scholarly journals Functional Analysis of Sterol O-Acyltransferase Involved in the Biosynthetic Pathway of Pachymic Acid in Wolfiporia cocos

Molecules ◽  
2021 ◽  
Vol 27 (1) ◽  
pp. 143
Author(s):  
Wenjun Zhu ◽  
Ying Liu ◽  
Jing Tang ◽  
Heping Liu ◽  
Naliang Jing ◽  
...  

Pachymic acid from Wolfiporia cocos possesses important medicinal values including anti-bacterial, anti-inflammatory, anti-viral, invigorating, anti-rejection, anti-tumor, and antioxidant activities. However, little is known about the biosynthetic pathway from lanostane to pachymic acid. In particular, the associated genes in the biosynthetic pathway have not been characterized, which limits the high-efficiency obtaining and application of pachymic acid. To characterize the synthetic pathway and genes involved in pachymic acid synthesis, in this study, we identified 11 triterpenoids in W. cocos using liquid chromatography tandem mass spectrometry (LC-MS/MS), and inferred the putative biosynthetic pathway from lanostane to pachymic acid based on analyzing the chemical structure of triterpenoids and the transcriptome data. In addition, we identified a key gene in the biosynthetic pathway encoding W. cocos sterol O-acyltransferase (WcSOAT), which catalyzes tumolusic acid to pachymic acid. The results show that silence of WcSOAT gene in W. cocos strain led to reduction of pachymic acid production, whereas overexpression of this gene increased pachymic acid production, indicating that WcSOAT is involved in pachymic acid synthesis in W. cocos and the biosynthesis of W. cocos pachymic acid is closely dependent on the expression of WcSOAT gene. In summary, the biosynthetic pathway of pachymic acid and the associated genes complement our knowledge on the biosynthesis of W. cocos pachymic acid and other triterpenoids, and also provides a reference for target genes modification for exploring high-efficiency obtaining of active components.

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Huahe Zhu ◽  
Shun Wang ◽  
Cong Shan ◽  
Xiaoqian Li ◽  
Bo Tan ◽  
...  

AbstractXuan-bai-cheng-qi decoction (XCD), a traditional Chinese medicine (TCM) prescription, has been widely used to treat a variety of respiratory diseases in China, especially to seriously infectious diseases such as acute lung injury (ALI). Due to the complexity of the chemical constituent, however, the underlying pharmacological mechanism of action of XCD is still unclear. To explore its protective mechanism on ALI, firstly, a network pharmacology experiment was conducted to construct a component-target network of XCD, which identified 46 active components and 280 predicted target genes. Then, RNA sequencing (RNA-seq) was used to screen differentially expressed genes (DEGs) between ALI model rats treated with and without XCD and 753 DEGs were found. By overlapping the target genes identified using network pharmacology and DEGs using RNA-seq, and subsequent protein–protein interaction (PPI) network analysis, 6 kernel targets such as vascular epidermal growth factor (VEGF), mammalian target of rapamycin (mTOR), AKT1, hypoxia-inducible factor-1α (HIF-1α), and phosphoinositide 3-kinase (PI3K) and gene of phosphate and tension homology deleted on chromsome ten (PTEN) were screened out to be closely relevant to ALI treatment. Verification experiments in the LPS-induced ALI model rats showed that XCD could alleviate lung tissue pathological injury through attenuating proinflammatory cytokines release such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β. Meanwhile, both the mRNA and protein expression levels of PI3K, mTOR, HIF-1α, and VEGF in the lung tissues were down-regulated with XCD treatment. Therefore, the regulations of XCD on PI3K/mTOR/HIF-1α/VEGF signaling pathway was probably a crucial mechanism involved in the protective mechanism of XCD on ALI treatment.


2021 ◽  
Author(s):  
Jing Yang ◽  
Chao-Tao Tang ◽  
Ruiri Jin ◽  
Bixia Liu ◽  
Peng Wang ◽  
...  

