Chao Sun

Ganoderma lucidum is a widely used medicinal macrofungus in traditional Chinese medicine that creates a diverse set of bioactive compounds. Here we report its 43.3-Mb genome, encoding 16,113 predicted genes, obtained using next-generation sequencing and optical mapping approaches. The sequence analysis reveals an impressive array of genes encoding cytochrome P450s (CYPs), transporters and regulatory proteins that cooperate in secondary metabolism. The genome also encodes one of the richest sets of wood degradation enzymes among all of the sequenced basidiomycetes. In all, 24 physical CYP gene clusters are identified. Moreover, 78 CYP genes are coexpressed with lanosterol synthase, and 16 of these show high similarity to fungal CYPs that specifically hydroxylate testosterone, suggesting their possible roles in triterpenoid biosynthesis. The elucidation of the G. lucidum genome makes this organism a potential model system for the study of secondary metabolic pathways and their regulation in medicinal fungi.

Salvia miltiorrhiza is an important medicinal plant with great economic and medicinal value. The complete chloroplast (cp) genome sequence of Salvia miltiorrhiza, the first sequenced member of the Lamiaceae family, is reported here. The genome is 151,328 bp in length and exhibits a typical quadripartite structure of the large (LSC, 82,695 bp) and small (SSC, 17,555 bp) single-copy regions, separated by a pair of inverted repeats (IRs, 25,539 bp). It contains 114 unique genes, including 80 protein-coding genes, 30 tRNAs and four rRNAs. The genome structure, gene order, GC content and codon usage are similar to the typical angiosperm cp genomes. Four forward, three inverted and seven tandem repeats were detected in the Salvia miltiorrhiza cp genome. Simple sequence repeat (SSR) analysis among the 30 asterid cp genomes revealed that most SSRs are AT-rich, which contribute to the overall AT richness of these cp genomes. Additionally, fewer SSRs are distributed in the protein-coding sequences compared to the non-coding regions, indicating an uneven distribution of SSRs within the cp genomes. Entire cp genome comparison of Salvia miltiorrhiza and three other Lamiales cp genomes showed a high degree of sequence similarity and a relatively high divergence of intergenic spacers. Sequence divergence analysis discovered the ten most divergent and ten most conserved genes as well as their length variation, which will be helpful for phylogenetic studies in asterids. Our analysis also supports that both regional and functional constraints affect gene sequence evolution. Further, phylogenetic analysis demonstrated a sister relationship between Salvia miltiorrhiza and Sesamum indicum. The complete cp genome sequence of Salvia miltiorrhiza reported in this paper will facilitate population, phylogenetic and cp genetic engineering studies of this medicinal plant.

