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Spatial transcriptomics technologies permit the study of the spatial distribution of RNA at near-single-cell resolution genome-wide. However, the feasibility of studying spatial allele-specific expression (ASE) from these data remains uncharacterized. Here, we introduce spASE, a computational framework for detecting and estimating spatial ASE. To tackle the challenges presented by cell type mixtures and a low signal to noise ratio, we implement a hierarchical model involving additive mixtures of spatial smoothing splines. We apply our method to allele-resolved Visium and Slide-seq from the mouse cerebellum and hippocampus and report new insight into the landscape of spatial and cell type-specific ASE therein., (© 2024. The Author(s).)

A method based on ultra-high performance liquid chromatography coupled with triple quadrupole linear ion trap-tandem mass spectrometry(UHPLC-QTRAP-MS/MS) was developed for the simultaneous determination of 41 bioactive constituents of flavonoids, organic acids, nucleosides, and amino acids in Lysimachiae Herba. The content of multiple bioactive constituents was compared among the samples from different habitats. The chromatographic separation was performed in a Waters XBridge®C_(18) column(4.6 mm×100 mm, 3.5 μm) at 30 ℃. The gradient elution was performed with 0.4% methanol(A)-formic acid water(B) as the mobile phase at a flow rate of 0.8 mL·min~(-1), and the multiple-reaction monitoring(MRM) mode was adopted. According to the content of 41 constituents, hierarchical cluster analysis(HCA), orthogonal partial least squares discriminant analysis(OPLS-DA), and gray relational analysis(GRA) were perfomed to comprehensively evaluate the samples from different habitats. The results showed that the 41 constituents exhibited good linear relationship within the tested concentration ranges, with the correlation coefficients(r) greater than 0.999 4. The method featured good precision, repeatability, and stability with the relative standard deviations(RSDs) less than 5.0%. The average recoveries of the 41 constituents ranged from 98.06% to 101.9%, with the RSDs of 0.62%-4.6%. HCA and OPLS-DA separated 48 batches of Lysimachiae Herba samples from different habitats into three categories: the producing areas in Sichuan and Chongqing, the producing areas in Jiangsu, Zhejiang, and Jiangxi, and the producing areas in Guizhou. The content of 41 constituents varied among the Lysimachiae Herba samples from different habitats. The GRA results revealed that the Lysimachiae Herba sample from Nanchong City, Sichuan Province had the best comprehensive quality. The method developed in this study was accurate and reliable and thus can be used for comprehensive evaluation of Lysimachiae Herba quality and provide basic information for the selection of habitats.

A comprehensive analytical method based on ultra-fast liquid chromatography coupled with triple quadrupole/linear ion trap tandem mass spectrometry(UFLC-QTRAP-MS/MS) was established for simultaneous determination of the content of 45 bioactive constituents including flavonoids, alkaloids, amino acids, phenolic acids, and nucleosides in Epimedium brevicornum. The multiple bioactive constituents in leaves, petioles, stems and rhizomes of E. brevicornum were analyzed. The gradient elution was performed at 30 ℃ in an XBridge~® C_(18) column(4.6 mm×100 mm, 3.5 μm) with 0.4% formic acid aqueous solution-acetonitrile as the mobile phase at a flow rate of 0.8 mL·min~(-1). Single factor experiment and response surface methodology were employed to optimize the extraction conditions. Multivariate statistical analyses including systematic cluster analysis(SCA), principal component analysis(PCA), partial least squares discriminant analysis(PLS-DA), and one-way analysis of variance(One-way ANOVA) were carried out to classify the samples from different parts and identify different constituents. Grey relation analysis(GRA) and entropy weight-TOPSIS analysis were performed to build a multi-index comprehensive evaluation model for different parts of E. brevicornum. The results showed that there was a good relationship between the mass concentrations of 45 constituents and the corresponding peak areas, with the correlation coefficients(r) not less than 0.999 0. The precision, repeatability, and stability of the established method were good for all the target constituents in this study, with the relative standard deviations(RSDs) less than 5.0%(0.62%-4.9%) and the average recovery of 94.51%-105.7%. The above results indicated that the bioactive constituents varied in different parts of E. brevicornum, and the overall quality followed the trend of leaves > petioles > rhizomes > stems. This study verified the rationality of the Chinese Pharmacopoeia(2020 edition) stipulating that the medicinal part of E. brevicornum is the leaf. Moreover, our study indicated that the rhizome had the potential for medicinal development. The established method was accurate and reliable, which can be used to comprehensive evaluate and control the quality of E. brevicornum. This study provides data reference for clarifying the medicinal parts and rationally utilizing the resources of E. brevicornum.

