better the existing understand of intracellular signal transduction and reveal potential biomarkers of oocyte health in women undergoing assisted reproductive therapy. To study on follicular fluid metabolomics components at different ages based on lipid metabolism, we have adopted a new method of SWATH to MRM(the sequential window acquisition of all theoretical fragment-ion spectra to multiple reaction monitor)metabolomics to provide extensive coverage and excellent quantitative data. This was done to investigate the differences in follicular fluid of patients undergoing in vitro fertilization (IVF) and embryo transfer in different age groups and to further explore the relationship between follicular fluid, age and reproductive function. METHOD: A combination of Ultra-high-performance liquid chromatography and high resolution mass spectrometry techniques were used to analyze the follicular fluid of 230 patients enrolled for the IVF cycle. The patients were of different ages grouped into two groups:the younger and older patients.The obtained multidimensional chromatographic data were processed by principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). The charge ratios and mass numbers enabled for the identification of different fragments in the samples. Matching information obtained through database search and the fragment information obtained by fragment ion scan structurally identified substances in the samples. This was used to determine the differential compounds. RESULTS: The quality of oocytes decline with age,and the lipid composition in follicular fluid also changes,The lipid metabolism that changes with age may be related to the quality of oocytes.The main differences were in lipid metabolites. Some were up-regulated: Arachidonate, LysoPC(16:1), LysoPC(20:4) and LysoPC(20:3) while others were down-regulated: LysoPC(18:3) and LysoPC(18:1). CONCLUSIONS: Metabolomic analysis of follicular fluid revealed that with the increase in age, several differential metabolites are at play. Among these metabolites, lipid metabolism undergoes significant changes that affect the development of oocytes thus causing reduced fertility in older women. These differential metabolites related to follicular development may provide possible detection and treatment targets for promoting oocyte health, and provide scientific basis for understanding the environment of oocyte development.

Leaf Anatomy, Morphology and Photosynthesis of Three Tundra Shrubs after 7-Year Experimental Warming on Changbai Mountain.

Tundra is one of the most sensitive biomes to climate warming. Understanding plant eco-physiological responses to warming is critical because these traits can give feedback on the effects of climate-warming on tundra ecosystem. We used open-top chambers following the criteria of the International Tundra Experiment to passively warm air and soil temperatures year round in alpine tundra. Leaf size, photosynthesis and anatomy of three dominant species were investigated during the growing seasons after 7 years of continuous warming. Warming increased the maximal light-saturated photosynthetic rate (Pmax) by 43.6% for Dryas. octopetala var. asiatica and by 26.7% for Rhododendron confertissimum across the whole growing season, while warming did not significantly affect the Pmax of V. uliginosum. The leaf size of Dr. octopetala var. asiatica and Rh. confertissimum was increased by warming. No marked effects of warming on anatomical traits of Dr. octopetala var. asiatica were observed. Warming decreased the leaf thickness of Rh. confertissimum and Vaccinium uliginosum. This study highlights the species-specific responses to climate warming. Our results imply that Dr. octopetala var. asiatica could be more dominant because it, mainly in terms of leaf photosynthetic capacity and size, seems to have advantages over the other two species in a warming world.

SWATHHM-Based Metabolomics of Follicular Fluid in Patients Shows That Progesterone Adversely Affects Oocyte Quality.

