Cell atlas of CCl 4-induced progressive liver fibrosis reveals stage-specific responses.

Chronic liver injury leads to progressive liver fibrosis and ultimately cirrhosis, a major cause of morbidity and mortality worldwide. However, there are currently no effective anti-fibrotic therapies available, especially for late-stage patients, which is partly attributed to the major knowledge gap regarding liver cell heterogeneity and cell-specific responses in different fibrosis stages. To reveal the multicellular networks regulating mammalian liver fibrosis from mild to severe phenotypes, we generated a single-nucleus transcriptomic atlas encompassing 49 919 nuclei corresponding to all main liver cell types at different stages of murine carbon tetrachloride (CCl 4)-induced progressive liver fibrosis. Integrative analysis distinguished the sequential responses to injury of hepatocytes, hepatic stellate cells and endothelial cells. Moreover, we reconstructed cell-cell interactions and gene regulatory networks implicated in these processes. These integrative analyses uncovered previously overlooked aspects of hepatocyte proliferation exhaustion and disrupted pericentral metabolic functions, dysfunction for clearance by apoptosis of activated hepatic stellate cells, accumulation of pro-fibrotic signals, and the switch from an anti-angiogenic to a pro-angiogenic program during CCl 4-induced progressive liver fibrosis. Our dataset thus constitutes a useful resource for understanding the molecular basis of progressive liver fibrosis using a relevant animal model.

[Preparation and Evaluation of Intranasal in situ Gel of Bupleuri Radix Volatile Oil and Baicalin].

OBJECTIVE: To prepare and evaluate a new formulation of thermosensitive and ion-sensitive in situ gel for nasal administration, using the volatile oil of Bupleuri radix and baicalin, the effective component extracted from Scutellariae radix . METHODS: Formulation of in situ nasal gel of Bupleuri radix volatile oil and baicalin was prepared by using poloxamer 407 and deacetylated gellan gum as the gel base, 10% pharmasolve and 2% polysorbate 80 as the solubilizer, and 0.8% triethanolamine as the pH regulator. The physical appearance, phase transition temperature, and baicalin release performance of the prepared gel were examined. The pharmacodynamic evaluation was done with the rat fever model developed with dry yeast and the mouse auricle swelling inflammation model. RESULTS: The phase transition temperature of the gel was optimized to be 36 ℃. The release of baicalin from the gel showed obvious features of sustained release, which accorded well the zero-order kinetics equation. The results of experiments with the rat dry yeast fever model and the mouse xylene auricle swelling inflammation model showed that the gel had significant antipyretic and anti-inflammatory effects that were significantly better than those of the groups treated with the blank gel base and the Bupleuri radix and Scutellariae radix granule. Results from the cilia toxicity test showed that the gel did not have obvious toxic effect on toad palate mucosal cilia. CONCLUSION: The in situ nasal gel of Bupleuri radix volatile oil and baicalin prepared in the study had a rapid onset time, high efficiency, and prolonged release of active ingredients, thus showing promises for further applicational development.

Genome-wide characterization, expression analyses, and functional prediction of the NPF family in Brassica napus.

BACKGROUND: NITRATE TRANSPORTER 1/PEPTIDE TRANSPORTER (NRT1/PTR) family (NPF) members are essential transporters for many substrates in plants, including nitrate, hormones, peptides, and secondary metabolites. Here, we report the global characterization of NPF in the important oil crop Brassica napus, including that for phylogeny, gene/protein structures, duplications, and expression patterns. RESULTS: A total of 199 B. napus (BnaNPFs) NPF-coding genes were identified. Phylogenetic analyses categorized these genes into 11 subfamilies, including three new ones. Sequence feature analysis revealed that members of each subfamily contain conserved gene and protein structures. Many hormone-/abiotic stress-responsive cis-acting elements and transcription factor binding sites were identified in BnaNPF promoter regions. Chromosome distribution analysis indicated that BnaNPFs within a subfamily tend to cluster on one chromosome. Syntenic relationship analysis showed that allotetraploid creation by its ancestors (Brassica rapa and Brassica oleracea) (57.89%) and small-scale duplication events (39.85%) contributed to rapid BnaNPF expansion in B. napus. A genome-wide spatiotemporal expression survey showed that NPF genes of each Arabidopsis and B. napus subfamily have preferential expression patterns across developmental stages, most of them are expressed in a few organs. RNA-seq analysis showed that many BnaNPFs (32.66%) have wide exogenous hormone-inductive profiles, suggesting important hormone-mediated patterns in diverse bioprocesses. Homologs in a clade or branch within a given subfamily have conserved organ/spatiotemporal and hormone-inductive profiles, indicating functional conservation during evolution. qRT-PCR-based comparative expression analysis of the 12 BnaNPFs in the NPF2-1 subfamily between high- and low-glucosinolate (GLS) content B. napus varieties revealed that homologs of AtNPF2.9 (BnaNPF2.12, BnaNPF2.13, and BnaNPF2.14), AtNPF2.10 (BnaNPF2.19 and BnaNPF2.20), and AtNPF2.11 (BnaNPF2.26 and BnaNPF2.28) might be involved in GLS transport. qRT-PCR further confirmed the hormone-responsive expression profiles of these putative GLS transporter genes. CONCLUSION: We identified 199 B. napus BnaNPFs; these were divided into 11 subfamilies. Allopolyploidy and small-scale duplication events contributed to the immense expansion of BnaNPFs in B. napus. The BnaNPFs had preferential expression patterns in different tissues/organs and wide hormone-induced expression profiles. Four BnaNPFs in the NPF2-1 subfamily may be involved in GLS transport. Our results provide an abundant gene resource for further functional analysis of BnaNPFs.

