EGCG modulates PKD1 and ferroptosis to promote recovery in ST rats.

BACKGROUND: Spinal cord injury (SCI) causes devastating loss of function and neuronal death without effective treatment. (-)-Epigallocatechin-3-gallate (EGCG) has antioxidant properties and plays an essential role in the nervous system. However, the underlying mechanism by which EGCG promotes neuronal survival and functional recovery in complete spinal cord transection (ST) remains unclear. METHODS: In the present study, we established primary cerebellar granule neurons (CGNs) and a T10 ST rat model to investigate the antioxidant effects of EGCG via its modulation of protein kinase D1 (PKD1) phosphorylation and inhibition of ferroptosis. RESULTS: We revealed that EGCG significantly increased the cell survival rate of CGNs and PKD1 phosphorylation levels in comparison to the vehicle control, with a maximal effect observed at 50 µM. EGCG upregulated PKD1 phosphorylation levels and inhibited ferroptosis to reduce the cell death of CGNs under oxidative stress and to promote functional recovery and ERK phosphorylation in rats following complete ST. CONCLUSION: Together, these results lay the foundation for EGCG as a novel strategy for the treatment of SCI related to PKD1 phosphorylation and ferroptosis.

EGCG treats ICH via up-regulating miR-137-3p and inhibiting Parthanatos.

Intracranial hemorrhage (ICH) causes high mortality and disability without effective treatment in the clinical setting. (-)-Epigallocatechin-3-gallate (EGCG) exerts an essential role in the central nervous system and offers a promising therapeutic agent for the treatment of oxidative damage-related diseases. MiR-137 can inhibit the oxidative stress and apoptosis to attenuate neuronal injury. However, the role of EGCG in regulating miR-137-3p and neuronal Parthanatos remains to be unclear. In the present study, we build the ICH mice model to investigate the antioxidant effects of EGCG via upregulating miR-137-3p and inhibiting neuronal Parthanatos. We revealed that EGCG upregulated miR-137-3p and inhibited neuronal Parthanatos, and promoted the functional recovery, alleviated ICH-induced brain injury, and reduced oxidative stress in mice following ICH. However, following the inhibition of miR-137-3p and activation of Parthanatos, EGCG was unable to exert neuroprotective roles. These combined results suggest that EGCG may upregulate miR-137-3p and inhibit neuronal Parthanatos to accelerate functional recovery in mice after ICH, laying the foundation for EGCG to be a novel strategy for the treatment of neuronal injuries related to Parthanatos.

Full-length sequencing of ginkgo transcriptomes for an in-depth understanding of flavonoid and terpenoid trilactone biosynthesis.

Ginkgo biloba L. is regarded as the most ancient living tree, and its kernel has been used as a traditional Chinese medicine for more than 2,000 years. The leaf extracts of this tree have been among the bestselling herbal remedies in Western countries since the last century. To understand the biosynthesis of the pharmacologically active ingredients in G. biloba, flavonoids and terpenoid trilactones (TTLs), we sequenced the transcriptomes of G. biloba leaves, kernels and testae with Iso-Seq and RNA-Seq technologies and obtained 152,524 clean consensus reads. When these reads were used to improve the annotation of the G. biloba genome, 4,856 novel genes, 25,583 new isoforms of previously annotated genes and 4,363 lncRNAs were discovered. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses indicated that genes involved in growth, regulation and response to stress were more likely to be regulated by alternative splicing (AS) or alternative polyadenylation (APA), which represent the two most important posttranscriptional regulation mechanisms. It was found that some of the characterized genes involved in the biosynthesis of flavonoids and TTLs were also possibly regulated by AS and APA. Using phylogenetic and gene expression pattern analyses, some candidate genes for the biosynthesis of flavonoids and TTLs were screened. After qRT-PCR validation, the final candidate genes for flavonoid biosynthesis included three UDP-glycosyltransferases and one MYB transcription factor, while the candidate genes for TTL biosynthesis included two cytochrome P450 and one WRKY transcription factor. Our study suggested that Iso-Seq may play an important role in improving genome annotation, elucidating AS and APA mechanisms and discovering candidate genes involved in the biosynthesis of some secondary metabolites.