A central problem in spatial transcriptomics is detecting differentially expressed (DE) genes within cell types across tissue context. Challenges to learning DE include changing cell type composition across space and measurement pixels detecting transcripts from multiple cell types. Here, we introduce a statistical method, cell type-specific inference of differential expression (C-SIDE), that identifies cell type-specific DE in spatial transcriptomics, accounting for localization of other cell types. We model gene expression as an additive mixture across cell types of log-linear cell type-specific expression functions. C-SIDE's framework applies to many contexts: DE due to pathology, anatomical regions, cell-to-cell interactions and cellular microenvironment. Furthermore, C-SIDE enables statistical inference across multiple/replicates. Simulations and validation experiments on Slide-seq, MERFISH and Visium datasets demonstrate that C-SIDE accurately identifies DE with valid uncertainty quantification. Last, we apply C-SIDE to identify plaque-dependent immune activity in Alzheimer's disease and cellular interactions between tumor and immune cells. We distribute C-SIDE within the R package https://github.com/dmcable/spacexr ., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

A limitation of spatial transcriptomics technologies is that individual measurements may contain contributions from multiple cells, hindering the discovery of cell-type-specific spatial patterns of localization and expression. Here, we develop robust cell type decomposition (RCTD), a computational method that leverages cell type profiles learned from single-cell RNA-seq to decompose cell type mixtures while correcting for differences across sequencing technologies. We demonstrate the ability of RCTD to detect mixtures and identify cell types on simulated datasets. Furthermore, RCTD accurately reproduces known cell type and subtype localization patterns in Slide-seq and Visium datasets of the mouse brain. Finally, we show how RCTD's recovery of cell type localization enables the discovery of genes within a cell type whose expression depends on spatial environment. Spatial mapping of cell types with RCTD enables the spatial components of cellular identity to be defined, uncovering new principles of cellular organization in biological tissue. RCTD is publicly available as an open-source R package at https://github.com/dmcable/RCTD ., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Taxilli Herba (TAXH) is an important traditional Chinese medicine with a long history, dating from the Eastern Han Dynasty to the present times. However, the active constituents in it that parasitize different hosts vary, affecting its clinical efficacy. Given the complexity of the host origins, evaluating the quality of TAXH is critical to ensure the safety and effectiveness of clinical medication. In the present study, a quantitative method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was established, which simultaneously determined the content of 33 active constituents, including 12 flavonoids, 4 organic acids, 12 amino acids, and 5 nucleosides in 45 samples. Orthogonal partial least squares discriminant analysis (OPLS-DA) was employed to classify and distinguish between TAXH and its adulterants, Tolypanthi Herba (TOLH). A hierarchical clustering analysis (HCA) was conducted combined with a heatmap to visually observe the distribution regularity of 33 constituents in each sample. Furthermore, gray relational analysis (GRA) was applied to evaluate the quality of samples to get the optimal host. The results demonstrated that TAXH excelled TOLH in quality as a whole. The quality of TAXH parasitizing Morus alba was also better, while those that were parasitic on Cinnamomum camphora and Glyptostrobus pensilis had relatively poor quality. This study may provide comprehensive information that is necessary for quality control and supply a scientific basis for further exploring the quality formation mechanism of TAXH.

Epigenetic editing is an emerging technology that uses artificial transcription factors (aTFs) to regulate expression of a target gene. Although human genes can be robustly upregulated by targeting aTFs to promoters, the activation induced by directing aTFs to distal transcriptional enhancers is substantially less robust and consistent. Here we show that long-range activation using CRISPR-based aTFs in human cells can be made more efficient and reliable by concurrently targeting an aTF to the target gene promoter. We used this strategy to direct target gene choice for enhancers capable of regulating more than one promoter and to achieve allele-selective activation of human genes by targeting aTFs to single-nucleotide polymorphisms embedded in distally located sequences. Our results broaden the potential applications of the epigenetic editing toolbox for research and therapeutics., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