Evolutionary and Comparative Expression Analyses of TCP Transcription Factor Gene Family in Land Plants.

The plant-specific Teosinte-branched 1/Cycloidea/Proliferating (TCP) transcription factor genes are involved in plants' development, hormonal pathways, and stress response but their evolutionary history is uncertain. The genome-wide analysis performed here for 47 plant species revealed 535 TCP candidates in terrestrial plants and none in aquatic plants, and that TCP family genes originated early in the history of land plants. Phylogenetic analysis divided the candidate genes into Classes I and II, and Class II was further divided into CYCLOIDEA (CYC) and CINCINNATA (CIN) clades; CYC is more recent and originated from CIN in angiosperms. Protein architecture, intron pattern, and sequence characteristics were conserved in each class or clade supporting this classification. The two classes significantly expanded through whole-genome duplication during evolution. Expression analysis revealed the conserved expression of TCP genes from lower to higher plants. The expression patterns of Class I and CIN genes in different stages of the same tissue revealed their function in plant development and their opposite effects in the same biological process. Interaction network analysis showed that TCP proteins tend to form protein complexes, and their interaction networks were conserved during evolution. These results contribute to further functional studies on TCP family genes.

Global Analysis of WOX Transcription Factor Gene Family in Brassica napus Reveals Their Stress- and Hormone-Responsive Patterns.

The plant-specific WUSCHEL-related homeobox (WOX) transcription factor gene family is important for plant growth and development but little studied in oil crops. We identified and characterized 58 putative WOX genes in Brassica napus (BnWOXs), which were divided into three major clades and nine subclades based on the gene structure and conserved motifs. Collinearity analysis revealed that most BnWOXs were the products of allopolyploidization and segmental duplication events. Gene structure analysis indicated that introns/exons and protein motifs were conserved in each subclade and RNA sequencing revealed that BnWOXs had narrow expression profiles in major tissues and/or organs across different developmental stages. The expression pattern of each clade was highly conserved and similar to that of the sister and orthologous pairs from Brassica rapa and Brassica oleracea. Quantitative real-time polymerase chain reaction showed that members of the WOX4 subclade were induced in seedling roots by abiotic and hormone stresses, indicating their contribution to root development and abiotic stress responses. 463 proteins were predicted to interact with BnWOXs, including peptides regulating stem cell homeostasis in meristems. This study provides insights into the evolution and expression of the WOX gene family in B. napus and will be useful in future gene function research.

On-line high-performance liquid chromatography coupled with biochemical detection method for screening of α-glucosidase inhibitors in green tea.

An on-line high-performance liquid chromatography-biochemical detection (HPLC-BCD) method, in which compounds separated by HPLC were on-line reacted with enzyme and substrate solutions delivered by flow injection and the enzyme inhibition signal was collected by UV detection, was developed to rapidly screen α-glucosidase inhibitors from green tea extracts in this study. The chromatographic fingerprints and enzyme inhibition profiles of the different brands of green tea could be simultaneously detected by the on-line HPLC-BCD method. Enzyme inhibition profiles were detected by the UV detector at 415 nm based on the reaction of α-glucosidase and p-nitrophenyl α-d-glucopyranoside (PNPG). PNPG (1.25 mm), α-glucosidase (0.4 U/mL) and the flow rate 0.07 mL/min were applied as optimized parameters to detect α-glucosidase inhibitors in green tea. Four components in green tea showed α-glucosidase inhibition action and three of them were identified as HHDP-galloyl glucose, (-)-epigallocatechin-3-gallate and (-)-epicatechin-3-gallate by HPLC-fourier-transform mass spectrometry (HPLC-FTMS). Two brands of green tea derived from Mengding and Enshi mountainous areas might be superior to the other samples in the prevention and treatment of diabetes owing to their stronger activities of enzyme inhibitors. The proposed on-line HPLC-BCD method could be used to rapidly identify the potential enzyme inhibitors in complex matrixes.

Expression of nattokinase in Escherichia coli and renaturation of its inclusion body.