Hybrid sequencing of the Gynostemma pentaphyllum transcriptome provides new insights into gypenoside biosynthesis.

BACKGROUND: Gypenosides are a group of triterpene saponins from Gynostemma pentaphyllum that are the same as or very similar to ginsenosides from the Panax species. Several enzymes involved in ginsenoside biosynthesis have been characterized, which provide important clues for elucidating the gypenoside biosynthetic pathway. We suppose that gypenosides and ginsenosides may have a similar biosynthetic mechanism and that the corresponding enzymes in the two pathways may have considerable similarity in their sequences. To further understand gypenoside biosynthesis, we sequenced the G. pentaphyllum transcriptome with a hybrid sequencing-based strategy and then determined the candidate genes involved in this pathway using phylogenetic tree construction and gene expression analysis. RESULTS: Following the PacBio standard analysis pipeline, 66,046 polished consensus sequences were obtained, while Illumina data were assembled into 140,601 unigenes with Trinity software. Then, these output sequences from the two analytical routes were merged. After removing redundant data with CD-HIT software, a total of 140,157 final unigenes were obtained. After functional annotation, five 2,3-oxidosqualene cyclase genes, 145 cytochrome P450 genes and 254 UDP-glycosyltransferase genes were selected for the screening of genes involved in gypenoside biosynthesis. Using phylogenetic analysis, several genes were divided into the same subfamilies or closely related evolutionary branches with characterized enzymes involved in ginsenoside biosynthesis. Using real-time PCR technology, their expression patterns were investigated in different tissues and at different times after methyl jasmonate induction. Since the genes in the same biosynthetic pathway are generally coexpressed, we speculated that GpOSC1, GpCYP89, and GpUGT35 were the leading candidates for gypenoside biosynthesis. In addition, six GpWRKYs and one GpbHLH might play a possible role in regulating gypenoside biosynthesis. CONCLUSIONS: We developed a hybrid sequencing strategy to obtain longer length transcriptomes with increased accuracy, which will greatly contribute to downstream gene screening and characterization, thus improving our ability to elucidate secondary metabolite biosynthetic pathways. With this strategy, we found several candidate genes that may be involved in gypenoside biosynthesis, which laid an important foundation for the elucidation of this biosynthetic pathway, thus greatly contributing to further research in metabolic regulation, synthetic biology and molecular breeding in this species.

Iso-Seq analysis of the Taxus cuspidata transcriptome reveals the complexity of Taxol biosynthesis.

BACKGROUND: Taxus cuspidata is well known worldwide for its ability to produce Taxol, one of the top-selling natural anticancer drugs. However, current Taxol production cannot match the increasing needs of the market, and novel strategies should be considered to increase the supply of Taxol. Since the biosynthetic mechanism of Taxol remains largely unknown, elucidating this pathway in detail will be very helpful in exploring alternative methods for Taxol production. RESULTS: Here, we sequenced Taxus cuspidata transcriptomes with next-generation sequencing (NGS) and third-generation sequencing (TGS) platforms. After correction with Illumina reads and removal of redundant reads, more than 180,000 nonredundant transcripts were generated from the raw Iso-Seq data. Using Cogent software and an alignment-based method, we identified a total of 139 cytochrome P450s (CYP450s), 31 BAHD acyltransferases (ACTs) and 1940 transcription factors (TFs). Based on phylogenetic and coexpression analysis, we identified 9 CYP450s and 7 BAHD ACTs as potential lead candidates for Taxol biosynthesis and 6 TFs that are possibly involved in the regulation of this process. Using coexpression analysis of genes known to be involved in Taxol biosynthesis, we elucidated the stem biosynthetic pathway. In addition, we analyzed the expression patterns of 12 characterized genes in the Taxol pathway and speculated that the isoprene precursors for Taxol biosynthesis were mainly synthesized via the MEP pathway. In addition, we found and confirmed that the alternative splicing patterns of some genes varied in different tissues, which may be an important tissue-specific method of posttranscriptional regulation. CONCLUSIONS: A strategy was developed to generate corrected full-length or nearly full-length transcripts without assembly to ensure sequence accuracy, thus greatly improving the reliability of coexpression and phylogenetic analysis and greatly facilitating gene cloning and characterization. This strategy was successfully utilized to elucidate the Taxol biosynthetic pathway, which will greatly contribute to the goals of improving the Taxol content in Taxus spp. using molecular breeding or plant management strategies and synthesizing Taxol in microorganisms using synthetic biological technology.