A comprehensive analytical method based on ultra-fast liquid chromatography coupled with triple quadrupole/linear ion trap tandem mass spectrometry(UFLC-QTRAP-MS/MS) was established for simultaneous determination of the content of 38 active components in Abelmoschi Corolla, including flavonoids, organic acids, nucleosides and amino acids, so as to investigate the effects of different harvesting and processing methods on multi-active components in Abelmoschi Corolla. The chromatographic separation was performed on a XBridg®C_(18) column(4.6 mm×100 mm, 3.5 μm) with(0.1% formic acid water) methanol-acetonitrile(1∶1) as the mobile phase for gradient elution at 30 ℃. The flow rate was 0.5 mL·min~(-1). The components were detected in a multiple-reaction monitoring(MRM) mode. The gray relational analysis(GRA) was used to comprehensively evaluate the multiple active components of Abelmoschi Corolla at different harvesting times and drying temperatures. The results showed that 38 components had a good linearity with correlation coefficients all above 0.999 0. The method featured a good precision, repeatability and stability with the relative stan-dard deviations(RSDs) of less than 5.0%. Recoveries ranged from 98.06% to 104.4% with RSD between 0.22% and 4.9%. The results of GRA indicated that a better quality in the samples collected on September 9 th. Samples dried at 90 ℃ had a better quality. The established method is accurate and reliable, and can be used to assess the internal quality of Abelmoschi Corolla. This study can provide basic materials for determining appropriate harvesting time and processing method of Abelmoschi Corolla.

Widespread changes to DNA methylation and chromatin are well documented in cancer, but the fate of higher-order chromosomal structure remains obscure. Here we integrated topological maps for colon tumors and normal colons with epigenetic, transcriptional, and imaging data to characterize alterations to chromatin loops, topologically associated domains, and large-scale compartments. We found that spatial partitioning of the open and closed genome compartments is profoundly compromised in tumors. This reorganization is accompanied by compartment-specific hypomethylation and chromatin changes. Additionally, we identify a compartment at the interface between the canonical A and B compartments that is reorganized in tumors. Remarkably, similar shifts were evident in non-malignant cells that have accumulated excess divisions. Our analyses suggest that these topological changes repress stemness and invasion programs while inducing anti-tumor immunity genes and may therefore restrain malignant progression. Our findings call into question the conventional view that tumor-associated epigenomic alterations are primarily oncogenic., Competing Interests: Declaration of Interests N.H. is an equity holder of BioNTech and a consultant for Related Sciences. M.J.A. declares outside interest in Excelsior Genomics. B.E.B. declares outside interests in Fulcrum Therapeutics, 1CellBio, HiFiBio, Arsenal Biosciences, Cell Signaling Technologies, BioMillenia, and Nohla Therapeutics., (Copyright © 2020 Elsevier Inc. All rights reserved.)

A method was established for simultaneous determination of 21 active constituents including flavanols, isoflavones, flavonols, dihydroflavones, dihydroflavonols, chalcones, pterocarpan, anthocyanidins and phenolic acids in Spatholobi Caulis by ultra fast liquid chromatography with triple quadrupole linear ion trap mass spectrometry(UFLC-QTRAP-MS/MS). Then, it was employed to analyze and evaluate the dynamic accumulation of multiple bioactive constituents in Spatholobi Caulis. The chromatographic separation was performed on a XBridge®C_(18)(4.6 mm×100 mm, 3.5 μm) at 30 ℃ with a gradient elution of 0.3% formic acid aqueous solution-methanol, and the flow rate was 0.8 mL·min~(-1), using multiple-reaction monitoring(MRM) mode. A comprehensive evaluation of the multiple bioactive constituents was carried out by gray correlation analysis(GRA). The 21 target components showed good linearity(r>0.999 0) in the range of the tested concentrations. The average recovery rates of the 21 components were from 97.46% to 103.6% with relative standard deviations less than 5.0%. There were differences in the contents of 21 components in Spatholobi Caulis at diffe-rent harvest periods. Spatholobi Caulis had high quality from early November to early December, which is consistent with the local tradi-tional harvest period. This study reveals the rule of the dynamic accumulation of 21 components in Spatholobi Caulis and provides basic information for the suitable harvest time. At the same time, it provides a new method reference for the comprehensive evaluation of the internal quality of Spatholobi Caulis.