Salvia miltiorrhiza Bunge (Danshen) is a medicinal plant of the Lamiaceae family, and its dried roots have long been used in traditional Chinese medicine with hydrophilic phenolic acids and tanshinones as pharmaceutically active components (Zhang et al., 2014Zhang Y. Yan Y.P. Wu Y.C. Hua W.P. Chen C. Ge Q. Wang Z.Z. Pathway engineering for phenolic acid accumulations in Salvia miltiorrhiza by combinational genetic manipulation.Metab. Eng. 2014; 21: 71-80Crossref PubMed Scopus (60) Google Scholar, Xu et al., 2016Xu Z. Ji A. Zhang X. Song J. Chen S. Biosynthesis and regulation of active constituents in medicinal model plant Salvia miltiorrhiza.Chin. Herbal Med. 2016; 8: 3-11Crossref Google Scholar). The first step of tanshinone biosynthesis is bicyclization of the general diterpene precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) to copalyl diphosphate (CPP) by CPP synthases (CPSs), which is followed by a cyclization or rearrangement reaction catalyzed by kaurene synthase-like enzymes (KSL). The resulting intermediate is usually an olefin, which requires the insertion of oxygen by cytochrome P450 mono-oxygenases (CYPs) for the final production of diterpenoids (Zi et al., 2014Zi J. Mafu S. Peters R.J. To gibberellins and beyond! Surveying the evolution of (di)terpenoid metabolism.Annu. Rev. Plant Biol. 2014; 65: 259-286Crossref PubMed Scopus (187) Google Scholar). While the CPS, KSL, and several early acting CYPs (CYP76AH1, CYP76AH3, and CYP76AK1) for tanshinone biosynthesis have been identified in S. miltiorrhiza (Gao et al., 2009Gao W. Hillwig M.L. Huang L. Cui G. Wang X. Kong J. Yang B. Peters R.J. A functional genomics approach to tanshinone biosynthesis provides stereochemical insights.Org. Lett. 2009; 11: 5170-5173Crossref PubMed Scopus (212) Google Scholar, Guo et al., 2013Guo J. Zhou Y.J. Hillwig M.L. Shen Y. Yang L. Wang Y. Zhang X. Liu W. Peters R.J. Chen X. et al.CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts.Proc. Natl. Acad. Sci. USA. 2013; 110: 12108-12113Crossref PubMed Scopus (272) Google Scholar, Guo et al., 2016Guo J. Ma X. Cai Y. Ma Y. Zhan Z. Zhou Y.J. Liu W. Guan M. Yang J. Cui G. et al.Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones.New Phytol. 2016; 210: 525-534Crossref PubMed Scopus (133) Google Scholar, Zi and Peters, 2013Zi J. Peters R.J. Characterization of CYP76AH4 clarifies phenolic diterpenoid biosynthesis in the Lamiaceae.Org. Biomol. Chem. 2013; 11: 7650-7652Crossref PubMed Scopus (76) Google Scholar), the majority of the overall biosynthetic pathway, as well as the relevant regulatory factors associated with tanshinone production, remains elusive (Figure 1B ). Here we report the draft sequence and analysis of the S. miltiorrhiza genome by a hybrid assembly approach. First, genomic DNA was extracted from S. miltiorrhiza line 99-3, a strain cultivated by IMPLAD, and 158.2 Gb Illumina data were generated on a Hiseq 2000 platform (250-fold genome coverage; Supplemental Table 1) and assembled with Phusion2 (Mullikin and Ning, 2003Mullikin J.C. Ning Z. The phusion assembler.Genome Res. 2003; 13: 81-90Crossref PubMed Scopus (163) Google Scholar), which resulted in a draft assembly of 558 Mb, with contig N50 of 2.47 kb. Attempts with other assemblers, such as SOAPdenovo and Fermi, gave similar assembly metrics, suggesting intrinsic complexity of this plant genome. We then generated 8.19 Gb of data with the PacBio RS platform (3.74 kb read length on average) and 8.65 Gb Roche/454 data (Supplemental Table 1). Celera Assembler (v7.0) was used for PacBio reads assembly after base-error correction with Roche/454 data, and the resultant contigs were combined with 454 reads for re-assembly. Finally, Illumina reads were mapped onto these contigs to correct single nucleotide polymorphisms (SNPs) and small insertions/deletions (indels) in homozygotes, which were presumably introduced by sequencing chemistry bias. This led to a final genome assembly of 538 Mb, with contig and scaffold N50 of 12.38 kb and 51.02 kb, respectively (Supplemental Table 2). Compared with the estimated genome size of 615 Mb by flow cytometry analysis (Supplemental Figure 1), the relatively small size of the assembled genome might result from the high repeat content of this species, as multiple copies of repetitive elements are presumably collapsed together. By mapping the Illumina reads onto the draft assembly, 1 486 270 heterozygous SNPs (and 302 217 short indels) were identified, corresponding to 2.76 SNPs per kb (Supplemental Table 3). This heterozygosity value was comparable with that of Populus (2.6 polymorphisms per kb) and grape (3.6 SNPs per kb). Sequence annotation revealed that repetitive elements accounted for 54.44% of the genome (Supplemental Table 4), twice that of sesame, another species from the order Lamiales (Wang et al., 2014Wang L. Yu S. Tong C. Zhao Y. Liu Y. Song C. Zhang Y. Zhang X. Wang Y. Hua W. et al.Genome sequencing of the high oil crop sesame provides insight into oil biosynthesis.Genome Biol. 2014; 15: R39Crossref PubMed Scopus (188) Google Scholar). Long terminal repeats were the most abundant, spanning 18.03% of the genome, while 55.58% of the repeats (30.26% of the genome) were unclassified, implying lineage-specific repeat expansion. We predicted 30 478 protein-coding genes in the S. miltiorrhiza genome using ab initio and homology-based gene prediction methods (Supplemental Table 4), which were further validated by RNA-seq data (Xu et al., 2015Xu Z. Peters R.J. Weirather J. Luo H. Liao B. Zhang X. Zhu Y. Ji A. Zhang B. Hu S. et al.Full-length transcriptome sequences and splice variants obtained by a combination of sequencing platforms applied to different root tissues of Salvia miltiorrhiza and tanshinone biosynthesis.Plant J. 2015; 82: 951-961Crossref PubMed Scopus (255) Google Scholar). Most of these genes (91.2%) had homologs in the non-redundant (nr) database at GenBank (E value = 1e−5), and more than half (56.60%) of them could be assigned to KEGG pathways. Among them, 1620 genes encode transcription factors (TFs), including 171 APETALA2, 139 bHLH, 291 MYB, and 78 WRKY family TFs (Supplemental Table 5). Several of these TFs have been previously revealed to be involved in the biosynthesis of tanshinone and phenolic acid (Xu et al., 2016Xu Z. Ji A. Zhang X. Song J. Chen S. Biosynthesis and regulation of active constituents in medicinal model plant Salvia miltiorrhiza.Chin. Herbal Med. 2016; 8: 3-11Crossref Google Scholar). In addition, 82 terpene synthase genes (TPS; Supplemental Table 6) that are involved in the production of hemi-, mono-, sesqui-, or di-terpenes, along with 437 CYPs (Supplemental Table 7) that catalyze various oxidation reactions, were identified. Gene family evolution among eight plant species, including rice, Arabidopsis, grape, tomato, potato, bladderwort, sesame, and S. miltiorrhiza, was analyzed by CAFÉ (version 2.1). The result suggests that gene family contraction outnumbered expansion along each lineage (Figure 1A). Intriguingly, families undergoing significant expansion in S. miltiorrhiza (P < 0.01) were primarily involved in stilbenoid, diarylheptanoid or gingerol biosynthesis (Ko00945), and terpenoid biosynthesis (Ko00902) or steroid biosynthesis (Ko00100), which is consistent with the high production of tanshinones and phenolic acids by this medicinal plant. Phylogenomic analysis revealed that S. miltiorrhiza was most closely related to sesame, with an estimated divergence time of approximately 67 million years ago (Figure 1A). Physical clustering of TPSs and CYPs is frequently associated with consecutive enzymatic actions in terpenoid biosynthesis (Boutanaev et al., 2015Boutanaev A.M. Moses T. Zi J. Nelson D.R. Mugford S.T. Peters R.J. Osbourn A. Investigation of terpene diversification across multiple sequenced plant genomes.Proc. Natl. Acad. Sci. USA. 2015; 112: E81-E88Crossref PubMed Scopus (183) Google Scholar), and was investigated here. Four TPS/CYP pairs were found in the draft S. miltiorrhiza genome (Figure 1C–1F). Three of them have been previously characterized, with SmCPS1 and SmCPS2 involved in tanshinone biosynthesis in the roots and leaves, respectively, while SmCPS5 is required for gibberellin phytohormone metabolism (Cui et al., 2015Cui G. Duan L. Jin B. Qian J. Xue Z. Shen G. Snyder J.H. Song J. Chen S. Huang L. et al.Functional divergence of diterpene syntheses in the medicinal plant Salvia miltiorrhiza Bunge.Plant Physiol. 2015; 169: 1607-1618PubMed Google Scholar). Interestingly, both SmCPS1 and SmCPS2 are flanked by genes from the CYP76AH sub-family. Notably, this includes the previously characterized CYP76AH1 (Guo et al., 2013Guo J. Zhou Y.J. Hillwig M.L. Shen Y. Yang L. Wang Y. Zhang X. Liu W. Peters R.J. Chen X. et al.CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts.Proc. Natl. Acad. Sci. USA. 2013; 110: 12108-12113Crossref PubMed Scopus (272) Google Scholar). More strikingly, one of CYP76AH sub-family members, CYP76AH3, was reported to be involved in tanshinone biosynthesis (Guo et al., 2016Guo J. Ma X. Cai Y. Ma Y. Zhan Z. Zhou Y.J. Liu W. Guan M. Yang J. Cui G. et al.Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones.New Phytol. 2016; 210: 525-534Crossref PubMed Scopus (133) Google Scholar), further confirming the association of these biosynthetic gene clusters with tanshinone biosynthesis. Phylogenetic analysis suggests that the SmCPS1 and SmCPS2 clusters originated from a duplication event of an ancestral CPS/CYP76AH pair (Supplemental Figure 2). To further investigate the role of these clusters in tanshinone biosynthesis, the tissue-specific expression of the genes was analyzed using RNA-seq data. As previously reported (Cui et al., 2015Cui G. Duan L. Jin B. Qian J. Xue Z. Shen G. Snyder J.H. Song J. Chen S. Huang L. et al.Functional divergence of diterpene syntheses in the medicinal plant Salvia miltiorrhiza Bunge.Plant Physiol. 2015; 169: 1607-1618PubMed Google Scholar), SmCPS1 and SmCPS2 are most highly expressed in the roots and leaves/flowers, respectively. However, the expression patterns of the CYP76AH sub-family members do not simply follow that of the co-clustered CPSs. Instead, despite being clustered with the root-specific SmCPS1, CYP76AH12 is equally expressed in both the roots and leaves, although the linked CYP76AH13 is more specifically expressed in roots. In addition, despite being clustered with the more aerial tissue-specific SmCPS2, CYP76AH1 and CYP76AH3 are quite specifically expressed in roots, although the linked CYP76AH28P is more highly expressed in the leaves. All of these expression patterns were validated by qRT–PCR (Figure 1C–1F). Taken together, it seemed to imply that the decoupling of expression between CPSs and their flanking CYPs had occurred after the gene cluster duplication event. The SmCPS7/CYP cluster contains two members of the CYP71 family (Figure 1E), CYP71AT88 and CYP71BS4. Given that a number of CYPs from the CYP71 family are involved in (di)terpenoid biosynthesis (Zi et al., 2014Zi J. Mafu S. Peters R.J. To gibberellins and beyond! Surveying the evolution of (di)terpenoid metabolism.Annu. Rev. Plant Biol. 2014; 65: 259-286Crossref PubMed Scopus (187) Google Scholar), this raises the possibility that this cluster might participate in a common diterpenoid biosynthetic pathway. For the SmCPS5/CYP cluster (Figure 1F), previous work had suggested that SmCPS5 is involved in gibberellin metabolism (Cui et al., 2015Cui G. Duan L. Jin B. Qian J. Xue Z. Shen G. Snyder J.H. Song J. Chen S. Huang L. et al.Functional divergence of diterpene syntheses in the medicinal plant Salvia miltiorrhiza Bunge.Plant Physiol. 2015; 169: 1607-1618PubMed Google Scholar), while CYP735A25v1 has no known function in such phytohormone metabolism. Thus, this particular pair of enzymes seems unlikely to operate together in a common pathway. We then compared the tissue-specific expression patterns of all 437 annotated CYP genes with that of SmCPS1. Thirty-two CYPs exhibited similar expression patterns to SmCPS1 across different organs examined (R2 > 0.85) (Supplemental Table 8). As expected, this includes CYP76AH1, whose role in tanshinone biosynthesis was firstly suggested on the basis of the similar co-expression analysis (Guo et al., 2013Guo J. Zhou Y.J. Hillwig M.L. Shen Y. Yang L. Wang Y. Zhang X. Liu W. Peters R.J. Chen X. et al.CYP76AH1 catalyzes turnover of miltiradiene in tanshinones biosynthesis and enables heterologous production of ferruginol in yeasts.Proc. Natl. Acad. Sci. USA. 2013; 110: 12108-12113Crossref PubMed Scopus (272) Google Scholar), as well as CYP76AH3 and CYP76AK1, which were recently demonstrated to play important roles in tanshinone biosynthesis using the same approach (Guo et al., 2016Guo J. Ma X. Cai Y. Ma Y. Zhan Z. Zhou Y.J. Liu W. Guan M. Yang J. Cui G. et al.Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones.New Phytol. 2016; 210: 525-534Crossref PubMed Scopus (133) Google Scholar). Hence, the remaining co-regulated CYPs provide additional candidates for dissecting the tanshinone biosynthetic pathway. The traditional use of Danshen involves decoction with water, indicating an important role for the hydrophilic phenolic acids. These include rosmarinic acid (RA), salvianolic acid, and lithospermic acid B, whose biosynthesis involves both general phenylpropanoid metabolism and the more specific tyrosine-derived pathway. As reported previously, the genome contains 29 genes from nine families potentially involved in S. miltiorrhiza phenolic acid biosynthesis. Notably, most families had multiple genes with distinct expression patterns, implying diversified roles for these natural products. In addition, from the 80 laccases genes, five were potentially involved in the conversion of RA to salvianolic acid, based on their specific expression in the root phloem and xylem tissues. Thus, the genome sequence reported here provides important insights into the biosynthesis of these water-soluble natural products as well. Compared with the S. miltiorrhiza variety with purple flowers that was used for genome sequencing, the white-flowered landrace of S. miltiorrhiza is known for better medical quality. To evaluate the genetic differences between these varieties, a white-flowered plant was selected for sequencing and subsequent comparative analysis. The number of homozygous SNPs (1 719 024) was roughly twice than that of heterozygotes, corresponding to a fixed polymorphism level of 3.87 SNPs per kb. Overall, 49 521 non-synonymous SNPs were identified, among which 580 protein-coding genes were affected through the formation of premature stop codons. Nine KEGG pathways were significantly enriched with non-synonymous amino acid changes, including pathways for diterpenoid, flavonoid, and phenylpropanoid biosynthesis, as well as those for Toll-like receptor signaling and plant–pathogen interactions (Supplemental Figure 3). While the average sequencing depth for the white-flower plant was 42X, more than 10% of the genome had no coverage at all. For further investigation, 28.6 Mb genomic regions longer than 1 kb with no mapping coverage were analyzed. Interestingly, only 12.68% of these regions were composed of repetitive sequences, a much lower proportion than the genome average (54.44%). In total, these regions contained 107 genes, which appear to have been lost in the white-flower landrace, including 11 disease-resistance genes, four CYPs, and 13 TF encoding genes. At least some of this intergenomic diversity is hypothesized to contribute to the phenotypic differences between these two varieties, such as flower coloration, and tanshinone content, which require future investigations. In summary, we present a draft assembly of the S. miltiorrhiza genome using long reads from the PacBio RS platform to supplement short Illumina reads, which resulted in significant improvement of the assembly quality. This hybrid approach is proved to be effective for the highly repetitive and complex genome of S. miltiorrhiza, enabling the assembly of sufficiently large enough scaffolds for the identification of potential biosynthetic gene clusters. The four CPS/CYP gene clusters revealed here, along with other genes potentially encoding biosynthetic enzymes (e.g., in tashinone biosynthesis; Supplemental Table 9), provide a strong foundation for understanding the biochemical diversity and pharmaceutical qualities of S. miltiorrhiza. Moreover, access to the genome sequence is further expected to aid molecular breeding with this important traditional medicinal herb. This work was supported by the National Natural Science Foundation of China (81130069, 81573398, 31400278), the National Key Technology R&D Program (2012BAI29B01), the Key Project of Chinese National Programs for Fundamental Research and Development (2013CB127000), and the US National Institutes of Health (GM109773).