Single nucleotide polymorphism of rs2596542 and the risk of hepatocellular carcinoma development: A meta-analysis.

BACKGROUND: Major histocompatibility complex class I-related chain A (MICA) is considered as a tumor antigen, and its expression is affected by its genetic polymorphisms. However, the relationship between rs2596542 polymorphisms in MICA promoter region and hepatocellular carcinoma (HCC) is not fully elucidated so far. This study aims to explore the relationship between single nucleotide polymorphism of rs2596542 and the risk of HCC development through meta-analysis. METHODS: MEDLINE, Web of Science, and EMBASE databases were systematically searched to identify relevant studies. A meta-analysis was performed to examine the association between MICA rs2596542 polymorphism and susceptibility to HCC. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. RESULTS: Fourteen case-control studies involving 4,900 HCC cases and 19,519 controls were included. The MICA rs2596542C allele was significantly associated with decreased risk of HCC based on allelic contrast (OR = 0.76, 95% CI = 0.69-0.83, P < .001), homozygote comparison (OR = 0.57, 95% CI = 0.48-0.69, P < .001), and a recessive genetic model (OR = 0.77, 95% CI = 0.65-0.91, P < .001), whereas patients carrying the MICA rs2596542TT genotype had significantly higher risk of HCC than those with the CT or CC genotype (TT vs CT + CC, OR = 1.57, 95% CI = 1.36-1.81, P < .001). Subgroups analyses based on the ethnic or the source of control groups found very similar findings. CONCLUSION: The C allele in MICA rs2596542 is a protective factor for hepatocarcinogenesis, whereas the T allele is a risk factor. Further large and well-designed studies are needed to confirm this conclusion.

Adjuvant transarterial chemoembolization to improve the prognosis of hepatocellular carcinoma following curative resection.

The present study aimed to investigate the prognostic factors for recurrence of hepatocellular carcinoma (HCC) following curative resection, and evaluate the efficacy of postoperative adjuvant transarterial chemoembolization (TACE) in improving prognosis. A total of 166 patients who underwent curative resection followed by adjuvant TACE, and 190 patients who underwent curative resection alone were studied retrospectively. Univariate and multivariate analyses were performed to investigate the risk factors of recurrence. Separated based on risk factors, subgroup studies were conducted and the association between TACE and recurrence rates was examined. Postoperative overall survival rates were determined using the Kaplan-Meier method and differences between the two therapeutic strategies were identified through log-rank analysis. Computerized tomography (CT)/magnetic resonance imaging (MRI) images were used to evaluate the function of postoperative adjuvant TACE for enhancing the efficacy of CT/MRI in detecting recurrence. The results of the univariate and multivariate analyses revealed that tumor capsule invasion, vascular invasion, and multiple nodules were risk factors of early recurrence. For patients with these risk factors, recurrence rates were markedly decreased at 6 and 12 months, but not at 18 and 24 months, if TACE was added to curative resection. The hepatitis B virus (HBV) was a risk factor for late recurrence. Postoperative TACE was not effective in reducing the recurrence rate in patients with HBV. Survival increased following curative resection with TACE compared with curative resection alone. Furthermore, adjuvant TACE enhanced the efficacy of CT/MRI in detecting recurrence. Postoperative adjuvant TACE may improve the prognosis of HCC following curative resection.