Objective: Type 2 diabetes (T2D) is a complex disease characterized by pancreatic islet dysfunction, insulin resistance, and disruption of blood glucose levels. Genome-wide association studies (GWAS) have identified > 400 independent signals that encode genetic predisposition. More than 90% of associated single-nucleotide polymorphisms (SNPs) localize to non-coding regions and are enriched in chromatin-defined islet enhancer elements, indicating a strong transcriptional regulatory component to disease susceptibility. Pancreatic islets are a mixture of cell types that express distinct hormonal programs, so each cell type may contribute differentially to the underlying regulatory processes that modulate T2D-associated transcriptional circuits. Existing chromatin profiling methods such as ATAC-seq and DNase-seq, applied to islets in bulk, produce aggregate profiles that mask important cellular and regulatory heterogeneity., Methods: We present genome-wide single-cell chromatin accessibility profiles in >1,600 cells derived from a human pancreatic islet sample using single-cell combinatorial indexing ATAC-seq (sci-ATAC-seq). We also developed a deep learning model based on U-Net architecture to accurately predict open chromatin peak calls in rare cell populations., Results: We show that sci-ATAC-seq profiles allow us to deconvolve alpha, beta, and delta cell populations and identify cell-type-specific regulatory signatures underlying T2D. Particularly, T2D GWAS SNPs are significantly enriched in beta cell-specific and across cell-type shared islet open chromatin, but not in alpha or delta cell-specific open chromatin. We also demonstrate, using less abundant delta cells, that deep learning models can improve signal recovery and feature reconstruction of rarer cell populations. Finally, we use co-accessibility measures to nominate the cell-specific target genes at 104 non-coding T2D GWAS signals., Conclusions: Collectively, we identify the islet cell type of action across genetic signals of T2D predisposition and provide higher-resolution mechanistic insights into genetically encoded risk pathways., (Published by Elsevier GmbH.)

The analysis of Forsythia suspensa was performed on Waters Symmetry C 18 column( 4. 6 mm×250 mm,5 μm) and mobile phase was methanol( A)-0. 1% formic acid aqueous solution( B) with the elution gradient. Column temperature was maintained at 30℃,and the flow rate was 1. 0 m L·min -1 with detection wavelength 265 nm. The HPLC-PDA fingerprint of F. suspensa was optimized.Chemical constituents in F. suspensa were analyzed by UFLC-Q-TOF-MS in positive and negative ion mode. The quality of 48 batches of F. suspensa from different habitats,processing methods and specifications was evaluated by similarity evaluation and cluster analysis.The 18 common peaks were confirmed. The similarity of F. suspensa from different habitats was more than 0. 98,and 56 chemical constituents were identified. Different processing methods had great influence on the quality of F. suspensa. Compared with boiled and direct drying,the quality of F. suspensa processed by sun-drying was obviously decreased. The similarity was about 0. 58. Different specifications of F. suspensa also had obvious distinction,and the similarity was about 0. 78. The effective components of grown F. suspensa,such as forsythoside A and phillyrin,were significantly reduced. The results of cluster analysis were basically consistent with the results of similarity evaluation. The establishment of fingerprint and the recognition of chemical pattern of F. suspensa can provide a more comprehensive reference for the quality control of herbs.

A method, for determination of saponins, amino acids and nucleosides in Panacis Japonici Rhizoma of ultra fast liquid chromatography with triple quadrupole linear ion trap mass spectrometry (UFLC-QTRAP-MS/MS), was established to investigate the effect of different processing methods on the target components of Panacis Japonici Rhizoma. The chromatographic separation was performed on a XBridgeC₁₈(4.6 mm×100 mm, 3.5 μm) at 30 °C with a gradient elution of 0.1% formic acid solution-0.1% formic acid acetonitrile, and the flow rate was 0.8 mL·min⁻¹, using multiple-reaction monitoring (MRM) mode. The grey relational analysis was adopted for the analysis of different processing samples. The results showed that the thirty-three constituents were in a good linear range and the correlation coefficient was greater than 0.999 0; the precision, repeatability and stability were good; the average recovery rates were between 95.33% and 101.8%, and the relative standard deviations were less than 5%. The result of grey relational analysis showed that the complete rhizomes without peeling, which were adopted for the microwave dried method, had the best quality. The established method was accurate and reliable, which could be used to appraise the quality of Panacis Japonici Rhizoma. Our study may lay the way for the processing method of Panacis Japonici Rhizoma in optimization,normalization and standardization., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