Nattokinase is an important fibrinolytic enzyme with therapeutic applications for cardiovascular diseases. The full-length and mature nattokinase genes were cloned from Bacillus subtilis var. natto and expressed in pQE30 vector in Escherichia coli. The full-length gene expressed low nattokinase activity in the intracellular soluble and the medium fractions. The mature gene expressed low soluble nattokinase activity and large amount insoluble protein in inclusion bodies without enzyme activity. Large amount of refolding solutions (RSs) at different pH values were screening and RS-10 and RS-11 at pH 9 were selected to refold nattokinase inclusion bodies. The recombinant cells were lysed with 0.1mg/mL lysozyme and ultrasonic treatment. After centrifugation, the pellete was washed twice with 20mM Tris-HCl buffer (pH 7.5) containing 1% Triton X-100 to purify the inclusion bodies. The inclusion bodies were dissolved in water at pH 12.0 and refolded with RS-10. The refolded proteins showed 42.8IU/mg and 79.3IU/mg fibrinolytic activity by the traditional dilution method (20-fold dilution into RS-10) and the directly mixing the protein solution with equal volume RS-10, respectively, compared to the 52.0IU/mg of total water-soluble proteins from B. subtilis var. natto. This work demonstrated that the inclusion body of recombinant nattokinase expressed in E. coli could be simply refolded to the natural enzyme activity level by directly mixing the protein solution with equal volume refolding solution.

Complete mitochondrial genome of the Spotted dove (Streptopelia chinensis).

The Spotted dove (Streptopelia chinensis) is a member of the bird family Columbidae. In this study, we report the complete mitochondrial genome of this species. The mitochondrial genome of Spotted dove is a circular molecule of 16,966 bp in size and contains 13 protein-coding genes, two rRNA genes, 22 tRNA genes, and one control region. The total base composition is 30.1% for A, 32.1% for C, 13.9% for G, and 23.9% for T. These data will be useful for the phylogenetic and population diversity analyses of birds, especially Columbidae species.

The complete sequence of the mitochondrial genome of Arctic fox (Alopex lagopus).

In the present study, the complete mitochondrial genome sequence of Arctic fox (Alopex lagopus) was determined for the first time. It has a total length of 16,656 bp, and contains 13 protein-coding genes, 22 tRNA genes, 2 ribosome RNA genes and 1 control region. The nucleotide composition is 31.3% for A, 26.2% for C, 14.8% for G and 27.7% for T, respectively. The D-loop region located between tRNAPro and tRNAPhe contains a (ACACGTACACGCAT)18 tandem repeat array. The data will be useful for the investigation of the genetic structure and diversity in the natural and farmed population of Arctic foxes.

[Two endmember extraction algorithms with combined spatial and spectral domain TM image].

Based on a few bands and unabundant spectral information of TM remote sensing image, two endmember extraction algorithms are put forward. First, spatial split endmember extraction algorithm, which firstly browses the image, based on the complexity of objects, divides the image into different blocks, then uses hourglass algorithm to extract endmembers. Second, region continuity algorithm, also based on dividing-into-blocks idea, which uses extraction and classification of homogenous object algorithm and spectral correlation energy level matching algorithm to extract endmembers. Finally, comparing the two algorithms, spatial split endmember extraction algorithm runs fast, with little prior knowledge, however, the probability of error extraction endmembers exists; and region continuity algorithm's precision is higher, needs for prior knowledge, and the segment process is slow. Experimental results show that both spatial-and-spectral combined endmember extraction algorithms can effectively solve the large regional scale, multispectral endmember extraction problem, and have broad application prospects.

Occurrence, function and potential medicinal applications of the phytohormone abscisic acid in animals and humans.

Abscisic acid (ABA) is an important phytohormone that regulates plant growth, development, dormancy and stress responses. Recently, it was discovered that ABA is produced by a wide range of animals including sponges (Axinella polypoides), hydroids (Eudendrium racemosum), human parasites (Toxoplasma gondii), and by various mammalian tissues and cells (leukocytes, pancreatic cells, and mesenchymal stem cells). ABA is a universal signaling molecule that stimulates diverse functions in animals through a signaling pathway that is remarkably similar to that used by plants; this pathway involves the sequential binding of ABA to a membrane receptor and the activation of ADP-ribose cyclase, which results in the overproduction of the intracellular cyclic ADP-ribose and an increase in intracellular Ca²âº concentrations. ABA stimulates the stress response (heat and light) in animal cells, immune responses in leukocytes, insulin release from pancreatic ß cells, and the expansion of mesenchymal and colon stem cells. ABA also inhibits the growth and induces the differentiation of cancer cells. Unlike some drugs that act as cell killers, ABA, when functioning as a growth regulator, does not have significant toxic side effects on animal cells. Research indicated that ABA is an endogenous immune regulator in animals and has potential medicinal applications for several human diseases. This article summarizes recent advances involving the discovery, signaling pathways and functions of ABA in animals.