American ginseng (Panax quinquefolius L.) is one of the most widely used herbal remedies in the world. Its major bioactive constituents are the triterpene saponins known as ginsenosides. However, little is known about ginsenoside biosynthesis in American ginseng, especially the late steps of the pathway.In this study, a one-quarter 454 sequencing run produced 209,747 high-quality reads with an average sequence length of 427 bases. De novo assembly generated 31,088 unique sequences containing 16,592 contigs and 14,496 singletons. About 93.1% of the high-quality reads were assembled into contigs with an average 8-fold coverage. A total of 21,684 (69.8%) unique sequences were annotated by a BLAST similarity search against four public sequence databases, and 4,097 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Based on the bioinformatic analysis described above, we found all of the known enzymes involved in ginsenoside backbone synthesis, starting from acetyl-CoA via the isoprenoid pathway. Additionally, a total of 150 cytochrome P450 (CYP450) and 235 glycosyltransferase unique sequences were found in the 454 cDNA library, some of which encode enzymes responsible for the conversion of the ginsenoside backbone into the various ginsenosides. Finally, one CYP450 and four UDP-glycosyltransferases were selected as the candidates most likely to be involved in ginsenoside biosynthesis through a methyl jasmonate (MeJA) inducibility experiment and tissue-specific expression pattern analysis based on a real-time PCR assay.We demonstrated, with the assistance of the MeJA inducibility experiment and tissue-specific expression pattern analysis, that transcriptome analysis based on 454 pyrosequencing is a powerful tool for determining the genes encoding enzymes responsible for the biosynthesis of secondary metabolites in non-model plants. Additionally, the expressed sequence tags (ESTs) and unique sequences from this study provide an important resource for the scientific community that is interested in the molecular genetics and functional genomics of American ginseng.

