Four Markers Useful for the Distinction of Intrauterine Growth Restriction in Sheep.

Intrauterine growth restriction (IUGR) is a common perinatal complication in animal reproduction, with long-lasting negative effects on neonates and postnatal animals, which seriously negatively affects livestock production. In this study, we aimed to identify potential genes associated with the diagnosis of IUGR through bioinformatics analysis. Based on the 73 differentially expressed related genes obtained by differential analysis and weighted gene co-expression network analysis, we used three machine learning algorithms to identify 4 IUGR-related hub genes (IUGR-HGs), namely, ADAM9, CRYL1, NDP52, and SERPINA7, whose ROC curves showed that they are a good diagnostic target for IUGR. Next, we identified two molecular subtypes of IUGR through consensus clustering analysis and constructed a gene scoring system based on the IUGR-HGs. The results showed that the IUGR score was positively correlated with the risk of IUGR. The AUC value of IUGR scoring accuracy was 0.970. Finally, we constructed a new artificial neural network model based on the four IUGR-HGs to diagnose sheep IUGR, and its accuracy reached 0.956. In conclusion, the IUGR-HGs we identified provide new potential molecular markers and models for the diagnosis of IUGR in sheep; they can better diagnose whether sheep have IUGR. The present findings provide new perspectives on the diagnosis of IUGR.

Exploration and optimization of conditions for quantitative analysis of lignans in Schisandra chinensis by an online supercritical fluid extraction with supercritical fluid chromatography system.

An online supercritical fluid extraction with supercritical fluid chromatography system could provide sequential extraction and quantitative analysis of lignans in Schisandra chinensis. Supercritical fluid extraction conditions were optimized at 15 MPa, 50°C, and 4 min with supercritical CO2 adding 1% methanol; the elution volume and flow rate were set at 6 mL and 2 mL/min to blow extract out of the tank completely. The split-flow rate was confirmed at 2.5%, which determines injection volume and accuracy of quantitative detection. The factors having negative influences on supercritical fluid chromatography retention in the online system, including sample loading forms and backpressure settings, are discussed in the paper. At last, an extraction-quantitative method for lignans in Schisandra chinensis was developed, which could be finished within 19.5 min. The total content percentage of four lignans (Schisandrin, Schisandrin A, Schisandrin B and Schisandrol B) in four batches was respectively measured to be 1.42, 1.54, 1.62, and 1.90%.

Quantification of isoflavone glycosides and aglycones in rat plasma by LC-MS/MS: Troubleshooting of interference from food and its application to pharmacokinetic study of Semen Sojae Praeparatum extract.

The isoflavones widely exist in the daily diets and interferences are usually inevitable in the determination of the in vivo level of the same analytes. A new strategy to eliminate the dietary interference was established to evaluate the exposure of isoflavones including daidzin, glycitin, genistin, daidzein, glycitein, and genistein in rats fed with Semen Sojae Praeparatum (SSP) extract. Plasma samples were pretreated by liquid-liquid extraction with ethyl acetate using quercetin as the internal standard (IS). The chromatographic separation was achieved on a Symmetry C18 column (100 mm × 3.0 mm) using a gradient mobile phase consisting of acetonitril and water (containing 0.1% formic acid) with a run time of 13.0 min at a flow rate of 0.4ml/min. The detection was carried out by a triple-quadrupole tandem mass spectrometer in multiple reaction monitoring (MRM) mode via polarity switching between negative (for and positive (for daidzin glycitin) ionization mode. All calibration curves exhibited good linearity (r> 0.99) over a wide concentration range for all components. The lower limit of quantitation (LLOQ) was in the range of 0.1-0.4 ng/ml. The intra-day and inter-day precisions (RSD) at three different levels were both less than 14.9% and the accuracies (RE) ranged from -9.3% to 14.5%. The extraction recoveries of the analytes and the IS ranged from 85.7% to 100.2%. The validated method was first successfully applied to pharmacokinetic study of the six isoflavones in rat plasma after oral administration of SSP extract. The dynamic baseline levels of six isoflavones in blank plasma from rats consuming food containing dietary isoflavones were measured for the correction of the plasma concentrations. The principle pharmacokinetic parameters were calculated from rats with or without regular commercial food, and found to be altered by the dietary food containing some isoflavones.

Diversity and bioprospecting of culturable actinomycetes from marine sediment of the Yellow Sea, China.

Marine actinomycetes are a potential source of a wide variety of bioactive natural products. In this work, seven pretreatments, three selective isolation media, and five artificial seawater concentrations were used to isolate actinomycetes from the sediments collected from Yellow Sea, China. Statistical analysis showed that only the isolation medium strongly affected the total and bioactive numbers of actinomycete isolates. A total of 613 actinobacterial strains were isolated and screened for antimicrobial activities; 154 isolates showed activity against at least one of nine test drug-resistant microorganisms. Eighty-nine representatives with strong antimicrobial activity were identified phylogenetically based on 16S rRNA gene sequencing, which were assigned to five different actinomycete genera Streptomyces, Kocuria, Saccharomonospora, Micromonospora, and Nocardiopsis. Using PCR-based screening for six biosynthetic genes of secondary metabolites, all 45 isolates with acute activity have at least one biosynthetic gene, 28.8 % of which possess more than three biosynthetic genes. As a case, strain SMA-1 was selected for antimicrobial natural product discovery. Three diketopiperazine dimers including a new compound iso-naseseazine B (1) and two known compounds naseseazine B (2) and aspergilazine A (3) were isolated by bioassay-guided separation. These results suggested that actinomycetes from marine sediments are a potential resource of novel secondary metabolites and drugs.