An analytical method based on UFLC-QTRAP-MS/MS was established for simultaneous determination of thirty-three components including steroidal saponins, homoisoflavonoids, amino acids and nucleosides in Ophiopogonis Radix. Thirty-three target components of commercial medicinal materials of Maidong were comparative analysis. Synergi™ Hydro-RP 100 column (2.0 mm × 100 mm, 2.5 μm) was used with 0.1% formic acid solution-0.1% formic acid acetonitrile for gradient elution at a flow rate of 0.4 mL·min⁻¹. In addition, multiple reaction monitoring (MRM) mode was employed. The data were comprehensively processed and analyzed with hierarchical clustering analysis(HCA), principal component analysis(PCA) and partial least squares discriminant analysis(PLS-DA) methods. All components showed good linearity( r >0.999 0) within the tested ranges. The average recoveries were between 96.23%-102.0%, and the relative standard deviation(RSD) were less than 5%. The results showed that there were significant differences in components between Ophiopogonis Radix and Liriopes Radix, with seven components obviously different. This method was useful for providing basis for the comprehensive evaluation and intrinsic quality control of Ophiopogonis Radix and Liriopes Radix , and may provide a new method reference for the identification of Ophiopogonis Radix and Liriopes Radix., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Dao-di herbs have been recognized as "quality models" with a firmly stable status. The formation of Dao-di herbs quality is involved from the genetic inheritance on the molecular level to the metabolic phenotype of final products, and the full material-based biosynthetic pathway remains unknown. In recent years, an increasing variety of omics technologies has provided new methods and ideas for the analysis of complex life systems and are suitable for explanation of quality formation in Dao-di herbs as well. In order to alleviate the scarcity of natural resources and offer scientific guidance of transplanting varieties, achievements of omics in the aspects of Dao-di herbs from genetics to phenotyping, the biosynthetic pathway of secondary metabolites, the interaction with human body and the new methods of quality evaluation have been summarized. It will be a fundamental work for protection and utilization of Chinese medicine resources., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Background: Bisulfite sequencing is widely employed to study the role of DNA methylation in disease; however, the data suffer from biases due to coverage depth variability. Imputation of methylation values at low-coverage sites may mitigate these biases while also identifying important genomic features associated with predictive power., Results: Here we describe BoostMe, a method for imputing low-quality DNA methylation estimates within whole-genome bisulfite sequencing (WGBS) data. BoostMe uses a gradient boosting algorithm, XGBoost, and leverages information from multiple samples for prediction. We find that BoostMe outperforms existing algorithms in speed and accuracy when applied to WGBS of human tissues. Furthermore, we show that imputation improves concordance between WGBS and the MethylationEPIC array at low WGBS depth, suggesting improved WGBS accuracy after imputation., Conclusions: Our findings support the use of BoostMe as a preprocessing step for WGBS analysis.

An analytical method based on UFLC-QTRAP-MS/MS was developed for simultaneous determination of fifteen components including eleven lignans (schizantherin B, schisandrol B, schizandrin C, γ-schisandrin, deoxyschizandrin, schisantherin, schisandrin, schisanhenol, gomisin D, gomisin J, and angeloylgomisin H) and organic acids (S)-malic acid, D(-)-tartaric acid, protocatechuic acid, and quinic acid) in Schisandrae Chinensis Fructus. Samples from different product specifications were evaluated and analyzed. The chromatographic separation was performed on a Synergi™ Hydro-RP 100Å column (2.0 mm×100 mm, 2.5 μm) at 40 °C with a gradient elution by employing 0.1% aqueous formic acid (A)-acetonitrile (B) as the mobile phase, and the flow rate was 0.4 mL·min⁻¹, using an electrospray ionization (ESI) source and multiple reaction monitoring (MRM) mode. Fifteen components were evaluated synthetically by TOPSIS and gray related degree. The results showed that fifteen components had good linearity (r>0.999 90), and the limits of detection were all satisfactory. The average recoveries of standard addition for the compounds were between 95.42 % and 98.86 %, and the relative standard deviations were less than 5%. The greatest difference of ri in grey related degree was 58.1%, whilst the greatest difference of Ci value in TOPSIS method was 94.8%. The results of these two methods showed that the holistic quality of No. 14 sample was the best. The developed method was accurate and reliable, which was suitable for the simultaneous determination of multiple functional substances and able to provide a new basis for the comprehensive assessment and overall control of the quality of Schisandrae Chinensis Fructus., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