Panax notoginseng (Burk) F.H. Chen is important medicinal plant of the Araliacease family. Triterpene saponins are the bioactive constituents in P. notoginseng. However, available genomic information regarding this plant is limited. Moreover, details of triterpene saponin biosynthesis in the Panax species are largely unknown.Using the 454 pyrosequencing technology, a one-quarter GS FLX titanium run resulted in 188,185 reads with an average length of 410 bases for P. notoginseng root. These reads were processed and assembled by 454 GS De Novo Assembler software into 30,852 unique sequences. A total of 70.2% of unique sequences were annotated by Basic Local Alignment Search Tool (BLAST) similarity searches against public sequence databases. The Kyoto Encyclopedia of Genes and Genomes (KEGG) assignment discovered 41 unique sequences representing 11 genes involved in triterpene saponin backbone biosynthesis in the 454-EST dataset. In particular, the transcript encoding dammarenediol synthase (DS), which is the first committed enzyme in the biosynthetic pathway of major triterpene saponins, is highly expressed in the root of four-year-old P. notoginseng. It is worth emphasizing that the candidate cytochrome P450 (Pn02132 and Pn00158) and UDP-glycosyltransferase (Pn00082) gene most likely to be involved in hydroxylation or glycosylation of aglycones for triterpene saponin biosynthesis were discovered from 174 cytochrome P450s and 242 glycosyltransferases by phylogenetic analysis, respectively. Putative transcription factors were detected in 906 unique sequences, including Myb, homeobox, WRKY, basic helix-loop-helix (bHLH), and other family proteins. Additionally, a total of 2,772 simple sequence repeat (SSR) were identified from 2,361 unique sequences, of which, di-nucleotide motifs were the most abundant motif.This study is the first to present a large-scale EST dataset for P. notoginseng root acquired by next-generation sequencing (NGS) technology. The candidate genes involved in triterpene saponin biosynthesis, including the putative CYP450s and UGTs, were obtained in this study. Additionally, the identification of SSRs provided plenty of genetic makers for molecular breeding and genetics applications in this species. These data will provide information on gene discovery, transcriptional regulation and marker-assisted selection for P. notoginseng. The dataset establishes an important foundation for the study with the purpose of ensuring adequate drug resources for this species.

Panax ginseng C. A. Meyer is one of the most widely used medicinal plants. Complete genome information for this species remains unavailable due to its large genome size. At present, analysis of expressed sequence tags is still the most powerful tool for large-scale gene discovery. The global expressed sequence tags from P. ginseng tissues, especially those isolated from stems, leaves and flowers, are still limited, hindering in-depth study of P. ginseng.Two 454 pyrosequencing runs generated a total of 2,423,076 reads from P. ginseng roots, stems, leaves and flowers. The high-quality reads from each of the tissues were independently assembled into separate and shared contigs. In the separately assembled database, 45,849, 6,172, 4,041 and 3,273 unigenes were only found in the roots, stems, leaves and flowers database, respectively. In the jointly assembled database, 178,145 unigenes were observed, including 86,609 contigs and 91,536 singletons. Among the 178,145 unigenes, 105,522 were identified for the first time, of which 65.6% were identified in the stem, leaf or flower cDNA libraries of P. ginseng. After annotation, we discovered 223 unigenes involved in ginsenoside backbone biosynthesis. Additionally, a total of 326 potential cytochrome P450 and 129 potential UDP-glycosyltransferase sequences were predicted based on the annotation results, some of which may encode enzymes responsible for ginsenoside backbone modification. A BLAST search of the obtained high-quality reads identified 14 potential microRNAs in P. ginseng, which were estimated to target 100 protein-coding genes, including transcription factors, transporters and DNA binding proteins, among others. In addition, a total of 13,044 simple sequence repeats were identified from the 178,145 unigenes.This study provides global expressed sequence tags for P. ginseng, which will contribute significantly to further genome-wide research and analyses in this species. The novel unigenes identified here enlarge the available P. ginseng gene pool and will facilitate gene discovery. In addition, the identification of microRNAs and the prediction of targets from this study will provide information on gene transcriptional regulation in P. ginseng. Finally, the analysis of simple sequence repeats will provide genetic makers for molecular breeding and genetic applications in this species.

