Single-cell transcriptomic dissection of the cellular and molecular events underlying the triclosan-induced liver fibrosis in mice.

BACKGROUND: Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS], a common antimicrobial additive in many personal care and health care products, is frequently detected in human blood and urine. Therefore, it has been considered an emerging and potentially toxic pollutant in recent years. Long-term exposure to TCS has been suggested to exert endocrine disruption effects, and promote liver fibrogenesis and tumorigenesis. This study was aimed at clarifying the underlying cellular and molecular mechanisms of hepatotoxicity effect of TCS at the initiation stage. METHODS: C57BL/6 mice were exposed to different dosages of TCS for 2 weeks and the organ toxicity was evaluated by various measurements including complete blood count, histological analysis and TCS quantification. Single cell RNA sequencing (scRNA-seq) was then carried out on TCS- or mock-treated mouse livers to delineate the TCS-induced hepatotoxicity. The acquired single-cell transcriptomic data were analyzed from different aspects including differential gene expression, transcription factor (TF) regulatory network, pseudotime trajectory, and cellular communication, to systematically dissect the molecular and cellular events after TCS exposure. To verify the TCS-induced liver fibrosis, the expression levels of key fibrogenic proteins were examined by Western blotting, immunofluorescence, Masson's trichrome and Sirius red staining. In addition, normal hepatocyte cell MIHA and hepatic stellate cell LX-2 were used as in vitro cell models to experimentally validate the effects of TCS by immunological, proteomic and metabolomic technologies. RESULTS: We established a relatively short term TCS exposure murine model and found the TCS mainly accumulated in the liver. The scRNA-seq performed on the livers of the TCS-treated and control group profiled the gene expressions of > 76,000 cells belonging to 13 major cell types. Among these types, hepatocytes and hepatic stellate cells (HSCs) were significantly increased in TCS-treated group. We found that TCS promoted fibrosis-associated proliferation of hepatocytes, in which Gata2 and Mef2c are the key driving TFs. Our data also suggested that TCS induced the proliferation and activation of HSCs, which was experimentally verified in both liver tissue and cell model. In addition, other changes including the dysfunction and capillarization of endothelial cells, an increase of fibrotic characteristics in B plasma cells, and M2 phenotype-skewing of macrophage cells, were also deduced from the scRNA-seq analysis, and these changes are likely to contribute to the progression of liver fibrosis. Lastly, the key differential ligand-receptor pairs involved in cellular communications were identified and we confirmed the role of GAS6_AXL interaction-mediated cellular communication in promoting liver fibrosis. CONCLUSIONS: TCS modulates the cellular activities and fates of several specific cell types (including hepatocytes, HSCs, endothelial cells, B cells, Kupffer cells and liver capsular macrophages) in the liver, and regulates the ligand-receptor interactions between these cells, thereby promoting the proliferation and activation of HSCs, leading to liver fibrosis. Overall, we provide the first comprehensive single-cell atlas of mouse livers in response to TCS and delineate the key cellular and molecular processes involved in TCS-induced hepatotoxicity and fibrosis.

Deciphering Nonbioavailable Substructures Improves the Bioavailability of Antidepressants by Serotonin Transporter.

Inadequate bioavailability is one of the most critical reasons for the failure of oral drug development. However, the way that substructures affect bioavailability remains largely unknown. Serotonin transporter (SERT) inhibitors are first-line drugs for major depression disorder, and improving their bioavailability may be able to decrease side-effects by reducing daily dose. Thus, it is an excellent model to probe the relationship between substructures and bioavailability. Here, we proposed the concept of "nonbioavailable substructures", referring to substructures that are unfavorable to bioavailability. A machine learning model was developed to identify nonbioavailable substructures based on their molecular properties and shows the accuracy of 83.5%. A more potent SERT inhibitor DH4 was discovered with a bioavailability of 83.28% in rats by replacing the nonbioavailable substructure of approved drug vilazodone. DH4 exhibits promising anti-depression efficacy in animal experiments. The concept of nonbioavailable substructures may open up a new venue for the improvement of drug bioavailability.