Site-specific accumulation of flavonoids in Apocyni Veneti Folium was determined by laser scanning confocal microscope (LSCM) and the localization of catechins also was observed via vanillin-HCl staining under the conventional optical microscope. The contents of five flavonoids in Apocyni Veneti Folium from different harvest times and growth parts were measured using HPLC method. LSCM observation showed that flavonoids are accumulated in cuticle of epidermal cells and vessel walls, especially in protoplasts and nucleolus of the collenchyma cells and the epidermal cells. Catechins are localized in the palisade parenchyma cells and vessel walls, particularly in the laticifers found in the phloem. On the basis of the difference of the maximal emission wavelength between quercetin and kaempferol derivatives which have fluorescence behavior by appropriate treatment, kaempferol and its derivatives are localized exclusively in the cuticle. Results showed that the content of astragalin in Apocyni Veneti Folium from different parts revealed the decreasing trend, while hyperin and isoquercitrin were higher in June and July analyzed by HPLC. In summary, the site-specific accumulation of flavonoids in Apocyni Veneti Folium can be determined by LSCM and vanillin-HCl staining. The contents of flavonoids in Apocyni Veneti Folium are correlated with harvest times and growth parts., (© 2017 Wiley Periodicals, Inc.)

Objective and Design: The goal of the study was to determine whether the mutational profile of early colorectal polyps correlated with growth behaviour. The growth of small polyps (6-9 mm) that were first identified during routine screening of patients was monitored over time by interval imaging with CT colonography. Mutations in these lesions with known growth rates were identified by targeted next-generation sequencing. The timing of mutational events was estimated using computer modelling and statistical inference considering several parameters including allele frequency and fitness., Results: The mutational landscape of small polyps is varied both within individual polyps and among the group as a whole but no single alteration was correlated with growth behaviour. Polyps carried 0-3 pathogenic mutations with the most frequent being in APC , KRAS/NRAS , BRAF , FBXW7 and TP53 . In polyps with two or more pathogenic mutations, allele frequencies were often variable, indicating the presence of multiple populations within a single tumour. Based on computer modelling, detectable mutations occurred at a mean polyp size of 30±35 crypts, well before the tumour is of a clinically detectable size., Conclusions: These data indicate that small colon polyps can have multiple pathogenic mutations in crucial driver genes that arise early in the existence of a tumour. Understanding the molecular pathway of tumourigenesis and clonal evolution in polyps that are at risk for progressing to invasive cancers will allow us to begin to better predict which polyps are more likely to progress into adenocarcinomas and which patients are at greater risk of developing advanced disease., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.)

Caveolin-1 is a major component protein of the caveolae-a type of flask shaped, 50-100 nm, nonclathrin-coated, microdomain present in the plasma membrane of most mammalian cells. Caveolin-1 functions as a scaffolding protein to organize and concentrate signaling molecules within the caveolae, which may be associated with its unique physicochemical properties including oligomerization, acquisition of detergent insolubility, and association with cholesterol. Here we demonstrate that caveolin-1 is detected in all brain areas examined and recovered in both detergent-soluble and -insoluble fractions. Surprisingly, the recovered molecules from the two different fractions share a similar molecular size ranging from 200 to 2,000 kDa, indicated by gel filtration. Furthermore, both soluble and insoluble caveolin-1 molecules generate a proteinase K (PK)-resistant C-terminal core fragment upon the PK-treatment, by removing ˜36 amino acids from the N-terminus of the protein. Although it recognizes caveolin-1 from A431 cell lysate, an antibody against the C-terminus of caveolin-1 fails to detect the brain protein by Western blotting, suggesting that the epitope in the brain caveolin-1 is concealed. No significant differences in the physicochemical properties of caveolin-1 between uninfected and prion-infected brains are observed., Competing Interests: CONFLICTS OF INTEREST There is no conflict of interest.