Dendrobium officinale Kimura et Migo (Orchidaceae) is a traditional Chinese medicinal plant. The stem contains an alkaloid that is the primary bioactive component. However, the details of alkaloid biosynthesis have not been effectively explored because of the limited number of expressed sequence tags (ESTs) available in GenBank. In this study, we analyzed RNA isolated from the stem of D. officinale using a single half-run on the Roche 454 GS FLX Titanium platform to generate 553,084 ESTs with an average length of 417 bases. The ESTs were assembled into 36,407 unique putative transcripts. A total of 69.97% of the unique sequences were annotated, and a detailed view of alkaloid biosynthesis was obtained. Functional assignment based on Kyoto Encyclopedia of Genes and Genomes (KEGG) terms revealed 69 unique sequences representing 25 genes involved in alkaloid backbone biosynthesis. A series of qRT-PCR experiments confirmed that the expression levels of 5 key enzyme-encoding genes involved in alkaloid biosynthesis are greater in the leaves of D. officinale than in the stems. Cytochrome P450s, aminotransferases, methyltransferases, multidrug resistance protein (MDR) transporters and transcription factors were screened for possible involvement in alkaloid biosynthesis. Furthermore, a total of 1061 simple sequence repeat motifs (SSR) were detected from 36,407 unigenes. Dinucleotide repeats were the most abundant repeat type. Of these, 179 genes were associated with a metabolic pathway in KEGG. This study is the first to produce a large volume of transcriptome data from D. officinale. It extends the foundation to facilitate gene discovery in D. officinale and provides an important resource for the molecular genetic and functional genomic studies in this species.

Summary A circular consensus sequencing ( CCS ) strategy involving single molecule, real‐time ( SMRT ) DNA sequencing technology was applied to de novo assembly and single nucleotide polymorphism ( SNP ) detection of chloroplast genomes. Chloroplast DNA was purified from enriched chloroplasts of pooled individuals to construct a shotgun library for each species. The sequencing reactions were performed on a PacBio RS platform. CCS sub‐reads were generated from polymerase reads that passed the native dumbbell‐shaped DNA templates multiple times. The complete chloroplast genome sequence was generated by mapping all reads to the draft sequence constructed in a step‐by‐step manner. The full‐chain, PCR ‐free approach eliminates the possible context‐specific biases in library construction and sequencing reaction. The chloroplast genome was easily and completely assembled using the data generated from one SMRT Cell without requiring a reference genome. Comparisons of the three assembled Fritillaria genomes to 34.1 kb of validation Sanger sequences revealed 100% concordance, and the detected intraspecies SNP s at a minimum variant frequency of 15% were all confirmed. This simple approach with potential for parallel sequencing yields high‐quality chloroplast genomes for sensitive SNP detection and comparative analyses. We recommend this approach for its powerful applicability for evolutionary genetics and genomics studies in plants based on the sequences of chloroplast genomes.

Salvia miltiorrhiza is a Chinese herb with significant pharmacologic effects because of the bioactive compounds of tanshinones and phenolic acids. Methyl jasmonate ( MeJA ) has been used as an effective elicitor to enhance the production of these compounds. However, the molecular mechanism of MeJA ‐mediated tanshinone and salvianolic acid biosynthesis remains unclear. The transcriptional profiles of S. miltiorrhiza leaves at 12 h ( T12 ) after MeJA elicitation and mock‐treated leaves ( T0 ) were generated using the Illumina deep RNA sequencing ( RNA ‐seq) strategy to detect the changes in gene expression in response to MeJA . In total, 37 647 unique sequences were obtained from about 21 million reads, and 25 641 (71.53%) of these sequences were annotated based on the blast searches against the public databases. A total of 5287 unique sequences were expressed differentially between the samples of T0 and T12 , which covered almost all the known genes involved in tanshinone and phenolic acid biosynthesis in S. miltiorrhiza . Many of the transcription factors (e.g. MYB , bHLH and WRKY ) and genes involved in plant hormone biosynthesis and signal transduction were expressed differentially in response to the MeJA induction. Importantly, three and four candidate cytochrome P450s ( P450 s) that could be involved in the tanshinone and phenolic acid biosynthesis, respectively, were selected from the RNA ‐seq data based on co‐expressed pattern analysis with SmCPS1 / SmKSL1 and SmRAS , which are the key genes responsible for biosynthesis. This comprehensive investigation of MeJA ‐induced gene expression profiles can shed light on the molecular mechanisms of the MeJA ‐mediated bioactive compound biosynthesis and regulation in S. miltiorrhiza .