Abstract Huanglian jiedu decoction (HLJDD) is a heat-clearing and detoxifying agent composed of four kinds of Chinese herbal medicine. Previous studies have shown that HLJDD can improve the inflammatory response of ulcerative colitis (UC) and maintain intestinal barrier function. However, its molecular mechanism is not completely clear. In this study, we verified the bioactive components (BCI) and potential targets of HLJDD in the treatment of UC by means of network pharmacology and molecular docking, and constructed the pharmacological network and PPI network. Then the core genes were enriched by GO and KEGG. Finally, the bioactive components were docked with the key targets to verify the binding ability between them. A total of 54 active components related to UC were identified. Ten genes are considered to be very important to PPI network. Functional analysis showed that these target genes were mainly involved in the regulation of cell response to different stimuli, IL-17 signal pathway and TNF signal pathway. The results of molecular docking showed that the active components of HLJDD had good affinity with Hub gene. This study systematically elucidates the "multi-component, multi-target, multi-pathway" mechanism of anti-UC with HLJDD for the first time, suggesting that HLJDD or its active components may be candidate drugs for the treatment of ulcerative colitis.


2021 ◽  
Author(s):  
Xiaoen Huang ◽  
Nian Wang

Sweet orange (Citrus sinensis) is the most economically important species for the citrus industry. However, it is susceptible to many diseases including citrus bacterial canker caused by Xanthomonas citri subsp. citri (Xcc) that triggers devastating effects on citrus production. Conventional breeding has not met the challenge to improve disease resistance of sweet orange due to the long juvenility and other limitations. CRISPR-mediated genome editing has shown promising potentials for genetic improvements of plants. Generation of biallelic/homozygous mutants remains difficult for sweet orange due to low transformation rate, existence of heterozygous alleles for target genes and low biallelic editing efficacy using the CRISPR technology. Here, we report improvements in the CRISPR/Cas9 system for citrus gene editing. Based on the improvements we made previously (dicot codon optimized Cas9, tRNA for multiplexing, a modified sgRNA scaffold with high efficiency, CsU6 to drive sgRNA expression), we further improved our CRISPR/Cas9 system by choosing superior promoters (CmYLCV or CsUbi promoter) to drive Cas9 and optimizing culture temperature. This system was able to generate a biallelic mutation rate of up to 89% for Carrizo citrange and 79% for Hamlin sweet orange. Consequently, this system was used to generate canker resistant Hamlin sweet orange by mutating the effector binding element (EBE) of canker susceptibility gene CsLOB1, which is required for causing canker symptoms by Xcc. Six biallelic Hamlin sweet orange mutant lines in the EBE were generated. The biallelic mutants are resistant to Xcc. Biallelic mutation of the EBE region abolishes the induction of CsLOB1 by Xcc. This study represents a significant improvement in sweet orange gene editing efficacy and generating disease resistant varieties via CRISPR-mediated genome editing. This improvement in citrus genome editing makes genetic studies and manipulations of sweet orange more feasible.


2021 ◽  
Author(s):  
Ken Takao ◽  
Katsumi Iizuka ◽  
Yanyan Liu ◽  
Teruaki Sakurai ◽  
Sodai Kubota ◽  
...  

Carbohydrate response element binding protein (ChREBP) is critical in the regulation of fatty acid and triglyceride synthesis in the liver. Interestingly, Chrebp-/- mice show reduced levels of plasma cholesterol, which is critical for steroid hormone synthesis in adrenal glands. Furthermore, Chrebp mRNA expression was previously reported in human adrenal glands. Thus, it remains to be investigated whether ChREBP plays a role directly or indirectly in steroid hormone synthesis and release in adrenal glands. In the present study, we find that Chrebp mRNA is expressed in mouse adrenal glands and that ChREBP binds to carbohydrate response elements. Histological analysis of Chrebp-/- mice shows no adrenal hyperplasia and less oil red O staining compared with that in wild-type mice. In adrenal glands of Chrebp-/- mice, expression of Fasn and Scd1, two enzymes critical for fatty acid synthesis, was substantially lower and triglyceride content was reduced. Expression of Srebf2, a key transcription factor controlling synthesis and uptake of cholesterol and the target genes was upregulated, while cholesterol content was not significantly altered in the adrenal glands of Chrebp-/- mice. Adrenal corticosterone content and plasma adrenocorticotropic hormone and corticosterone levels were not significantly altered in Chrebp-/- mice. Consistently, expression of genes related to steroid hormone synthesis was not altered. Corticosterone secretion in response to two different stimuli, namely 24-h starvation and cosyntropin administration, were also not altered in Chrebp-/- mice. Taking these results together, corticosterone synthesis and release were not affected in Chrebp-/- mice despite reduced plasma cholesterol levels.