Evolution of volatile profile and aroma potential of 'Gold Finger' table grapes during berry ripening.

BACKGROUND: 'Gold Finger' is a grape cultivar with a finger-like shape and a milk flavor. The process by which its aroma profile evolves during ripening is unclear. Thus, changes in the free and bound volatile compounds present in 'Gold Finger' grapes during ripening were investigated using headspace sampling-solid-phase microextraction-gas chromatography-mass spectroscopy (HS-SPME-GC-MS). RESULTS: A total of 83 volatile aroma components were identified in the grapes, with aldehydes, esters, acids, and alcohols being the main components. The total aroma compound content exhibited significant differences between the bound and free forms. The total content of bound volatile compounds did not change significantly during fruit development, although the free aroma compound content was significantly higher than the bound content. The total content of free aldehydes, free alcohols, bound norisoprenoids, and ketones gradually increased for up to 70 days after flowering (DAF), while the total free ester, terpene, and acid content decreased. The characteristic aroma compounds of 'Gold Finger' grapes were identified as hexanal, (E)-2-hexenal, and ethyl hexanoate. CONCLUSIONS: These results give a foundation for the further development of 'Gold Finger' grapes and provide a theoretical basis for the selection and breeding of novel aromatic grape varieties. © 2021 Society of Chemical Industry.

Supersensitive and reusable perovskite nanocomposite fiber paper for time-resolved single-droplet detection.

Traditional test paper cannot be reusable and needs much sample solution. In this study, a reusable perovskite nanocomposite fiber paper consisting of CsPbBr3 quantum dots in-situ growing in the solid polymer fibers with high concentration is fabricated via microwave and electrospinning methods. RhoB is used as the sample solution because it is a hazardous matter but often occurs in printing and dyeing wastewater or appears in food as additives, and traditional detection system generally requires much sample solution (>1 ml) to concentrate for higher concentrations due to the low detection sensitivity. Just need a droplet of sample solution (<25 µl) can this perovskite fiber paper achieve 0.01 ppm of supersensitive detection, which is superior to a majority of reported detection limit. Different from traditional detection based on luminescence intensity, this detection is a new kind of time-resolved method, so that it gets rid of complex and time-consuming calibration (>1 h) usually in traditional detection, and this time-resolved detection can be achieved within ~3 min. Moreover, this perovskite fiber paper is endowed with recyclable property without losing advantages of supersensitive detection (~0.01 ppm), rapid measuring speed (<3 min), and tiny dosage (<25 µl), which is another advantage than conventional detection systems.

Computational Fragment-Based Design Facilitates Discovery of Potent and Selective Monoamine Oxidase-B (MAO-B) Inhibitor.

Parkinson's disease (PD) is one of the most common age-related neurodegenerative diseases. Inhibition of monoamine oxidase-B (MAO-B), which is mainly found in the glial cells of the brain, may lead to an elevated level of dopamine (DA) in patients. MAO-B inhibitors have been used extensively for patients with PD. However, the discovery of the selective MAO-B inhibitor is still a challenge. In this study, a computational strategy was designed for the rapid discovery of selective MAO-B inhibitors. A series of (S)-2-(benzylamino)propanamide derivatives were designed. In vitro biological evaluations revealed that (S)-1-(4-((3-fluorobenzyl)oxy)benzyl)azetidine-2-carboxamide (C3) was more potent and selective than safinamide, a promising drug for regulating MAO-B. Further studies revealed that the selectivity mechanism of C3 was due to the steric clash caused by the residue difference of Phe208 (MAO-A) and Ile199 (MAO-B). Animal studies showed that compound C3 could inhibit cerebral MAO-B activity and alleviate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neuronal loss.

Cluster bagging promotes melatonin biosynthesis in the berry skins of Vitis vinifera cv. Cabernet Sauvignon and Carignan during development and ripening.