Complete chloroplast genome sequence of the medicinal plant Gynostemma pentaphyllum.

We report the complete chloroplast genome sequence of Gynostemma pentaphyllum, a well-known traditional Chinese medicine, which produces triterpenoid saponins similar to Panax ginseng. The assembled chloroplast genome (cpDNA) was 157,654 bp in length and structurally divided into four distinct regions, namely, large single copy region (86,794 bp), small single copy region (18,654 bp) and a pair of inverted repeat regions (26,103 bp). A total of 143 genes were annotated, including 87 protein-coding genes, 10 tRNA genes and 46 rRNA genes. Phylogenetic analysis revealed that the chloroplast genome sequence of G. pentaphyllum is most closely related to Cucumis melo.

[Analysis of codon usage bias based on Fritillaria cirrhosa transcriptome].

Understanding of codon usage bias of Fritillaria cirrhosa can provide theoretical basis for heterologous biosynthesis of F. cirrhosa alkaloids by genetic engineering technology. A total of 9 843 full length coding sequences (CDS) from the F. cirrhosa transcriptome data were used for the analysis of codon usage bias. The GC and GC3s contents, effective number of codons(ENC) and relative synonymous codon usage (RSCU) were calculated using the CodonW software. The results show that the codon usage bias value is low in the CDS of F. cirrhosa. A total of 15 codons, including UUG, CUU, AUU, GUU, UCA, CCU, CCA, ACU, ACA, GCA, UAU, CAU, AAU, AGA and GGA, were identified as optimal codons in F. cirrhosa. The optimal codons generally end with A/T at the third codon position. By the transcriptome annotation, we found 26 CDSs possibly involved in the biosynthesis of alkaloids in the F. cirrhosa. The proportion of rare codons of Escherichia coli and Saccharomyces cerevisiae are low in these CDSs. We also proposed a method for the codonoptimization in these target genes. Our work lays the foundation for further study on the biosynthesis of alkaloids of the F. cirrhosa in heterologous species.

[Key technologies in synthetic biology of natural products].

Natural products with complex and diverse structures are the major sources of new drugs. The biosynthesis of natural products is considered to be one of the best ways to solve the problems of complex and scarce natural products. DNA assembly technology and genome editing technology are two key technologies in the emerging interdisciplinary field of synthetic biology. A number of novel DNA assembly methods developed in the last few years have paved the way for the engineering of high molecular weight DNA molecules, including whole genomes, hence, it can realize the reconstruction of the metabolic pathways and speed up optimization process. A wide variety of new tools for microbial genome editing will be applied widely to modify the chassis genome to increase its adaptation with the exogenetic pathways. This article summarized the latest advance with respect to DNA assembly and genome editing, which aims to provide help for reconstruction and optimization of the synthetic biological systems of natural products.

[Strategies of elucidation of biosynthetic pathways of natural products].

Elucidation of the biosynthetic pathways of natural products is not only the major goal of herb genomics, but also the solid foundation of synthetic biology of natural products. Here, this paper reviewed recent advance in this field and put forward strategies to elucidate the biosynthetic pathway of natural products. Firstly, a proposed biosynthetic pathway should be set up based on well-known knowledge about chemical reactions and information on the identified compounds, as well as studies with isotope tracer. Secondly, candidate genes possibly involved in the biosynthetic pathway were screened out by co-expression analysis and/or gene cluster mining. Lastly, all the candidate genes were heterologously expressed in the host and then the enzyme involved in the biosynthetic pathway was characterized by activity assay. Sometimes, the function of the enzyme in the original plant could be further studied by RNAi or VIGS technology. Understanding the biosynthetic pathways of natural products will contribute to supply of new leading compounds by synthetic biology and provide "functional marker" for herbal molecular breeding, thus but boosting the development of traditional Chinese medicine agriculture.

[Advance in flavonoids biosynthetic pathway and synthetic biology].