Acta Medica ◽  
2019 ◽  
Vol 50 (4) ◽  
pp. 48-56
Author(s):  
Ufuk Bozkurt Obuz ◽  
Incilay Lay

Bile acids are synthesized from cholesterol through 17 different enzymes located in different intracellular compartments of hepatocytes. Defects have been identified in the genes encoding the enzymes involved in the bile acid synthesis pathways and nine different diseases have been identified so far. In this review, four different biosynthetic pathway of bile acids together with disorders of bile acid synthesis is described. In inborn errors of bile acid synthesis clinical findings can range from liver failure to cirrhosis in infancy or progressive neuropathy in adolescence / adulthood. Laboratory analysis of urine profiling of bile acids is important in early diagnosis and early treatment.


2021 ◽  
Vol 5 (Supplement_1) ◽  
pp. A48-A48
Author(s):  
Maria del Carmen Vazquez Borrego ◽  
Mercedes del Rio Moreno ◽  
Andre Sarmento-Cabral ◽  
Mariyah Mahmood ◽  
Papasani V Subbaiah ◽  
...  

Abstract A reduction in GH, as well as IGF1, is associated with non-alcoholic fatty liver disease (NAFLD). However, the relative contribution of changes in circulating GH and IGF1, to hepatic triglyceride accumulation (steatosis), remains to be clearly defined. To study the direct actions of GH on hepatocyte metabolism, we have utilized a mouse model of adult-onset, hepatocyte-specific, GHR knockdown (aHepGHRkd; 10–12 week-old, GHRfl/fl male mice, treated with AAV8-TBGp-Cre). In this and previous reports, we have observed that aHepGHRkd male mice rapidly develop steatosis (after 7 days) associated with enhanced de novo lipogenesis (DNL; measured by deuterated H2O labeling, 10h after 0800h food removal), and low ketone levels, suggestive of reduced hepatic β-oxidation. Of note, aHepGHRkd also reduces plasma IGF1 levels to >80% of GHR-intact controls (GHRfl/fl mice treated with AAV8-TBGp-Null), leading to a rise in GH, due to loss of IGF1 negative feedback to the pituitary/hypothalamus. This reciprocal shift in IGF1/GH is associated with an increase in insulin levels. Therefore, it is possible that the steatosis that develops in aHepGHRkd mice is the consequence of systemic insulin resistance supplying excess substrates (glucose and NEFA) for hepatic lipogenesis. However, inconsistent with this theory is the fact that glucose and NEFA levels are not altered after aHepGHRkd. To tease out the indirect (perhaps driven by high insulin levels) vs. direct effects of GH on hepatocyte lipid accumulation, male aHepGHRkd mice were injected with a vector expressing rat IGF1 (AAV8-TBGp-rIGF1). Reconstitution of hepatocyte IGF1 in aHepGHRkd mice, raised plasma IGF1 and normalized GH, insulin and ketone levels, but hepatic steatosis and DNL remained greater than that of GHR-intact controls, indicating GH directly suppresses hepatic fat accumulation. RNAseq analysis of livers from aHepGHRkd mice showed expression of genes related to carbohydrate metabolism (Gck, Khk) and fatty acid synthesis (Fasn, Srebf1, Usf1), processing (Scd1) and uptake (Cd36) were increased, while genes related to gluconeogenesis (Pck1, Fbp1, G6pc) were reduced. Remarkably, IGF1 reconstitution had no major impact on the hepatic transcriptome of aHepGHRkd mice, with the exception of reducing the expression of Srebf1, consistent with the reduction in circulating insulin levels. Interestingly, carbohydrate-responsive element-binding protein (CHREBP) levels, but not mRNA levels, were greater in aHepGHRkd mice with or without IGF1 reconstitution, consistent with upregulation of CHREBP target genes (Khk and Fasn among others). Taken together, these results suggest GH directly regulates steatosis, at least in part, by suppressing carbohydrate-driven DNL, where additional studies are underway to test this hypothesis.