OBJECTIVE: We reveal the relationship between progesterone level in follicular fluid and oocyte quality based on sequential window acquisition of all theoretical fragment-ion spectra (SWATH™), a powerful high-resolution mass spectrometric data independent acquisition technique. METHOD: Follicular fluid samples were collected from 22 subjects (the level of progesterone > 1.5 ng/mL) of progesterone group, as well as from 22 subjects (the level of progesterone < 1.5 ng/mL) of control group, and analyzed using UPLC-Q-TOF. All methods were performed in accordance with ISO 9001:2008. Novel SWATH acquisition mode on an ultra-high performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (with resolving power 20,000-40,000) was investigated for the analysis of human follicular fluid. The principal component variable grouping detects intersample variable correlation and groups variables with similar profiles which simplifies interpretation and highlights related ions and fragments. It can also extract product ion spectra from the data collected by fragmenting a wide precursor ion window. RESULTS: Follicular fluid from the two groups differed with respect to five metabolites. Follicular fluid from the progesterone group contained elevated levels of 8-hydroxyguanosine and 4-hydroxynonenal and reduced levels of ATP, estradiol, and L-carnitine. The increased progesterone level on the day of HCG injection could negatively impact oocyte quality, thus reducing the pregnancy rate of IVF patients.

Novel SWATHTM technology for follicular fluid metabolomics in patients with endometriosis.

AIM OF THE STUDY: Sequential window acquisition of all theoretical fragment-ion spectra (SWATHTM), a powerful high-resolution mass spectrometric data independent acquisition technique, was used to identify differences that relate certain metabolites to endometriosis (EMT) in follicular fluid collected from EMT patients and a control group. METHODS: A case-control study was conducted to analyze the EMT-related metabolites and the IVF clinical data of 33 subjects. Subjects were divided between the observation group (17 cases, infertility due to EMT) and the control group (16 cases, infertility due to male factor, such as obstructive azoospermia). RESULTS: Analysis revealed three metabolites including phytosphingosine, LysoPC(18:2(9Z,12Z)) and LysoPC(18:0), which were closely related to infertility associated withEMT. In the EMT group, LysoPC(18:2(9Z,12Z)) and LysoPC(18:0) were upregulated, while phytosphingosine was downregulated. CONCLUSIONS: This study employed, for the first time, the SWATHTM data acquisition mode for the metabolomics study of human follicular fluid in patients with EMT. The differential metabolite profiles of follicular fluid were identified and mapped. These differential metabolites are involved in cell proliferation and apoptosis, energy metabolism, inflammatory responses and angiogenesis. The differential metabolite profile may be a new tool for early noninvasive assessment of the developmental potential of oocytes in patients with EMT.

Pharmacokinetic Profiling of Butylidenephthalide and Alisol B in Danggui-Shaoyao-San in Rats.

BACKGROUND AND OBJECTIVE: Danggui-Shaoyao-San (DSS), a famous Chinese formula, has been widely used to treat gynecological disorders since ancient times and has recently showed efficacy in treating Alzheimer's disease. Butylidenephthalide (BDPH) and alisol B (ALI) are recognized as the primary active ingredients of DSS. The objectives of the present study were to evaluate the pharmacokinetic comparative study of BDPH and ALI in herbal extracts and their purified forms. METHOD: A sensitive and specific high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) methodology was developed to determine the concentration level of BDPH and ALI in rat plasma. This approach enables a real-time pharmacokinetics profiling of BDPH and ALI in DSS extracts as well as their purified forms in rats after oral administration. RESULTS: The validated method showed an evident linearity over a wide range of dosages (r > 0.99) with sensitivity down to 1.0 ng/mL for each analyte. The extraction recovery of the analyte ranged from 80.8 to 99.1%. The pharmacokinetic parameters were significantly different in herbal extracts and their purified forms. The results showed that the absorption of both BDPH and ALI from DSS extracts was significantly greater compared with their purified forms. CONCLUSIONS: A highly specific, sensitive and rapid HPLC-MS/MS method was developed and applied for the determination of BDPH and ALI in rat plasma. It was found that BDPH and ALI had higher bioavailability in the DSS extract compared with their purified forms.

Fractal scaling of particle-size distribution and associations with soil properties of Mongolian pine plantations in the Mu Us Desert, China.