Melatonin, a tryptophan derivative, is an important functional component in grape berries. We investigated the effect of cluster bagging on melatonin biosynthesis in the berries of two wine grape cultivars, Cabernet Sauvignon and Carignan, during fruit development and ripening. Cluster bagging delayed fruit coloring and ripening, and bag-treated berries of both grape cultivars synthesized more melatonin and most of the precursor compounds including L-tryptophan, N-acetylserotonin, tryptamine, and serotonin compared to those exposed to light (control) conditions. Interestingly, 5-methoxytryptamine was only detected in the berries of Carignan and not of Cabernet Sauvignon, both in the cluster bagging and control groups. In addition, melatonin and most of its precursors, decreased after veraison. VvSNAT1 and VvT5H expression levels were positively correlated with melatonin content. Our findings suggested that melatonin synthesis pathways differ among grape cultivars, and that VvSNAT1 and VvT5H may show key regulatory roles in the melatonin synthesis of grape berries.

The pretreatment thrombocytosis may predict prognosis of patients with colorectal cancer: a systematic review and meta-analysis.

AIM: Recently, several studies have reported that thrombocytosis may be associated with the poor prognosis of colorectal cancer (CRC). Nevertheless, their conclusions were still controversial. Results & methodology: We searched PubMed, Embase, Cochrane Library and Web of Science up to April 2016. A total of 30 studies including 9129 patients were included in this meta-analysis. Thrombocytosis had a close relationship with the poor overall survival of CRC compared with normal platelet counts, with the pooled hazard ratios being 1.89 (95% CI: 1.45-2.47; p < 0.00001) and 1.83 (95% CI: 1.33-2.53; p = 0.0002), with univariate and multivariate analyses, respectively. DISCUSSION & CONCLUSION: This meta-analysis indicated that thrombocytosis may be a cost-effective and noninvasive indicator for poor prognosis of patients with CRC, especially for overall survival.

Palladium-catalyzed dearomative arylalkynylation of indoles.

A palladium-catalyzed indole dearomative bisfunctionalization via a domino arylation/alkynylation sequence has been developed, which provides a reliable approach to a series of structurally diverse tetracyclic indolines bearing vicinal tertiary and quaternary stereocenters in moderate to good yields and excellent diastereoselectivities.

Lewis Acid Catalyzed Friedel-Crafts Alkylation of Alkenes with Trifluoropyruvates.

A Friedel-Crafts alkylation reaction of styrenes with trifluoropyruvates has been developed, which delivered allylic alcohols in excellent yields (up to 98%) using the Ni(ClO4)2·6H2O/bipyridine complex as a catalyst. The asymmetric reaction was catalyzed by the chiral Cu(OTf)2/bisoxazoline complex to afford the corresponding chiral allylic alcohols bearing trifluoromethylated quaternary stereogenic centers in moderate enantioselectivities (up to 75% ee).

Melatonin treatment of pre-veraison grape berries to increase size and synchronicity of berries and modify wine aroma components.

A comprehensive investigation was carried out to determine the effect of exogenous melatonin treatment of pre-veraison grapes on grape berries and its wines. Two melatonin treatments of pre-veraison grape berries increased the weight of the berries by approximately 6.6%. Meanwhile, this melatonin treatment could be beneficial in the reduction of underripe and overripe fruits and in enhancing the synchronicity of the berries. In addition, there were significant differences in the volatile compound composition between the wine produced from the melatonin-treated berries and the wines made from untreated berries. The wine from melatonin-treated pre-veraison grape berries had stronger fruity, spicy, and sweet sensory properties, compared to the wines made from untreated berries. Prolonging the treatment through repeated applications can enhance these effects and under different seasonal conditions, more pronounced effects on the grape quality and wine properties can be observed.

Enantioselective Arylative Dearomatization of Indoles via Pd-Catalyzed Intramolecular Reductive Heck Reactions.

A highly enantioselective intramolecular arylative dearomatization of indoles via palladium-catalyzed reductive Heck reactions was developed. The new strategy led to a series of optically active indolines bearing C2-quaternary stereocenters in modest to good yields with excellent enantioselectivities (up to 99% ee).

The ameliorative effects of exogenous melatonin on grape cuttings under water-deficient stress: antioxidant metabolites, leaf anatomy, and chloroplast morphology.