In order to study the influence of ecological environment regarding the synthesis and accumulation of metabolites in Eucommiae Cortex, LC-QTOF MS/MS method combined with multivariate statistical analysis was used to analyze the differences of chemical constituents in Eucommiae Cortex from different habitats. Through the analysis of the multistage tandem mass spectrometry, the characteristic peaks were extracted with mass spectrometry data peak matching, peak alignment, and noise filtering. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were used for data processing. The chemical constituents were identified or tentative presumed according to MS accurate mass and MS/MS spectrometry fragmentation information, combined with the software of database search, comparison with reference standards and literature. The results show the differences among samples of Eucommiae Cortex from different habitats are distinguishable. A total of 23 chemical constituents in Eucommiae Cortex were identified or tentative presumed. Among of them, 14 kinds of common differential chemical constituents (aucubin, geniposidic acid, neochlorogenic acid, syringin, olivil-4',4'-di-O-β-D-glucopyranoside, chlorogenic acid, cryptochlorogenic acid, 1-hydroxypinoresinol- 4',4'-di-O-β-D-glucopyranoside, caffeic acid, pinoresinol-di-O-β-D-glucopyranoside, syringaresionl-di-O-β-D-glucopyranoside, pinoresinol-4'-O-β-D-glucopyranoside, eucommiol, isochlorogenic acid C and asiatic acid) presented different changing laws. This study provides basic information for revealing the influence law of ecological environment on the biosynthesis of metabolites in Eucommiae Cortex., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

To explore rapidly the potential chemical markers for differentiating Pseudostellariae Radix from different cultivated fields :and germplasms, a method is proposed based on ultra-performance liquid chromatographytriple time-of-flight mass/mass spectrometry (UPLC-Triple TOF-MS/MS) coupled with multivariate statistical analysis. Peak matching, peak alignment, and noise filtering were used in analyzing mass spectrometric data. Accurate m/z value analysis of MS data based on software of database search and MS/MS fragment analysis were applied to identify constituents. The obtained. data were statistically analyzed with hierarchical cluster analysis (HCA), principal component analysis (PCA), and partial least squared-discriminant analysis (PLS-DA) to compare the differences among these samples. The PLS-DA loading plot obtained from all mass data showed that 21 compounds were identified as the potential chemical markers to characterize the samples. The results provide experimental data to reveal the influence of ecological environments and germplasms on metabolite biosynthesis in Pseudostellariae Radix.

Objective: To analyze the correlation between index components and climatic factors of Pseudostellariae Radix., Methods: Correlation analysis ( CA), partial least square (PLS) and PCA ordination were applied to analyze the correlation of index components in Pseudostellariae Radix and climatic factors, and to explore the main climatic factors affecting the accumulation of index components in Pseudostellariae Radix., Results: The origin and harvest time had influence on index components. The monthly minimum temperature, monthly average humidity, monthly precipitation and the annual precipitation were the dominant factors influencing the content of index components., Conclusion: Through the positive and negative correlation between climatic factors and index components, it could explain the rationality of traditional harvest time and the reason of high content of heterophyllin B in Pseudostellariae Radix from Jiangsu Jurong.

This work was designed to investigate proteins differentially expressed in cultivated Pseudostellaria heterophylla and its wild type using i TRAQ proteomics approach. The extracted proteins were digested using FASP method and identified by i TRAQ coupled with LC-MS/MS technology and then analyzed by Protein Pilot 5.0 search engine. Proteins differentially expressed were searched through comparison of relatively quantified proteins. The analysis was conducted using GO(gene ontology), KEGG and STRING. A total of 3 775 proteins were detected, among them, 3 676 proteins can be quantified, of which 127 proteins were up-regulated and 205 were down-regulated in cultivated Pseudostellaria heterophylla. We found 71 significantly differentially expressed proteins for further analysis. These proteins were classified into nine categories: heat shock proteins, transferases, oxidoreductases, lyases, isomerases, ligases, hydrolases, tubulin and translocases. The results indicated that the carbohydrate and cellular amino acids metabolism of cultivated Pseudostellaria heterophylla were weaker than its wild type and its ability of responding to stress was much stronger. GWD1, PHS1, GBE1, PGM, and BAM1 are the important proteins to regulate sucrose; met E and CYS are the key proteins that regulate amino acids in cultivated and wild Pseudostellaria heterophylla. This will provide the basic information for exploring the cause of secondary metabolites differences in different ecotype of Pseudostellaria heterophylla and the protein mechanism of its quality formation.