Mongolian pine plantations (MPPs) composed of Pinus sylvestris var. mongolica (P. sylvestris) are used for desertification control and restoration of degraded land in arid and semi-arid regions. We studied soil changes associated with P. sylvestris by comparing top (0-20 cm) and sub-top (20-40 cm) soil properties across 8 stand density gradients of MPPs ranging from 900 ± 5-2700 ± 50 trees ha-1. The study was conducted in the uncovered Sandy Land in the southern Mu Us Desert, China. The relationships between the volume fractal dimensions (D) of soil particle size distribution and soil physicochemical properties were evaluated. D was determined using a laser diffraction technique and soil properties were measured. In the top layer, P. sylvestris significantly positively affected soil physicochemical properties except for bulk density and total nitrogen. These effects were not observed in the sub-top soil layer. D values ranged from 1.52 ± 0.29-2.08 ± 0.06 and were significantly correlated with stand density. Significant correlations were observed between D and soil properties (except total nitrogen) in the top soil layer. Given these results, we concluded that D is a sensitive and useful index because it quantifies changes in soil properties that additionally implies desertification in the studied area.

Determination of a novel B-Raf(V600E) and EGFR dual inhibitor in rat plasma by HPLC-MS/MS and its application in a pharmacokinetic study.

The EGFR and B-Raf(V600E) dual inhibition is a promising strategy in treatment of colorectal cancer patients with B-Raf(V600E) mutation. Previously, compound 3 was designed and synthesized as a novel B-Raf(V600E) and EGFR dual inhibitor with highly potency in both kinase and cell based assay. Herein, a sensitive and rapid HPLC-MS/MS quantitative method was developed and validated for the further pharmacokinetic evaluation of compound 3 in rats.

Determination of a novel dipeptidyl peptidase IV inhibitor in monkey plasma by HPLC-MS/MS and its application in a pharmacokinetics study.

A lot of attention has been given to novel diabetes treatment, which is used to replace injectable insulin. A novel dipeptidyl peptidase IV inhibitor (HWH-10d) for treating type 2 diabetes was previously determined to have comparable efficacy to the marketed drug (alogliptin) in ICR male mice. Therefore, a sensitive and rapid liquid chromatography-tandem mass spectrometric method was developed and validated for the further evaluation of HWH-10d in monkey. The analytes were extracted through a liquid-liquid extraction with ethyl acetate. The linear detection range for HWH-10d in monkey plasma was from 0.5 to 2000 ng/mL with the lower limit of quantification of 0.5 ng/mL. The relative standard deviation was measured to be less than 10.4% for determination of inter- and intra-day precisions, and the relative error was determined to be within ±7.2% for all accuracy measurements. The simple and rapid LC-MS/MS method for HWH-10d in monkey plasma could be used for the pharmacokinetics studies of HWH-10d in monkeys. The oral bioavailability of HWH-10d in monkeys is 57.8%.

Development of an optimized cytotoxicity assay system for CYP3A4-mediated metabolic activation via modified piggyBac transposition.

Drug-induced hepatotoxicity is often caused by cytochrome P450 (CYP)-dependent metabolism of drugs into reactive metabolites. Assessment of hepatotoxicity induced by bioactive compounds is hampered by low CYP expression within in vitro cell lines. To overcome this limitation, piggyBac transposition and monoclonal expansion were used to generate a HepG2 cell line with stable and homogenously high expression of CYP3A4, a prominent CYP isoform. Our studies demonstrate the generated line's constant CYP3A4 expression and activity for over 40 cell passages; to date, it has been in subculture for more than a year without addition of Puromycin. This cell line was utilized to evaluate cytotoxicity of two bioactive (troglitazone and acetaminophen) and two non-bioactive (citrate and galactosamine) compounds by MTT assay. Cell viability significantly decreased upon treatment with bioactive drugs. Moreover, cell lines used in the present study were more sensitive to toxic effects of troglitazone than previously reported. Therefore, this HepG2 cell-based assay system may provide a suitable hepatic model for predicting CYP3A4-mediated hepatotoxicity during preclinical drug development.

The Sap Flow Dynamics and Response of Hedysarum scoparium to Environmental Factors in Semiarid Northwestern China.