Glycyrrhiza uralensis is one of the most popular medicinal plants in the world and is also widely used in the flavoring of food and tobacco. Due to limited genomic and transcriptomic data, the biosynthetic pathway of glycyrrhizin, the major bioactive compound in G. uralensis, is currently unclear. Identification of candidate genes involved in the glycyrrhizin biosynthetic pathway will significantly contribute to the understanding of the biosynthetic and medicinal chemistry of this compound.We used the 454 GS FLX platform and Titanium regents to produce a substantial expressed sequence tag (EST) dataset from the vegetative organs of G. uralensis. A total of 59,219 ESTs with an average read length of 409 bp were generated. 454 ESTs were combined with the 50,666 G. uralensis ESTs in GenBank. The combined ESTs were assembled into 27,229 unique sequences (11,694 contigs and 15,535 singletons). A total of 20,437 unique gene elements representing approximately 10,000 independent transcripts were annotated using BLAST searches (e-value <or= 1e-5) against the SwissProt, KEGG, TAIR, Nr and Nt databases. The assembled sequences were annotated with gene names and Gene Ontology (GO) terms. With respect to the genes related to glycyrrhizin metabolism, genes encoding 16 enzymes of the 18 total steps of the glycyrrhizin skeleton synthesis pathway were found. To identify novel genes that encode cytochrome P450 enzymes and glycosyltransferases, which are related to glycyrrhizin metabolism, a total of 125 and 172 unigenes were found to be homologous to cytochrome P450s and glycosyltransferases, respectively. The cytochrome P450 candidate genes were classified into 32 CYP families, while the glycosyltransferase candidate genes were classified into 45 categories by GO analysis. Finally, 3 cytochrome P450 enzymes and 6 glycosyltransferases were selected as the candidates most likely to be involved in glycyrrhizin biosynthesis through an organ-specific expression pattern analysis based on real-time PCR.Using the 454 GS FLX platform and Titanium reagents, our study provides a high-quality EST database for G. uralensis. Based on the EST analysis, novel candidate genes related to the secondary metabolite pathway of glycyrrhizin, including novel genes encoding cytochrome P450s and glycosyltransferases, were found. With the assistance of organ-specific expression pattern analysis, 3 unigenes encoding cytochrome P450s and 6 unigenes encoding glycosyltransferases were selected as the candidates most likely to be involved in glycyrrhizin biosynthesis.

Camptotheca acuminata is a Nyssaceae plant, often called the "happy tree", which is indigenous in Southern China. C. acuminata produces the terpenoid indole alkaloid, camptothecin (CPT), which exhibits clinical effects in various cancer treatments. Despite its importance, little is known about the transcriptome of C. acuminata and the mechanism of CPT biosynthesis, as only few nucleotide sequences are included in the GenBank database.From a constructed cDNA library of young C. acuminata leaves, a total of 30,358 unigenes, with an average length of 403 bp, were obtained after assembly of 74,858 high quality reads using GS De Novo assembler software. Through functional annotation, a total of 21,213 unigenes were annotated at least once against the NCBI nucleotide (Nt), non-redundant protein (Nr), Uniprot/SwissProt, Kyoto Encyclopedia of Genes and Genomes (KEGG), and Arabidopsis thaliana proteome (TAIR) databases. Further analysis identified 521 ESTs representing 20 enzyme genes that are involved in the backbone of the CPT biosynthetic pathway in the library. Three putative genes in the upstream pathway, including genes for geraniol-10-hydroxylase (CaPG10H), secologanin synthase (CaPSCS), and strictosidine synthase (CaPSTR) were cloned and analyzed. The expression level of the three genes was also detected using qRT-PCR in C. acuminata. With respect to the branch pathway of CPT synthesis, six cytochrome P450s transcripts were selected as candidate transcripts by detection of transcript expression in different tissues using qRT-PCR. In addition, one glucosidase gene was identified that might participate in CPT biosynthesis. For CPT transport, three of 21 transcripts for multidrug resistance protein (MDR) transporters were also screened from the dataset by their annotation result and gene expression analysis.This study produced a large amount of transcriptome data from C. acuminata by 454 pyrosequencing. According to EST annotation, catalytic features prediction, and expression analysis, novel putative transcripts involved in CPT biosynthesis and transport were discovered in C. acuminata. This study will facilitate further identification of key enzymes and transporter genes in C. acuminata.

Ophiocordyceps sinensis is a highly valuable and popular medicinal fungus used as a tonic and roborant for thousands of years in traditional Asian medicine. However, unsustainable harvesting practices have endangered this species and very little is known about its developmental programming, its biochemistry and genetics. To begin to address this, the transcriptome of the medicinal O. sinensis fruiting body was analyzed by high-throughput. In this O. sinensis 454-EST dataset, four mating type genes and 121 genes that may be involved in fruiting body development, especially in signal transduction and transcription regulation, were discovered. Moreover, a model was developed for the synthesis of the primary medicinal compound, cordycepin, and the putative biosynthetic enzymes identified. This transcriptome dataset provides a significant new resource for gene discovery in O. sinensis and dissection of its valuable biosynthetic and developmental pathways.

Pre-mRNA splicing is a fundamental process that plays a considerable role in generating protein diversity. Pre-mRNA splicing is also the key to the pathology of numerous diseases, especially cancers. In this review, we discuss how aberrant splicing isoforms precisely regulate three basic functional aspects in cancer: proliferation, metastasis and apoptosis. Importantly, clinical function of aberrant splicing isoforms is also discussed, in particular concerning drug resistance and radiosensitivity. Furthermore, this review discusses emerging strategies how to modulate pathologic aberrant splicing isoforms, which are attractive, novel therapeutic agents in cancer. Last we outline current and future directions of isoforms diagnostic methodologies reported so far in cancer. Thus, it is highlighting significance of aberrant splicing isoforms as markers for cancer and as targets for cancer therapy.

Panax notoginseng (Burk) F. H. Chen, an economically significant medicinal plant with hemostatic and health tonic activities, has been used in Traditional Chinese Medicine (TCM) for more than 3,000 years. Triterpene saponins are responsible for most of the pharmacological activities of P. notoginseng. Here, we cloned five cDNA sequences encoding the key enzymes involved in triterpene saponin biosynthesis, namely, PnFPS, PnSS, PnSE1, PnSE2, and PnDS, and analyzed the conserved domains and phylogenetics of their corresponding proteins. Their organ-specific expression patterns in four-year-old P. notoginseng were detected by real-time PCR, showing that they were all most highly expressed in flowers. In addition, four of the genes, excluding PnSE2, were upregulated in leaves following stimulation with methyl jasmonate. This study is the first comprehensive analysis of the expression patterns of pivotal genes for triterpene saponin biosynthesis in P. notoginseng and provides a basis to further elucidate the molecular mechanism for the biosynthesis of these medically important compounds.

