[Metabolic characteristics of active parts of lipid-lowering flavonoid extract of Daidai in liver and intestinal microsomes of rats].

The paper studies and compares the metabolic difference of active ingredients of lipid-lowering flavonoid extract of Daidai in rat livers and intestinal microsomes,in order to explore the phase Ⅰ metabolism characteristics of active ingredients in livers and intestines. UPLC-MS/MS was used to establish a quantitative analysis method for active ingredients,neohesperidin and narngin,in a phase Ⅰ metabolism incubation system of liver and intestinal microsomes. Differential centrifugation was used to make liver and intestinal microsomes of rats. A phase Ⅰ metabolism incubation system was established,and the concentrations of the residual at different incubation time points were analyzed. Graphs were plotted to calculate the metabolic elimination half-life of the main active parts,with the natural logarithm residual percentage values ln( X) at different time points as the y axis,and time t as the x axis. The metabolism characteristics of the active ingredients were compared. The established UPLC-MS/MS quantitative analysis method has a good specialization,standard curve and linear range,accuracy and precision,with a satisfactory lower quantitative limit. The method allows quantitative detection of the active ingredients in a phase Ⅰ metabolism incubation system of liver and intestinal microsomes of rats. In the rats liver microsomes incubation system,the metabolic elimination half-life of neohesperidin and narngin were( 2. 20 ± 0. 28) h and( 1. 97±0. 28) h respectively. The elimination half-life of neohesperidin was larger than that of narngin,but with no statistically significant difference. In the rats intestinal microsomes incubation system,the metabolic elimination half-lives of neohesperidin and narngin were( 3. 68±0. 54) h and( 2. 26±0. 13) h respectively. The elimination half-life of neohesperidin was larger than that of narngin,with statistically significant differences( P<0. 05). The elimination half-lives of the active ingredients in liver microsomes were smaller than those in intestinal microsomes. The experiment results showed that the active ingredients of lipid-lowering flavonoid extract of Daidai had different elimination half-lives in phase Ⅰ rats liver and intestinal microsomes incubation system. This implied that they had different metabolic characteristics in rats liver and intestine,and liver may be the main metabolism site of the active ingredients. The phaseⅠ metabolism of narngin was stronger than that of neohesperidin. The differences between their metabolic characteristics may be related to the binding sites of B-ring hydroxyl in flavonoid glycosides and the number of methoxyl group. The results provided an important experimental basis for further development and clinical application of lipid-lowering flavonoid extract preparation of Daidai.

[Binding characteristics of plasma protein in active parts of Daidai lipid-lowering flavonoid extract].

To study the binding capacity of active ingredients of Daidai lipid-lowering flavonoid extract and plasma protein,investigate the ways to improve the traditional formula for calculating protein binding rates based on ultrafiltration,and increase the stability and reliability of the experimental results. UPLC-MS/MS was used to establish a quantitative analysis method for simultaneous determination of active ingredients( neohesperidin and narngin) in ultrafiltrate. The protein binding rates were calculated by the traditional ultrafiltration formula. The correction factors( F) were introduced later,and the binding rates calculated with the correction factors were compared with those without the correction factors. The binding capacity of the extract and plasma protein was evaluated. The quantitative analysis method established by UPLC-MS/MS had a good specificity. The standard curve and linear range,method accuracy,precision and lower limit of quantitation all met the requirements. The method met the requirement for quantitative detection of the active ingredients in ultrafiltrate after the rat plasma was filtrated in the ultrafiltration tube. Under the experimental conditions,the binding rates of both active ingredients( neohesperidin and narngin) were higher than 90%. The active ingredients and rat plasma protein were bound in a concentration-dependent manner,with statistically significant differences( P<0. 01). There was no statistically significant difference between the protein binding abilities of the two active ingredients with rat plasma protein. Therefore,the active ingredients of Daidai lipid-lowering flavonoid extract had a relatively strong binding strength with rat plasma protein,and they were bound in a concentration-dependent manner. Additionally,when calculating protein binding rates by the traditional ultrafiltration formula,the correction factors could be introduced to effectively reflect the errors of multiple ingredient groups in traditional Chinese medicine extracts.This correction method could provide a reference thinking and practical reference for the improvement of the determination method of the traditional Chinese medicine plasma protein binding ability based on ultrafiltration.

High-throughput primer design by scoring in piecewise logistic model for multiple polymerase chain reaction variants.