Hedysarum scoparium is an important, fast-growing and drought-resistant shrub that has been extensively used for grassland restoration and preventing desertification in semiarid regions of northwestern China. The primary objective of this study was to investigate the diurnal and seasonal variations in stem sap flow (Js) and its relation to environmental factors. The stem heat balance method was applied to plants that were approximately 17 years old (with diameters of 25, 16, 13, and 9 mm at ground level and heights of 3.1, 1.8, 1.7 and 1.4 m) and growing under natural conditions. The vertical soil temperature profile (ST), soil surface heat flux (SoilG), volumetric soil moisture content (SWC) and meteorological variables such as solar radiation (Rn), air temperature (Ta), vapour pressure deficit (VPD), wind speed (Ws) relative humidity (RH) and precipitation (P) were simultaneously measured at a meteorological station on site. Results indicated that Js varied regularly during the diurnal and seasonal term. The nocturnal Js was substantial, with a seasonal variation similar to the patterns of daytime Js. The magnitude of Js changed considerably between sunny and rainy days. Redundancy (RDA) and Kendall's tau analysis suggested that daily Js in large plants was more sensitive to environmental factors, and the variation in daily Js during the growing season could be described by a multiple linear regression against environmental variables including Ta, VPD, Ws, RH, ST, and SoilG. While the nocturnal Js in smaller plants was more sensitive to meteorological factors. Ta, VPD, and Ws were significantly correlated with nighttime Js. The hourly nighttime sap flow rate of H. scoparium corresponded closely to Ta and VPD following a non-linear pattern. The results of this study can be used to estimate the transpiration of H. scoparium.

A rapid and sensitive HPLC-MS/MS method for determination of an aminopyridazine derived anti-neuroinflammatory agent (ZW14) in dog plasma: Application to a pharmacokinetic study.

The unclear etiology of Alzheimer's disease leaves a large space for drug exploration. A novel anti-neuroinflammation agent (ZW14) was previously determined to have comparable efficacy to the marketed drug (donepezil) in the Aß-induced model mice. Herein, a sensitive and rapid HPLC-MS/MS quantitative method was developed and validated for the further evaluation of ZW14 in dogs. Plasma samples were processed by liquid-liquid extraction with ethyl acetate and separated on Luna C18 column (2.1 mm × 50 mm, 1.7 µm) at room temperature with a flow rate of 0.2 mL/min. The analyte and IS were all detected by monitoring the precursor → product ion transition at unit resolution using multiple reaction monitoring (MRM) scan mode with positive ionization mode. No endogenous interference was observed and the linear range was 0.05-1500 ng/mL with the lower limit of quantification of 0.05 ng/mL. The intra- and inter-day precisions were within 10.9%, while the accuracy was all between 96.0% and 110%. The developed method was successfully applied to the pharmacokinetic study of ZW14 in beagle dogs after oral and intravenous administration of 2 mg/kg. The oral bioavailability of ZW14 was 26.3% with half-life of 2.6h.

Different binding sites of bovine organic anion-transporting polypeptide1a2 are involved in the transport of different fluoroquinolones.