Objective: To analyze nucleosides and nucleobases of Pseudostellariae Radix cultivated in different idibplasni resources and to compare the differences., Methods: QTRAP LC-MS/MS method was applied for the analysis of 13 kinds of nucleosides and nucleobases in Pseudostellariae Radix and the data obtained was analyzed by SPSS 16. 0 software., Results: There were some differences between Pseudostellariae Radix cultivated in different idioplasm resources. The highest amount of nucleosides and nucleobases was ZS2 which came from Zherong in Fujian Province. The total content of nucleosides and nucleobases in the sample from Shibing in Guizhou Province was the lowest. There was little difference between ZS1 (Zherong in Fujian Province) and XC(Xuancheng in Anhui Province)., Conclusion: This study provides evidence for the influence of eco-environment on the metabolites of Pseudostellariae Radix.

Background: To explore the expression of DcR3 protein and its clinicopathological significance in bladder urothelial carcinomas (BUC)., Materials and Methods: Immunohistochemistry was performed to detect the expression of DcR3, caspase-3, Bcl-2, VEGF, Ki-67, PCNA and P53 in 166 BUC and 56 normal bladder tissues. Western blotting was used to detect the expression of DcR3 in the supernatants of cultured BUC cells., Results: Overexpression of DcR3 was found in BUC tissues and cell lines, with significant elevation as compared to normal bladder tissues (p<0.0001). Higher DcR3 expression was related to the status of invasion, lymph node metastasis and recurrence. Furthermore, DcR3 expression was negatively correlated with caspase-3 and positively associated with Bcl-2, VEGF, Ki-67 labeling index (LI), PCNA LI and P53 (all p<0.0001), respectively., Conclusions: DcR3 may play a crucial role as an oncogene in tumorigenesis, deterioration and progress of BUC via influencing related pathways of apoptosis, proliferation and angiogenesis. The detection of DcR3 protein in the formalin- fixed and paraffin-embedded samples could assist to predict in prognosis of BUC patients.

Objective: To study the distribution law of epigoitrin in roots of Isatis indigotica of different breed types and provide a scientific basis for screening of high-quality Isatis indigotica breed., Methods: Determined the contents of epigoitrin in tap root and lateral root of Chinese-cabbage-leaf Isatis, cabbage-leaf Isatis, mustard-leaf Isatis and tetraploid Isatis by HPLC. Also, compared the contents of epigoitin in xylem and phloem of Isatis indigotica., Results: Contents of epigoitrin in the tap root and lateral root of Isatis indigotica of the different breed types were significant different. In four breed types of Isatis indigotica, contents of epigoitrin in the tap root and phloem were higher than those in the lateral root and xylem, respectively., Conclusion: Contents of epigoitrin in the lateral root of Isatis indigotica are higher than those of tap root and epigoitrin distributes mainly in phloem.

Muscovy duck parvovirus (MDPV) usually causes high morbidity and mortality in 1- to 3-wk-old Muscovy ducklings due to serious infections, which is an imminent threat to the commercial duck industry in China. The objectives of this study were to develop and evaluate a simple, rapid, and inexpensive loop-mediated isothermal amplification (LAMP) method for specific detection of MDPV and to compare it with the PCR method in rapidity, sensitivity, and accuracy. The novel LAMP assay used a set of 4 specific primers to recognize 6 distinct genomic sequences of capsid protein (VP3) from MDPV, which could be completed within 50 min at 63 degrees C in a simple water bath. The diagnostic results demonstrated that the LAMP assay detected all 7 preserved MDPV isolates, had no cross-reactivity with other duck pathogens (i.e., goose parvovirus, duck plague virus, H9N2 avian influenza virus, duck hepatitis type virus I, and Muscovy duck reovirus). The LAMP assay was at least 10-fold more sensitive than the routine PCR assay and obtained more sensitivity in 61 clinical samples. Therefore, the newly developed LAMP assay provides a specific and sensitive means for detecting MDPV and can be simply applied both in field conditions and in laboratory operations in a cost-effective manner with primary care facilities.