Fungi have evolved powerful genomic and chemical defense systems to protect themselves against genetic destabilization and other organisms. However, the precise molecular basis involved in fungal defense remain largely unknown in Basidiomycetes. Here the complete genome sequence, as well as DNA methylation patterns and small RNA transcriptomes, was analyzed to provide a holistic overview of secondary metabolism and defense processes in the model medicinal fungus, Ganoderma sinense. We reported the 48.96 Mb genome sequence of G. sinense, consisting of 12 chromosomes and encoding 15,688 genes. More than thirty gene clusters involved in the biosynthesis of secondary metabolites, as well as a large array of genes responsible for their transport and regulation were highlighted. In addition, components of genome defense mechanisms, namely repeat-induced point mutation (RIP), DNA methylation and small RNA-mediated gene silencing, were revealed in G. sinense. Systematic bioinformatic investigation of the genome and methylome suggested that RIP and DNA methylation combinatorially maintain G. sinense genome stability by inactivating invasive genetic material and transposable elements. The elucidation of the G. sinense genome and epigenome provides an unparalleled opportunity to advance our understanding of secondary metabolism and fungal defense mechanisms.

Diagnosis of bladder cancer, one of the most common types of human cancer, at an early (nonmuscle-invasive) stage is the best way to reduce the mortality rate. Tumor malignancy in general is closely associated with alterations of glycan expression. Glycosylation status, particularly global glycomes, in bladder cancer has not been well studied. We integrated lectin microarray and mass spectrometry (MS) methods to quantitatively analyze and compare glycan expression in four bladder cancer cell lines (KK47, YTS1, J82, T24) and one normal bladder mucosa cell line (HCV29). Glycopattern alterations were analyzed using lectin microarray analysis and confirmed by lectin staining and lectin blotting. Associations of glycopatterns with diverging stages were evaluated by lectin histochemistry on tissue microarrays. N-Glycans were derivatized by amidation of sialylated glycans with acetohydrazide and reductive amination with the stable isotope tags [(12)C6]- and [(13)C6]-aniline, and were quantitatively analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). N-Glycan biosynthesis-associated proteins were quantitatively analyzed by a stable isotope labeling by amino acids in cell culture (SILAC) proteomics method, which revealed significant differences in expression of 13 glycosyltransferases and 4 glycosidases. Our findings indicate that sialyl Lewis X (sLe(x)), terminal GalNAc and Gal, and high mannose-type N-glycans were more highly expressed in bladder cancer cells and tissues than in normal cells. Bladder cancer cells showed high expression of core-fucosylated N-glycans but low expression of terminally fucosylated N-glycans. Each of these glycome changes may be directly related to bladder cancer progression.

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. 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. 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.

Graphene, a new type of nanomaterial, has unique physical properties and important potential biological applications. However, few studies have been conducted on the environmental impact of graphene. Therefore, to explore the effect of graphene on plants, three-week-old, tissue-cultured ‘Gala’ apple plants (Malus domestica) were treated with different concentrations (0, 0.1, 1, 10 mg/L) of graphene oxide (GO) and examined after 40 days. Results indicated that adventitious root length, moisture content and the number of lateral roots were all inhibited by 0.1–10 mg/L GO. At 0.1 and 1 mg/L GO, however, the number of adventitious roots and the rooting rate exhibited a significant increase, relative to the control (no GO). Treatment with GO increased the activities of oxidative stress enzymes including catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in the apple plants, relative to controls. Malondialdehyde (MDA) levels were also significantly decreased at 10 mg/L GO. Treatment of apple plantlets with 0.1 mg/L GO increased the transcript abundance of auxin efflux carrier (PIN7, ABCB1) genes and auxin influx carrier (LAX2, LAX3) genes but inhibited the transcript levels of the ARR3 gene, which involved in cytokinin biosynthesis. Additionally, the transcript levels of ARRO1, ARF19, and TTG1, which play roles in the formation of adventitious roots, lateral roots, and root hairs, respectively, were all decreased in response to treatment with 1 and 10 mg/L GO. Collectively, the results indicate that treatment of ‘Gala’ apple plants with 0.1 mg/L GO had a positive effect on root formation but a negative effect on root growth. This response may be related to the negative impact of GO on cellular structure and function.

Cancer is a hyper-proliferative disease. Whether the proliferative state originates from the cell-of-origin or emerges later remains difficult to resolve. By tracking de novo transformation from normal hematopoietic progenitors expressing an acute myeloid leukemia (AML) oncogene MLL-AF9, we reveal that the cell cycle rate heterogeneity among granulocyte-macrophage progenitors (GMPs) determines their probability of transformation. A fast cell cycle intrinsic to these progenitors provide permissiveness for transformation, with the fastest cycling 3% GMPs acquiring malignancy with near certainty. Molecularly, we propose that MLL-AF9 preserves gene expression of the cellular states in which it is expressed. As such, when expressed in the naturally-existing, rapidly-cycling immature myeloid progenitors, this cell state becomes perpetuated, yielding malignancy. In humans, high CCND1 expression predicts worse prognosis for MLL fusion AMLs. Our work elucidates one of the earliest steps toward malignancy and suggests that modifying the cycling state of the cell-of-origin could be a preventative approach against malignancy.

Abstract The anthocyanin content in apple skin determines its red coloration, as seen in a Fuji apple mutant. Comparative RNA-seq analysis was performed to determine differentially expressed genes at different fruit development stages between the wild-type and the skin color mutant. A novel R2R3-MYB transcription factor, MdMYB90-like, was uncovered as the key regulatory gene for enhanced coloration in the mutant. The expression of MdMYB90-like was 21.3 times higher in the mutant. MdMYB90-like regulates anthocyanin biosynthesis directly through the activation of anthocyanin biosynthesis genes and indirectly through the activation of other transcription factors that activate anthocyanin biosynthesis. MdMYB90-like bound to the promoters of both structural genes ( MdCHS and MdUFGT ) and other transcription factor genes ( MdMYB1 and MdbHLH3 ) in the yeast one-hybrid system, electrophoretic mobility shift assay, and dual-luciferase assay. Transgenic analysis showed that MdMYB90-like was localized in the nucleus, and its overexpression induced the expression of other anthocyanin-related genes, including MdCHS , MdCHI , MdANS , MdUFGT , MdbHLH3 , and MdMYB1 . The mutant had reduced levels of DNA methylation in two regions (−1183 to −988 and −2018 to −1778) of the MdMYB90-like gene promoter, which might explain the enhanced expression of the gene and the increased anthocyanin content in the mutant apple skin.