Polymerase chain reaction (PCR) variants requiring specific primer types are widely used in various PCR experiments, including generic PCR, inverse PCR, anchored PCR, and ARMS PCR. Few tools can be adapted for multiple PCR variants, and many tools select primers by filtration based on the given parameters, which result in frequent design failures. Here we introduce PrimerScore2, a robust high-throughput primer design tool that can design primers in one click for multiple PCR variants. It scores primers using a piecewise logistic model and the highest-scored primers are selected avoiding the issue of design failure and the necessity to loosen parameters to redesign, and it creatively evaluates specificity by predicting the efficiencies of all target/non-target products. To assess the prediction accuracy of the scores and efficiencies, two next generation sequencing (NGS) libraries were constructed-a 12-plex and a 57-plex-and the results showed that 17 out of 19 (89.5%) low-scoring pairs had a poor depth, 18 out of 19 (94.7%) high-scoring pairs had a high depth, and the depth ratios of the products were linearly correlated with the predicted efficiencies with a slope of 1.025 and a coefficient of determination (R2) 0.935. 116-plex and 114-plex anchored PCR panels designed by PrimerScore2 were applied to 26 maternal plasma samples with male fetuses, the results showed that the predicted fetal DNA fractions were concordant with fractions measured in gold standard method (Y fractions). PrimerScore2 was also used to design 77 monoplex Sanger sequencing primers, the sequencing results indicated that all the primers were effective.

A Cell-free DNA Barcode-Enabled Single-Molecule Test for Noninvasive Prenatal Diagnosis of Monogenic Disorders: Application to ß-Thalassemia.

Noninvasive prenatal testing of common aneuploidies has become routine over the past decade, but testing of monogenic disorders remains a challenge in clinical implementation. Most recent studies have inherent limitations, such as complicated procedures, a lack of versatility, and the need for prior knowledge of parental genotypes or haplotypes. To overcome these limitations, a robust and versatile next-generation sequencing-based cell-free DNA (cfDNA) allelic molecule counting system termed cfDNA barcode-enabled single-molecule test (cfBEST) is developed for the noninvasive prenatal diagnosis (NIPD) of monogenic disorders. The accuracy of cfBEST is found to be comparable to that of droplet digital polymerase chain reaction (ddPCR) in detecting low-abundance mutations in cfDNA. The analytical validity of cfBEST is evidenced by a ß-thalassemia assay, in which a blind validation study of 143 at-risk pregnancies reveals a sensitivity of 99.19% and a specificity of 99.92% on allele detection. Because the validated cfBEST method can be used to detect maternal-fetal genotype combinations in cfDNA precisely and quantitatively, it holds the potential for the NIPD of human monogenic disorders.

The role of Mean Platelet Volume/platelet count Ratio and Neutrophil to Lymphocyte Ratio on the risk of Febrile Seizure.

Systemic inflammatory response has been implicated as a contributor to the onset of febrile seizures (FS). The four novel indices of the inflammatory response such as, neutrophil-to-lymphocyte ratio (NLR), mean platelet volume (MPV), platelet count (PLT) ratio and red blood cell distribution width (RDW) have been investigated in FS susceptibility and FS types (simple febrile seizure and complex febrile seizure). However, the potential role of these inflammatory markers and MPV/PLT ratio (MPR) in Chinese children with FS has yet to be fully determined. This study investigated the relevance of NLR, MPV, PLT, MPR and RDW in febrile children with and without seizures. 249 children with FS and 249 age matched controls were included in this study. NLR and MPR were calculated from complete blood cell counts prior to therapy. Differences in age, gender and these inflammatory markers between the FS group and the control group were evaluated using the chi-square test, t-test or logistic regression analysis. Receiver Operating Characteristic (ROC) curve was used to determine the optimal cut-off value of NLR and MPR for FS risk. Interactions between NLR and MPR on the additive scale were calculated by using the relative excess risk due to interaction (RERI), the proportion attributable to interaction (AP), and the synergy index (S). It has been shown that the elevated NLR and MPR levels were associated with increased risk of FS. The optimal cut-off values of NLR and MPR for FS risk were 1.13 and 0.0335 with an area under the curve (AUC) of 0.768 and 0.689, respectively. Additionally, a significant synergistic interaction between NLR and MPR was found on an additive scale. The mean levels of MPV were lower and NLR levels were higher in complex febrile seizure (CFS) than simple febrile seizure (SFS), and the differences were statistically significant. ROC analysis showed that the optimal cut-off value for NLR was 2.549 with 65.9% sensitivity and 57.5% specificity. However, no statistically significant differences were found regarding average values of MPR and RDW between CFS and SFS. In conclusion, elevated NLR and MPR add evidence to the implication of white cells subsets in FS risk, and our results confirmed that NLR is an independent, albeit limited, predictor in differentiating between CFS and SFS. Moreover, NLR and MPR may have a synergistic effect that can influence the occurrence of FS.