2019 ◽  
Author(s):  
Remi L. Gratacap ◽  
Tim Regan ◽  
Carola E. Dehler ◽  
Samuel A.M. Martin ◽  
Pierre Boudinot ◽  
...  

1AbstractGenome editing is transforming bioscience research, but its application to non-model organisms, such as farmed animal species, requires optimisation. Salmonids are the most important aquaculture species by value, and improving genetic resistance to infectious disease is a major goal. However, use of genome editing to evaluate putative disease resistance genes in cell lines, and the use of genome-wide CRISPR screens is currently limited by a lack of available tools and techniques. In the current study, an optimised protocol using lentivirus transduction for efficient integration of constructs into the genome of a Chinook salmon (Oncorhynchus tshwaytcha) cell line (CHSE-214) was developed. As proof-of-principle, two target genes were edited with high efficiency in an EGFP-Cas9 stable CHSE cell line; specifically, the exogenous, integrated EGFP and the endogenous RIG-I locus. Finally, the effective use of antibiotic selection to enrich the successfully edited targeted population was demonstrated. The optimised lentiviral-mediated CRISPR method reported here increases possibilities for efficient genome editing in salmonid cells, in particular for future applications of genome-wide CRISPR screens for disease resistance.


eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Fillip Port ◽  
Claudia Strein ◽  
Mona Stricker ◽  
Benedikt Rauscher ◽  
Florian Heigwer ◽  
...  

Genetic screens are powerful tools for the functional annotation of genomes. In the context of multicellular organisms, interrogation of gene function is greatly facilitated by methods that allow spatial and temporal control of gene abrogation. Here, we describe a large-scale transgenic short guide (sg) RNA library for efficient CRISPR-based disruption of specific target genes in a constitutive or conditional manner. The library consists currently of more than 2600 plasmids and 1700 fly lines with a focus on targeting kinases, phosphatases and transcription factors, each expressing two sgRNAs under control of the Gal4/UAS system. We show that conditional CRISPR mutagenesis is robust across many target genes and can be efficiently employed in various somatic tissues, as well as the germline. In order to prevent artefacts commonly associated with excessive amounts of Cas9 protein, we have developed a series of novel UAS-Cas9 transgenes, which allow fine tuning of Cas9 expression to achieve high gene editing activity without detectable toxicity. Functional assays, as well as direct sequencing of genomic sgRNA target sites, indicates that the vast majority of transgenic sgRNA lines mediate efficient gene disruption. Furthermore, we conducted the so far largest fully transgenic CRISPR screen in any metazoan organism, which further supported the high efficiency and accuracy of our library and revealed many so far uncharacterized genes essential for development.


2021 ◽  
Vol 17 ◽  
Author(s):  
Xingxing Zhuang ◽  
Li Zhou ◽  
Renhua Miao ◽  
Shoudong Ni ◽  
Meng Li