Grapes are an important economic crop and are widely cultivated around the world. Most grapes are grown in arid or semi-arid regions, and droughts take a heavy toll in grape and wine production areas. Developing effective drought-resistant cultivation measures is a priority for viticulture. Melatonin, an indoleamine, mediates many physiological processes in plants. Herein, we examined whether exogenously applied melatonin could improve the resistance of wine grape seedlings grown from cuttings to polyethylene glycol-induced water-deficient stress. The application of 10% polyethylene glycol (PEG) markedly inhibited the growth of cuttings, caused oxidative stress and damage from H2 O2 and O2∙-, and reduced the potential efficiency of Photosystem II and the amount of chlorophyll. Application of melatonin partially alleviated the oxidative injury to cuttings, slowed the decline in the potential efficiency of Photosystem II, and limited the effects on leaf thickness, spongy tissue, and stoma size after application of PEG. Melatonin treatment also helped preserve the internal lamellar system of chloroplasts and alleviated the ultrastructural damage induced by drought stress. This ameliorating effect may be ascribed to the enhanced activity of antioxidant enzymes, increased levels of nonenzymatic antioxidants, and increased amount of osmoprotectants (free proline). We conclude that the application of melatonin to wine grapes is effective in reducing drought stress.

A pathogenesis related protein, VpPR-10.1, from Vitis pseudoreticulata: an insight of its mode of antifungal activity.

Previously, VpPR-10.1 was isolated and characterized from a cDNA library of a fungus-resistant accession of Chinese wild grape (Vitis pseudoreticulata). We found that expression of VpPR-10.1 is affected by the fungal pathogen Erysiphe necator. To investigate the biochemical basis of the nuclease activity of VpPR-10.1 and its role in antifungal resistance, we generated recombinant VpPR-10.1 as well as site-directed mutations targeting three conserved amino acid residues among plant PR-10 s: Lys55, Glu149, and Tyr151. We showed that wild-type recombinant VpPR-10.1 exhibits both RNase and DNase activities. Mutant VpPR10.1-Y151H essentially retained all these activities. In contrast, VpPR10.1-K55N, where Lys55 in the P-loop region is mutated to Asn, and VpPR10.1-E149G, where Glu149 is mutated to Gly, lost their nuclease activity, indicating that both residues play a critical role in catalyzing RNA and DNA degradation. Furthermore, VpPR10.1 and VpPR10.1-Y151H inhibited the growth of the cultured fungal pathogen Alternaria alternate. Through transient expression in grapevine, we also demonstrated that VpPR10.1-K55N and VpPR10.1-E149G compromised resistance to E. necator. Finally, we further found that VpPR-10.1 can lead to programmed cell death and DNA degradation when incubated with tobacco BY-2 suspension cells. We show here that Lys55 and Glu149, but not Tyr151, are required for the RNase, DNase and antifungal activities of VpPR-10.1. The strong correlation between the level of VpPR-10.1 nuclease activity and its antifungal property indicates that the former is the biochemical basis for the latter. Taken together, our experiments revealed that VpPR-10.1 is critical in mediating fungal resistance in grape, potentially playing a dual role by degrading pathogen RNA and inducing programmed death of host cells.

Hydrolysates of lignocellulosic materials for biohydrogen production.

Lignocellulosic materials are commonly used in bio-H2 production for the sustainable energy resource development as they are abundant, cheap, renewable and highly biodegradable. In the process of the bio-H2 production, the pretreated lignocellulosic materials are firstly converted to monosaccharides by enzymolysis and then to H2 by fermentation. Since the structures of lignocellulosic materials are rather complex, the hydrolysates vary with the used materials. Even using the same lignocellulosic materials, the hydrolysates also change with different pretreatment methods. It has been shown that the appropriate hydrolysate compositions can dramatically improve the biological activities and bio-H2 production performances. Over the past decades, hydrolysis with respect to different lignocellulosic materials and pretreatments has been widely investigated. Besides, effects of the hydrolysates on the biohydrogen yields have also been examined. In this review, recent studies on hydrolysis as well as their effects on the biohydrogen production performance are summarized.

Cloning and expression of thermo-alkali-stable laccase of Bacillus licheniformis in Pichia pastoris and its characterization.