Endophytes in the plant Huperzia serrata: fungal diversity and discovery of a new pentapeptide.

Endophytic fungi are an underexploited resource of natural products and have a capacity to produce diverse classes of plant-derived secondary metabolites. Here, we investigated the diversity of endophytic fungi from Huperzia serrata and the potential for discovering novel fungal natural products. One hundred and fifty-five endophytic fungi isolates obtained from H. serrata, belonging to four classes Dothideomycetes (47.3 %), Sordariomycetes (36.8 %), Eurotiomycetes (10.6 %) and an undefined class (5.3 %, Mucoraceae), were grouped into nine genera based on morphological and molecular identification. Colletotrichum, Cladosporium, Sordariomycetes and Guignardia were the dominant genera, whereas the remaining genera were infrequent groups. To our knowledge, the fungal genera Mucor and Neurospora were first reported in Huperzia plant. Among these endophytic fungi isolates, strain B14, belonging to Penicillium oxalicum, gave a gray precipitate with Dragendorff's reagent. A new pentapeptide was isolated from the culture of strain B14, and its chemical structure was elucidated on the basis of spectroscopic data from (1)H NMR, (13)C NMR and ESI-MS/MS. Taken together, H. serrata has a significant diversity of endophytic fungi that could be a rich resource for the discovery of new natural products.

Synthesis and insecticidal evaluation of novel N-pyridylpyrazolecarboxamides containing an amino acid methyl ester and their analogues.

On the basis of the commercial insecticide chlorantraniliprole, a series of novel N-pyridylpyrazolecarboxamides containing an amino acid methyl ester and their analogues were designed and synthesized. Their chemical structures were established on the basis of corresponding (1) H NMR spectroscopy, (13)C NMR spectroscopy, elemental analysis, and single-crystal X-ray diffraction analysis. The insecticidal activities of the new compounds against oriental armyworm ( Mythimna separata ) and diamondback moth ( Plutella xylostella ) were evaluated. The results of bioassays indicated that most of the compounds showed moderate to high activities at the tested concentration, of which the compounds 6 containing a methyl acrylate substructure had excellent larvicidal activity; for example, 6a displayed 100% larvicidal activity against P. xylostella at the concentration of 0.005 mg/L, whereas the activities of both compounds 6g and 6h against M. separata were 100% at 2.5 mg/L. The calcium imaging technique experiment results showed that novel compound 6 could elevate the calcium concentration in the cytoplasm. Furthermore, this study also provided evidence that compound 6h activates inositol 1,4,5-trisphosphate (IP3) sensitive intracellular calcium release channels in the endoplasmic reticulum of Spodoptera exigua third-instar larva neurons.

Synthesis and insecticidal evaluation of novel N-pyridylpyrazolecarboxamides containing cyano substituent in the ortho-position.

In an attempt to search for potent insecticides targeting the ryanodine receptor (RyR), a series of novel N-pyridylpyrazolecarboxamides containing cyano substituent in the ortho-position were designed and synthesized. Their insecticidal activities of target compounds against oriental armyworm (Mythimna separata) and diamondback moth (Plutella xylostella) indicated that most of the compounds showed moderate to high activities at the tested concentrations. In particular, compound 6l and 6o showed 86% larvicidal activities against Plutella xylostella at the concentration of 0.1mg/L, while the activity of compound 6h against Mythimna separate was 80% at 1mg/L. The calcium imaging technique was applied to investigate the effects of some title compounds on the intracellular calcium ion concentration ([Ca(2+)](i)), experimental results demonstrated that compound 6h stimulates a transient elevation in [Ca(2+)](i) in the absence of external calcium after the central neurons dye loading with fluo-3 AM. However, when the central neurons were dyed with fluo-5N and incubated with 2-APB, [Ca(2+)]i decreased transiently by treated of compound 6h. All of the calcium imaging technique experiments demonstrated that these novel compounds deliver calcium from endoplasmic reticulum to cytoplasm, which proved that the title compounds were the possible activators of insect RyR.

Establishment of a search library about benzylisoquinoline alkaloids based on selective separation on the binaphthyl column and standard analysis on C18 column.