Isolation of a new flavanone from Daidai fruit and hypolipidemic activity of total flavonoids extracts.

In order to further discover the medicinal value of Daidai fruit, an exploration on the hypolipidemic activity of total flavonoids extracts of Daidai fruit (TFEODF) was conducted in high-fat diet-induced hyperlipemia rats. Results indicated that TFEODF exhibited significant hypolipidemic activity which resulted in the decline of serum total cholesterol, triglyceride, low density lipoprotein cholesterol, arteriosclerosis index and rise of high density lipoprotein cholesterol in hyperlipemia rats. For the purpose of expounding the chemical constituents of TFEODF, a phytochemical investigation of TFEODF was carried out for the first time. Research resulted in the isolation of a new compound together with 17 known compounds. This study lay a foundation for the development of a new hypolipidemic agent of traditional Chinese medicine whose chemical constituents were clarified.

Construction and analysis of high-density linkage map using high-throughput sequencing data.

Linkage maps enable the study of important biological questions. The construction of high-density linkage maps appears more feasible since the advent of next-generation sequencing (NGS), which eases SNP discovery and high-throughput genotyping of large population. However, the marker number explosion and genotyping errors from NGS data challenge the computational efficiency and linkage map quality of linkage study methods. Here we report the HighMap method for constructing high-density linkage maps from NGS data. HighMap employs an iterative ordering and error correction strategy based on a k-nearest neighbor algorithm and a Monte Carlo multipoint maximum likelihood algorithm. Simulation study shows HighMap can create a linkage map with three times as many markers as ordering-only methods while offering more accurate marker orders and stable genetic distances. Using HighMap, we constructed a common carp linkage map with 10,004 markers. The singleton rate was less than one-ninth of that generated by JoinMap4.1. Its total map distance was 5,908 cM, consistent with reports on low-density maps. HighMap is an efficient method for constructing high-density, high-quality linkage maps from high-throughput population NGS data. It will facilitate genome assembling, comparative genomic analysis, and QTL studies. HighMap is available at http://highmap.biomarker.com.cn/.

SLAF-seq: an efficient method of large-scale de novo SNP discovery and genotyping using high-throughput sequencing.

Large-scale genotyping plays an important role in genetic association studies. It has provided new opportunities for gene discovery, especially when combined with high-throughput sequencing technologies. Here, we report an efficient solution for large-scale genotyping. We call it specific-locus amplified fragment sequencing (SLAF-seq). SLAF-seq technology has several distinguishing characteristics: i) deep sequencing to ensure genotyping accuracy; ii) reduced representation strategy to reduce sequencing costs; iii) pre-designed reduced representation scheme to optimize marker efficiency; and iv) double barcode system for large populations. In this study, we tested the efficiency of SLAF-seq on rice and soybean data. Both sets of results showed strong consistency between predicted and practical SLAFs and considerable genotyping accuracy. We also report the highest density genetic map yet created for any organism without a reference genome sequence, common carp in this case, using SLAF-seq data. We detected 50,530 high-quality SLAFs with 13,291 SNPs genotyped in 211 individual carp. The genetic map contained 5,885 markers with 0.68 cM intervals on average. A comparative genomics study between common carp genetic map and zebrafish genome sequence map showed high-quality SLAF-seq genotyping results. SLAF-seq provides a high-resolution strategy for large-scale genotyping and can be generally applicable to various species and populations.

Draft genome of the kiwifruit Actinidia chinensis.

The kiwifruit (Actinidia chinensis) is an economically and nutritionally important fruit crop with remarkably high vitamin C content. Here we report the draft genome sequence of a heterozygous kiwifruit, assembled from ~140-fold next-generation sequencing data. The assembled genome has a total length of 616.1 Mb and contains 39,040 genes. Comparative genomic analysis reveals that the kiwifruit has undergone an ancient hexaploidization event (γ) shared by core eudicots and two more recent whole-genome duplication events. Both recent duplication events occurred after the divergence of kiwifruit from tomato and potato and have contributed to the neofunctionalization of genes involved in regulating important kiwifruit characteristics, such as fruit vitamin C, flavonoid and carotenoid metabolism. As the first sequenced species in the Ericales, the kiwifruit genome sequence provides a valuable resource not only for biological discovery and crop improvement but also for evolutionary and comparative genomics analysis, particularly in the asterid lineage.