A thermo-alkali-stable laccase gene from Bacillus licheniformis was cloned and expressed in Pichia pastoris. The recombinant laccase was secreted into the culture medium with a maximum activity of 227.9 U/L. The purified laccase is a monomeric glycoprotein, and its molecular weight was estimated to be 65 kDa on SDS-PAGE after deglycosylation. Optimal enzyme activity was observed at pH 6.2 and 70°C with syringaldazine as substrate. The recombinant laccase was highly stable in the pH range 7-9 after 10 days at 30°C. The enzyme displayed remarkable thermostability at 50-70°C, with a half-life of inactivation at 70°C of 6.9 h. It also exhibited high tolerance to NaCl and organic solvents like the native spore laccase. The purified laccase could rapidly decolorize reactive blue 19, reactive black 5 and indigo carmine in the presence of acetosyringone. More than 93% of the tested dyes were decolorized in 4 h at pH 9.0.

Effect of rain-shelter cultivation of Vitis vinifera cv. Cabernet Gernischet on the phenolic profile of berry skins and the incidence of grape diseases.

Rain-shelter cultivation is an effective cultural method to prevent rainfall damage during grape harvest and widely applied in the Chinese rainy regions. In this study we investigated the effect of rain-shelter cultivation on grape diseases and phenolic composition in the skins of Vitis vinifera cv. Cabernet Gernischet grape berries through the comparison with open-field cultivation at two vintages (2010 and 2011). The results showed that rain-shelter cultivation reduced the incidence of grape diseases significantly and delayed the maturation of Cabernet Gernischet fruits. With regards to most of the phenolic compounds identified in this study, their content in grape samples under rain-shelter cultivation was decreased compared to those under open-field cultivation. However, rain-shelter cultivation stimulated the accumulation of dihydroquercetin-3-O-rhamnoside in grape skins during grape maturation. These were related with micrometeorological alterations in vineyards by using plastic covering under rain-shelter cultivation. It suggests the rain-shelter cultivation makes possible the cultivation of "Cabernet Gernischet" grapes in an organic production system, for providing a decrease in the incidence of diseases and the dependence on chemical pesticides in the grape and wine industry.

[Application of three-dimensional reconstruction technique based on CT-MRI fusion in skull base surgery].

OBJECTIVE: To study the application of three-dimensional reconstruction technique based on CT-MRI fusion in skull base surgery. METHODS: To acquire the thin layer CT scan and MRI scanned images, to achieve image registration, fusion, segmentation and 3D visualization by using self-preparation software, to operate, observe and measure models by using methods of endoscopic observation, volume rendering segmentation, automatically and manually measure. RESULTS: The center of the eye and foramen magnum in CT-MRI were used as point registration. Good coincidence of important anatomic landmarks were formed in the image fusion. The boundary of spotted graphical was clear and complete. The models showed a complete, continuous, smooth surface. Virtual endoscopy could display the inside three-dimensional structures of skull from nasal with fluent operations of rotation and transparency. The boundary of skull stump segmented after volume rendering segmentation was clear and smooth, and it could show bone signs and soft tissue models together. Cooperation of automatic measurement method [(32.007 ± 15.311) mm] and the manual measurement method [(30.240 ± 15.169) mm] for measuring the maximum diameters of the tumor model, the difference was significant (t = 8.409, P < 0.05). CONCLUSIONS: The method of selecting the center of the eye and foramen magnum in point matching is scientific, simple and easy to operate. The models reconstructed based on CT-MRI fusion images can accurately reflect the size of the soft tissue and be better measured through the automatic measurement. Reconstruction models can be observed through the way of virtual endoscopic within the nasal cavity or volume rendering segmentation from outside to inside to frustrate the relationship of skull structures. Three-dimensional reconstruction techniques based on CT-MRI fusion in skull base surgery can be used to plan surgical approach, to assess the risk of surgery and to achieve space measurements, and it laid the foundation for the three-dimensional navigation.

[Construction of a shuttle plasmid encoding a chimeric gene of eglA p-Her2/neu ECD-IL-12].