Introduction:: Asari Radix et Rhizoma (ARR) and dried ginger (Zingiber officinalis) (DG) are often used together in drug preparations in traditional Chinese medicine (TCM) to treat respiratory diseases including cold, bronchitis and pneumonia. Previous studies suggested that ARR and/or DG may influence the pharmacokinetics of other herbal components. In the current study, we examined pharmacokinetic interactions between ARR and DG in rats after oral administration. Methods:: We developed a method based on ultra-high-performance liquid chromatography-tandem mass spectrometry to simultaneously measure serum concentrations of two active components each in ARR (L-asarinin and sesamin) and DG (6-gingerol and 6-shogaol). Adult Sprague-Dawley rats were starved overnight, then given ARR extract, DO extract, or a co-decoction of ARR and DG by gastric gavage (6 g raw material per kg body weight; n = 6 per group). Blood samples were collected prior to drug administration and at the following times (h) afterward: 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0 and 24.0. Pharmacokinetic parameters were compared using Student’s t test for independent samples. Results:: A simple, rapid, sensitive analytical method has been developed to detect four bioactive components simultaneously in the ARR-DG herbal pair. Pharmacokinetic parameters including Cmax, Tmax, T1/2 and AUC(0~t) were calculated using the non-compartmental model with the DAS 2.0 pharmacokinetic software. For L-asarinin, Tmax was 2.00 ± 0.00 h in ARR animals and 1.67±0.26 h in ARR-DG animals (P<0.05), T1/2 was 8.58 ± 1.75 h in ARR and 11.93 ± 2.13 h in ARR-DG (P<0.05). For 6-gingerol, Cmax was 350.48 ± 23.85 ng/mL in DG animals and 300.21 ± 20.02 ng/mL in ARR-DG (P<0.01), Tmax was 2.83 ± 0.41 h in DG and 2.17 ± 0.41 h in ARR-DG (P<0.05) and AUC(0~t) was 1.93 ± 0.15 mg/mL•h in ARR and 1.70 ± 0.15 mg/mL•h in ARR-DG (P<0.05). For 6-shogaol, Cmax was 390.28 ± 26.02 ng/mL in DG animals and 455.63 ± 31.01 ng/mL in ARR-DG (P<0.01), Tmax was 2.93 ± 0.10 h in DG and 1.92 ± 0.10 h in ARR-DG (P<0.01), T1/2 was 3.74 ± 0.29 h in DG and 3.28 ± 0.22 h in ARR-DG (P<0.01), and AUC(0~t) was 2.15 ± 0.18 mg/mL•h in DG and 2.73 ± 0.15 mg/mL•h in ARR-DG (P<0.01). Conclusions:: Pharmacokinetic interations between ARR and DG decrease Tmax, increase T1/2 but do not affect overall bioavailability of L-asarinin in ARR. The interactions in ARR-DG decrease Cmax and Tmax but increase T1/2 and AUC(0~t) of 6-gingerol in DG. The interactions increase Cmax and AUC(0~t) but decrease Tmax and T1/2 of 6- shogaol in DG. Interactions in ARR-DG do not affect the pharmacokinetics of sesamin.


2021 ◽  
Author(s):  
Xi Cen ◽  
Yan Wang ◽  
LeiLei Zhang ◽  
XiaoXiao Xue ◽  
Yan Wang ◽  
...  

Abstract BackgroundType 2 diabetes mellitus (T2DM) is regarded as Pi Dan disease in traditional Chinese medicine (TCM). Dahuang Huanglian Xiexin Decoction (DHXD), a classical TCM formula, has been used for treating Pi Dan disease in clinic, its pharmacological mechanism has not been elucidated. MethodsThis study used network pharmacological analysis and molecular docking approach to explore the mechanism of DHXD on T2DM. Firstly, the compounds in DHXD were obtained from TCMSP and TCMID databases, the potential targets were determined based on TCMSP and UniProt databases. Next, Genecards, Digenet and UniProt databases were used to identify the targets of T2DM. Then, the protein-protein interaction (PPI) network was established with overlapping genes of T2DM and compounds, and the core targets in the network were identified and analyzed. Then, the David database was used for GO and KEGG enrichment analysis. Finally, the target genes were selected and the molecular docking was completed by Autodock software to observe the binding level of active components with target genes.ResultsA total of 397 related components and 128 overlapping genes were identified. After enrichment analysis, it was found that HIF-1, TNF, IL-17 and other signaling pathways, as well as DNA transcription, gene expression, apoptosis and other cellular biological processes had the strongest correlation with the treatment of T2DM by DHXD, and most of them occurred in the extracellular space, plasma membrane and other places, which were related to enzyme binding and protein binding. In addition, 42 core genes of DHXD, such as VEGFA, TP53 and MAPK1, were considered as potential therapeutic targets, indicating the potential mechanism of DHXD on T2DM. Finally, the results of molecular docking showed that HIF-1 pathway had strong correlation with the target genes INSR and GLUT4, quercetin and berberine had the strongest binding power with them respectively.ConclusionThis study summarized the main components of DHXD in the treatment of T2DM, identified the core genes and pathways, and systematically analyzed the interaction of related targets, trying to lay the foundation for clarifying the potential mechanism of DHXD on T2DM, so as to carry out further research in the future.


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