Flavonoids are the valuable components in medicinal plants, which possess a variety of pharmacological activities, including anti-tumor, antioxidant and anti-inflammatory activities. There is an unambiguous understanding about flavonoids biosynthetic pathway, that is,2S-flavanones including naringenin and pinocembrin are the skeleton of other flavonoids and they can transform to other flavonoids through branched metabolic pathway. Elucidation of the flavonoids biosynthetic pathway lays a solid foundation for their synthetic biology. A few flavonoids have been produced in Escherichia coli or yeast with synthetic biological technologies, such as naringenin, pinocembrin and fisetin. Synthetic biology will provide a new way to get valuable flavonoids and promote the research and development of flavonoid drugs and health products, making flavonoids play more important roles in human diet and health.

[Advance in biosynthesis of terpenoid indole alkaloids and its regulation in Catharanthus roseus].

Catharanthus roseus can produce a variety of terpenoid indole alkaloids (TIA), most of which exhibit strong pharmacological activities. Hence, biosynthesis and regulation of TIA have received recent attention. 3α (S)-strictosidine is an important node in TIA biosynthesis, which is a condensation product of secologanin and tryptamine. The former is produced in iridoid pathway, and the latter is produced in indole pathway. Vindoline and catharanthine, which are produced respectively by 3α (S)-strictosidine via multi-step enzymatic reaction, can form α-3, 4-anhydrovinblastine by the condensation reaction. Then, vinblastine and vincristine are generated from α-3, 4-anhydrovinblastine. Many transcription factors are involved in the regulation of TIA synthesis, such as AP2/ERF and WRKY. Illumination of biosynthetic pathway has laid a foundation for the study of synthetic biology. Today, 3α (S)-strictosidine and vindoline have been synthesized in heterologous hosts Saccharomyces cerevisiae.Research about synthetic biology and the regulation mechanisms will provide a guidance for the production and development of TIA drugs in C. roseus.

[Advance in transcriptomic studies of ginseng species].

There are many valuable medicinal plants in Ginseng genus belonging to Araliaceae. Among them, Panax ginseng, P. quinquefolium and P. notoginseng are the most famous species. With the development of next-generation sequencing (NGS) technologies, sequencing and analysis of transcriptomes have become powerful tools for discovery of novel genes, screening molecular markers and elucidation of specific biosynthetic pathway of secondary metabolites. Their transcriptomes provided abundant genes for further study on functional genomics. Here this paper summarized the recent advances in the transcriptomic studies of these three medicinal plants, including discovery of novel genes and elucidation of metabolic regulation, which will contribute to functional genomics in ginseng species.

[Recent advances in biosynthetic pathway and synthetic biology of taxol].

Taxol, a kind of terpenoid secondary metabolite produced by Taxus brevifolia, is an effective anticancer drug that manufacture relies mainly on the extraction form plants. In order to solve the resource shortage, a lot of work has been done to develop the alternative method. Recently, using synthetic biology to realize heterologous biosynthesis of the precursors of taxol has become a hotspot. Now, the basic framework of taxol biosynthetic pathways has been confirmed, and most enzyme genes involved in taxol biosynthesis have been cloned and identified. The two taxol precursors, taxa-4(5),11(12)-diene and taxa-4(20),11(12)-dien-5α-ol, have been synthesized in Escherichia coli and Saccharomyces cerevisiae. Here this paper reviewed the recent advances in the biosynthetic pathway of taxol and the latest developments of synthetic biology, which aims to provide a guidance for the heterologous biosynthesis of taxol.

[Codon usage bias of Catharanthus roseus].

This study aimed to provide guidance for the heterogenous gene expression, gene prediction and species evolution by analyzing codon usage bias of Catharanthus roseus.The codon composition and usage bias of 30 437 high-confidence coding sequences from C.roseus were analyzed and the proportion of rare codons of Escherichia coli and Saccharomyces cerevisiae in 25 genes involved in the biosynthesis of terpenoid indole alkaloids (TIAs) in C.roseus were calculated.The results showed that the average GC content of the genes was 42.47%; the average GC content of the third bases in codon was 35.89%.The relative synonymous codon usage (RSCU) of 28 codons were greater than 1 and 26 of them ended with A or T.The above 25 genes involved in TIA biosynthesis contained much more rare condons of E.coli than that of S.cerevisiae.It was concluded that C.roseus mainly prefered the codons ending with A or T and the rule of codon usage was more different to E.coli than S.cerevisiae.Thus, S.cerevisiae may be more suitable host for heterologous expression of these genes.