AIM: To construct a shuttle plasmid encoding a chimeric gene of Clostridium saccharobutylicum eglA promoter(eglA p)-extracellular domain of human epidermal growth factor receptors 2(hHer2/neu ECD)-human Interleukin-12(rhIL-12), pIMP1 eglA p-hHer 2/neu ECD-rhIL-12. METHODS: The hHer2/neu ECD and the eglA p was amplified from the corresponding template: pcDNA3.1 hHer2/neu and 55 bp fragment in eglA p by PCR. pcDNA6 eglA p-hHer2/neu ECD-rhIL-12 was prepared by inserting the hHer2/neu ECD and eglAp fragment into the plasmid, pcDNA6 rhIL-12. Shuttle plasmid pIMP1 eglA p-hHer2/neu ECD-rhIL-12 was acquired by using in-fusion technique. subsequently, the recombinant shuttle plasmid was identified. RESULTS: All the fragments of hHer2/neu ECD , eglA p and eglAp-Her2/neu ECD-rhIL-12 were amplified, and the corresponding plasmids were prepared rightly. The shuttle plasmid of pIMP1 eglAp-hHer2/neu ECD-rhIL-12 was successfully const- ructed. No error was found both in the sequence and ORF of the acquired chimeric gene. CONCLUSIONS: A shuttle plasmid encoding a chimeric gene of Clostridium saccharobut- ylicum eglAp-hHer2/neu ECD-rhIL-12(pIMP1 eglA p-hHer2/neu ECD-rhIL- 12) was success- fully constructed.

[Construction of an eukaryotic plasmid encoding HER2 and screening of a cell line stably expressing clones].

AIM: To construct an eukaryotic vector encoding extracellular domain of human epidermal growth factor receptors (HER2), pcDNA6/v5-his-HER2, and to screen HER2 positive clones from mouse breast cancer cell line EMT6. METHODS: The extracellular domain of HER2 was amplified from pcDNA3.1-HER2 by PCR. pcDNA6/v5-his-HER2 was prepared by inserting the fragment into the plasmid pcDNA6/v5-his. Then the recombinant vector was identified by restriction enzyme and sequencing. Next, pcDNA6/v5-his-HER2 was transfected into the EMT6 cell line and the positive clones (EMT6/HER2) were screened with blasticidin. Finally, the expression of HER2 in EMT6/HER2 was detected by RT-PCR and immunohistochemistry. RESULTS: The fragment of HER2 was amplified and pcDNA6/v5-his-HER2 was prepared successfully. No errors were found both in the sequence and ORF of the acquired fragment. The expected fragment of HER2 (1896 bp) was amplified from EMT6/HER2 by RT-PCR and positive signals of HER2 were detected in EMT6/HER2 by immunohistochemistry. CONCLUSION: An eukaryotic plasmid encoding HER2 (pcDNA6/v5-his-HER2) has been constructed and a cell line expressing HER2 stably has been prepared successfully.

Interleukin-12 gene modification exerts anti-tumor effects on murine mammary sarcoma cell line in vivo.

The aim of this project was to investigate the anti-tumor effect of an IL-12 gene modified mammary sarcoma murine cell line, EMT6/IL-12, in mouse model. In this study, we transfected the recombinant eukaryotic plasmid encoding IL-12 gene (pcDNA6-p70) into EMT6 and obtained the IL-12 expressing EMT6/IL-12 cell line. Then EMT6/IL-12 cells were s.c. inoculated into mice. The recombinant vector treatment group was set as control. We then evaluated the inhibition of tumor growth and the anti-tumor immunity function in vivo such as cytotoxicity, proliferation of splenocytes and serial IFN-gamma level. And the percentage of IFN-gamma producing CD4 or CD8 T cells among splenocytes was also analyzed in tumor bearing mice. Our results showed that the growth of tumors was obviously inhibited in EMT6/IL-12 group. Moreover, the capacities of anti-tumor immunity were all significantly higher in EMT6/IL-12 group compared to the controls. The results of the present investigation support the notion that EMT6/IL-12 could exert gene therapy in tumor model by improving the anti-tumor cellular immunity.