A search library about benzylisoquinoline alkaloids was established based on preparation of alkaloid fractions from Rhizoma coptidis, Cortex phellodendri, and Rhizoma corydalis. In this work, two alkaloid fractions from each herbal medicine were first prepared based on selective separation on the "click" binaphthyl column. And then these alkaloid fractions were analyzed on C18 column by liquid chromatography coupled with tandem mass spectrometry. Many structure-related compounds were included in these alkaloids fractions, which led to easy separation and good MS response in further work. Therefore, a search library of 52 benzylisoquinoline alkaloids was established, which included eight aporphine, 19 tetrahydroprotoberberine, two protopine, two benzyltetrahydroisoquinoline, and 21 protoberberine alkaloids. The information of the search library contained compound names, structures, retention times, accurate masses, fragmentation pathways of benzylisoquionline alkaloids, and their sources from three herbal medicines. Using such a library, the alkaloids, especially those trace and unknown components in some herbal medicine could be accurately and quickly identified. In addition, the distribution of benzylisoquinoline alkaloids in the herbal medicines could be also summarized by searching the source samples in the library.

Preparation of a stationary phase with quaternary ammonium embedded group for selective separation of alkaloids based on ion-exclusion interaction.

A stationary phase (named QA C10) with quaternary ammonium embedded between a propyl and a decyl chain was synthesized by immobilization of N,N-dimethyldecylamine on chloropropyl-silica surface. A set of representative neutral, basic, and acidic compounds was employed to evaluate its chromatographic properties. The results illustrated that QA C10 was a mixed-mode stationary phase possessing both hydrophobic and ionic characteristics. The QA C10 stationary phase was further used for selective separation of alkaloids from Cortex phellodendri. Under acidic condition, alkaloids could be eluted in first 8 min, while other neutral and acidic fractions were retained better on QA C10 column. Then, obtained alkaloid fraction was analyzed by LC-MS/MS and 22 alkaloids were identified. Our study confirmed the advantages and application potential of the QA C10 stationary phase for alkaloids separation.

Selective separation of structure-related alkaloids in Rhizoma coptidis with "click" binaphthyl stationary phase and their structural elucidation with liquid chromatography-mass spectrometry.

It is a new task to separate structure-related compounds into a fraction according to their structural characteristics in a complex traditional Chinese medicine (TCM). This method makes separation of the components of the sample simple and structural elucidation easy. In this study, selective separation of alkaloids in Rhizoma coptidis was realized on a "click" binaphthyl column possessing a planar conjugate structure. Three kinds of alkaloids, aporphine, tetrahydroprotoberberine and protoberberine in Rhizoma coptidis showed better retention than other compounds by virtue of π-π interactions with the stationary phase. Moreover, the "click" binaphthyl column could distinguish the aporphine and tetrahydroprotoberberine alkaloids possessing two benzene rings from the protoberberine alkaloids possessing three benzene rings. After separating on the "click" binaphthyl column, the fractions containing the alkaloids were collected and then analyzed with liquid chromatography-mass spectrometry (LC-MS). Totally, 23 alkaloids were identified, and among these alkaloids, three tetrahydroprotoberberine, two aporphine and seven protoberberine alkaloids were first found in Rhizoma coptidis. These newly found alkaloids are minor compounds, and they are always neglected without eliminating the interference of compounds in large amounts by pre-separation on the "click" binaphthyl column. The typical fragmentation pathways of each class of alkaloids were summarized to illustrate their structures. In the MS(2) spectrum, the loss of a molecule of dimethylamine ((CH(3))(2)NH) was observed as the characteristic loss of aporphine alkaloids. All the tetrahydroprotoberberine alkaloids would undergo the Retro-Diels-Alder (RDA) fragmentation reaction in the MS(2) fragmentation. For protoberberine alkaloids, different characteristic fragmentations were observed with different skeleton structures.

Analysis of cephalosporins by hydrophilic interaction chromatography.

A simple hydrophilic interaction chromatography (HILIC) method was developed to analyze seven cephalosporins. These seven cephalosporins could be separated well on the Click ß-CD column and Atlantis HILIC Silica column. The effects of buffer concentration and pH on the retention under HILIC mode were studied. Except cefepime hydrochloride (4), the retention of other six cephalosporins increased with increasing buffer concentration, while decreased with increasing pH. Different separation selectivities could be observed on the Click ß-CD column and Atlantis HILIC Silica column, and changing pH also resulted in the changing of separation selectivity. The separations of cephalosporins by HILIC and reversed-phase high performance liquid chromatography (RP-HPLC) were compared, and the two separation modes had good orthogonality. In addition, cefotaxime sodium (1) and its degradation were separated well on the Click ß-CD column, which indicated that the Click ß-CD column by HILIC can be used for studying the stability of cephalosporins.

Enantioselective organocatalytic michael addition of malonates to alpha,beta-unsaturated ketones.

A novel type of primary amine thiourea organocatalysts derived from 1,2-diaminocyclohexane and 9-amino (9-deoxy) cinchona alkaloid was developed into asymmetric Michael addition of malonates to enones. A series of cyclic and acyclic enones could react very well with different malonates in the presence of 4 with 0.5-10 mol % catalyst loading affording chiral Michael adducts with excellent yields and ee values.