Because of their wide distribution and capability of transporting a large variety of compounds, organic anion-transporting polypeptides (OATPs) have been extensively recognized as crucial players in absorption, distribution, and excretion of various drugs. OATP1A2 was the first cloned human OATP and has been found to transport wide range of endogenous and exogenous compounds. Bovine Oatp1a2 (bOatp1a2) shares high homology with human OATP1A2 and is considered the functional ortholog of the latter. Previous study in our laboratory demonstrated that bOatp1a2 transport of estrone-3-sulfate (ES) exhibited biphasic saturation kinetics. In the present study, we investigated the transport function of bOatp1a2 for four different quinolone antibacterial agents (enrofloxacin, levofloxacin, norfloxacin, and ciprofloxacin) and found that all the tested fluoroquinolones can be transported by bOatp1a2. Further studies showed that different binding sites are responsible for the transport of different fluoroquinolones. Both ciprofloxacin and norfloxacin exhibited biphasic saturation kinetics. The Kms of the high- and low-affinity components for ciprofloxacin were 3.80 ± 0.85 µM and 182 ± 31 µM, respectively, while those for norfloxacin were 24.7 ± 0.1 µM and 393 ± 79 µM, respectively. Enrofloxacin and levofloxacin showed an inhibitory effect on the uptake of only the high concentration of ES and thus may be transported by the low-affinity site for ES. Interestingly, enrofloxacin and levofloxacin demonstrated an activation effect on ES uptake at the high-affinity binding site. These results suggested that multiple binding sites within the structure of bOatp1a2 may be responsible for the uptake of different quinolone antimicrobial agents.

Fractal scaling of particle size distribution and relationships with topsoil properties affected by biological soil crusts.

BACKGROUND: Biological soil crusts are common components of desert ecosystem; they cover ground surface and interact with topsoil that contribute to desertification control and degraded land restoration in arid and semiarid regions. METHODOLOGY/PRINCIPAL FINDINGS: To distinguish the changes in topsoil affected by biological soil crusts, we compared topsoil properties across three types of successional biological soil crusts (algae, lichens, and mosses crust), as well as the referenced sandland in the Mu Us Desert, Northern China. Relationships between fractal dimensions of soil particle size distribution and selected soil properties were discussed as well. The results indicated that biological soil crusts had significant positive effects on soil physical structure (P<0.05); and soil organic carbon and nutrients showed an upward trend across the successional stages of biological soil crusts. Fractal dimensions ranged from 2.1477 to 2.3032, and significantly linear correlated with selected soil properties (R(2) = 0.494∼0.955, P<0.01). CONCLUSIONS/SIGNIFICANCE: Biological soil crusts cause an important increase in soil fertility, and are beneficial to sand fixation, although the process is rather slow. Fractal dimension proves to be a sensitive and useful index for quantifying changes in soil properties that additionally implies desertification. This study will be essential to provide a firm basis for future policy-making on optimal solutions regarding desertification control and assessment, as well as degraded ecosystem restoration in arid and semiarid regions.

The highly potent and selective dipeptidyl peptidase IV inhibitors bearing a thienopyrimidine scaffold effectively treat type 2 diabetes.

New dipeptidyl peptidase IV inhibitors were designed based on Alogliptin using a scaffold-hopping strategy. All of the compounds constructed on a thienopyrimidine scaffold demonstrated good inhibition and selectivity for DPP-IV. Compound 10d exhibited subnanomolar (IC(50)=0.33nM) DPP-IV inhibitory activity, good in vivo efficacy and an acceptable pharmacokinetic profile. A pharmacokinetic-driven optimization of 10d may lead to a new class of clinical candidate DPP-IV inhibitors.

Simultaneous analysis of bambuterol and its active metabolite terbutaline enantiomers in rat plasma by chiral liquid chromatography-tandem mass spectrometry.

A chiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) simultaneous stereoselective analysis of bambuterol and its active metabolite terbutaline enantiomers in Wistar rat plasma has been developed and validated. All analytes and the internal standard were extracted from rat plasma samples by liquid-liquid extraction, separated on macrocyclic glycopeptide teicoplanin column with mobile phase constituted of 20mM ammonium acetate solution-methanol (10:90, v/v) at a flow-rate of 0.4 mL/min. Detection was performed on an API 3000 tandem mass spectrometer with positive electrospray ionization in multiple reaction monitoring mode. The calibration curves in the range 1-800 ng/mL were linear and the accuracy for each analyte was within 8.0%. The intra- and inter-day precision as determined from quality control samples was less than 10.1%. The validated assay was successfully used to determine the enantiomers of bambuterol and terbutaline in rat plasma samples in the pharmacokinetic studies of rac-bambuterol.