Transducin (Beta)-Like 1 X-Linked Receptor 1 Correlates with Clinical Prognosis and Epithelial-Mesenchymal Transition in Hepatocellular Carcinoma.

BACKGROUND: Recent studies have demonstrated that transducin (beta)-like 1 X-linked receptor 1 (TBLR1) is involved in tumor progression. However, the exact role and clinical significance of TBLR1 in hepatocellular carcinoma (HCC) are poorly understood. AIM: In this study, we aimed to investigate the expression and clinical significance of TBLR1 in HCC. METHODS: Quantitative polymerase chain reaction and immunohistochemical staining were performed to detect the expression levels of TBLR1 in HCC tissue and adjacent noncancerous tissue (ANT). The relationships between TBLR1 expression and clinicopathological factors were examined in this study. The effects of TBLR1 on epithelial-mesenchymal transition (EMT) of HCC cells were investigated in vitro. RESULTS: The expression levels of TBLR1 were elevated in HCC cell lines. TBLR1 mRNA in HCC tissue was markedly higher (P < 0.001) than that in ANT. High expression of TBLR1 is closely related to serum alpha fetoprotein (P = 0.047), BCLC stage (P < 0.001), maximum size of tumors (P < 0.001), tumor embolus (P < 0.001), and histological grade (P < 0.001). The disease-free survival and overall survival of HCC patients with high expression of TBLR1 were significantly shorter. Furthermore, we found that EMT of HCC cells could be induced by up-regulating TBLR1 and be inhibited by down-regulating TBLR1. ICG-001, the inhibitor of Wnt/ß-catenin signaling, could suppress induction of EMT mediated by TBLR1. CONCLUSIONS: Our finding suggested that TBLR1 is likely to be a potential prognostic indicator and therapeutic target for HCC and that TBLR1 may be implicated in EMT of HCC cells.

[Single-incision laparoscopic appendectomy versus conventional laparoscopic appendectomy for adult acute appendicitis: a Meta-analysis for randomized controlled trials].

OBJECTIVE: To evaluate the efficiency and safety for single-incision laparoscopic appendectomy (SILA) and conventional laparoscopic appendectomy (CLA). METHODS: The literature of randomized controlled trials (RCTs) concerning SILA versus CLA was retrieved by searching the electronic databases from the inception date to May 2014. Methodological quality of the included trials was assessed by using the Cochrane Reviewers' Handbook criteria, and the data were extracted and subjected to Meta-analysis by using RevMan 5.1 analysis software. RESULTS: A total of 1 183 patients were finally selected after the screening. Among them, 582 cases underwent SILA and 601 cases underwent CLA. Meta-analysis results demonstrated that SILA was associated with higher conversion rate (RR=4.38, 95% CI 1.96-9.79, Z=3.59, P=0.0003), longer operative time (RR=4.83, 95% CI 1.57-8.09, Z=2.90, P=0.004), shorter length of hospital stay (WMD=?0.11, 95%CI ?0.21-?0.01, Z=2.02, P=0.04), and better scores for the postoperative appearance of incision (WMD=0.94, 95%CI 0.49-1.40, Z=4.06, P< 0.001) compared with CLA; no significant difference was observed in postoperative complications and postoperative pain scores (RR=1.03, 95% CI 0.74-1.45, Z=0.18, P=0.86; WMD=?0.19, 95%CI ?0.59-0.20, Z=0.95, P=0.34) between the 2 groups. CONCLUSION: For patients under the conditions of the strict selection, SILA is a safe and effective procedure for the treatment of adult acute appendicitis, especially apply to those who care about incision appearance.