There is accumulating evidence that mitochondrial dysfunction is associated with the contribution of diabetes to Alzheimer disease (AD) progression. Neuronal mitochondrial proteins are found in plasma neuronal-derived exosomes (NDEs) at levels that reflect those in brain neurons. Here, we tested the performance of mitochondrial proteins in plasma NDEs to predict cognitive decline and brain injury in participants with diabetes. The study participants with type 2 diabetes mellitus (T2DM) included 41 cognitively normal control subjects, 97 individuals with mild cognitive impairment (MCI) (68 individuals with stable MCI; 29 individuals with progressive MCI), and 36 patients with AD dementia. Plasma neuroexosomal proteins were measured by ELISA kits. Spearman correlation was used to test associations between plasma neuroexosomal mitochondrial proteins and other core biomarkers of AD. Diagnostic accuracy for progressive MCI and AD was obtained for mitochondrial proteins using receiver operating characteristic curve analyses. The associations of mitochondrial proteins with the conversion from MCI to AD were assessed by Cox proportional hazard regression analysis. Plasma levels of neuroexosomal NADH ubiquinone oxidoreductase core subunit S3 (NDUFS3) and succinate dehydrogenase complex subunit B (SDHB) were significantly lower in patients with T2DM with AD dementia and progressive MCI than in cognitively normal subjects (P < 0.001 for both groups). We also found that plasma neuroexosomal NDUFS3 and SDHB levels were lower in progressive MCI subjects than in stable MCI subjects. Both plasma neuroexosomal NDUFS3 and SDHB offer diagnostic utility for AD. Low plasma neuroexosomal SDHB levels significantly predicted conversion from MCI to AD. In addition, low mitochondrial protein levels were associated with the rate of hippocampal and gray matter atrophy and reduced AD signature cortical thickness in progressive MCI over the follow-up period. These data suggest that both plasma neuroexosomal NDUFS3 and SDHB are already increased at the early clinical stage of AD, and indicate the promise of plasma neuroexosomal mitochondrial proteins as diagnostic and prognostic biomarkers for the earliest symptomatic stage of AD in participants with diabetes.

Plants of the Huperziaceae family, which comprise the two genera Huperzia and Phlegmariurus, produce various types of lycopodium alkaloids that are used to treat a number of human ailments, such as contusions, swellings and strains. Huperzine A, which belongs to the lycodine type of lycopodium alkaloids, has been used as an anti-Alzheimer's disease drug candidate. Despite their medical importance, little genomic or transcriptomic data are available for the members of this family. We used massive parallel pyrosequencing on the Roche 454-GS FLX Titanium platform to generate a substantial EST dataset for Huperzia serrata (H. serrata) and Phlegmariurus carinatus (P. carinatus) as representative members of the Huperzia and Phlegmariurus genera, respectively. H. serrata and P. carinatus are important plants for research on the biosynthesis of lycopodium alkaloids. We focused on gene discovery in the areas of bioactive compound biosynthesis and transcriptional regulation as well as genetic marker detection in these species.For H. serrata, 36,763 unique putative transcripts were generated from 140,930 reads totaling over 57,028,559 base pairs; for P. carinatus, 31,812 unique putative transcripts were generated from 79,920 reads totaling over 30,498,684 base pairs. Using BLASTX searches of public databases, 16,274 (44.3%) unique putative transcripts from H. serrata and 14,070 (44.2%) from P. carinatus were assigned to at least one protein. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) orthology annotations revealed that the functions of the unique putative transcripts from these two species cover a similarly broad set of molecular functions, biological processes and biochemical pathways.In particular, a total of 20 H. serrata candidate cytochrome P450 genes, which are more abundant in leaves than in roots and might be involved in lycopodium alkaloid biosynthesis, were found based on the comparison of H. serrata and P. carinatus 454-ESTs and real-time PCR analysis. Four unique putative CYP450 transcripts (Hs01891, Hs04010, Hs13557 and Hs00093) which are the most likely to be involved in the biosynthesis of lycopodium alkaloids were selected based on a phylogenetic analysis. Approximately 115 H. serrata and 98 P. carinatus unique putative transcripts associated with the biosynthesis of triterpenoids, alkaloids and flavones/flavonoids were located in the 454-EST datasets. Transcripts related to phytohormone biosynthesis and signal transduction as well as transcription factors were also obtained. In addition, we discovered 2,729 and 1,573 potential SSR-motif microsatellite loci in the H. serrata and P. carinatus 454-ESTs, respectively.The 454-EST resource allowed for the first large-scale acquisition of ESTs from H. serrata and P. carinatus, which are representative members of the Huperziaceae family. We discovered many genes likely to be involved in the biosynthesis of bioactive compounds and transcriptional regulation as well as a large number of potential microsatellite markers. These results constitute an essential resource for understanding the molecular basis of developmental regulation and secondary metabolite biosynthesis (especially that of lycopodium alkaloids) in the Huperziaceae, and they provide an overview of the genetic diversity of this family.

Recently, a non-synonymous (Gly307Ser) variant, rs763361, in the CD226 gene was shown to be associated with multiple autoimmune diseases (ADs) in European Caucasian populations. However, shared autoimmunity with CD226 has not been evaluated in non-European populations. The aim of the present study is to assess the association of this single nucleotide polymorphism (SNP) with ADs in non-European populations.To replicate this association in non-European populations, we evaluated case-control association between rs763361 and coeliac disease (CED) samples from Argentina; SLE, RA, type-1 diabetes (T1D) and primary SS (pSS) from Colombia; and SLE samples from China and Japan. We genotyped rs763361 and evaluated its genetic association with multiple ADs, using chi(2)-test. For each association, odds ratio (OR) and 95% CI were calculated.We show that rs763361 is significantly associated with Argentinean CED (P = 0.0009, OR = 1.60). We also observed a trend of possible association with Chinese SLE (P = 0.01, OR = 1.19), RA (P = 0.047, OR = 1.25), SLE (P = 0.0899, OR = 1.24) and pSS (P = 0.09, OR = 1.33) in Colombians. Meta-analyses for SLE (using our three populations) and T1D (our population and three published populations) yielded significant association with rs763361, P = 0.009 (OR = 1.16) and P = 1.1.46 x 10(-9) (OR = 1.14), respectively.Our results demonstrate that the coding variant rs763361 in CD226 gene is associated with multiple ADs in non-European populations.

Vitamin D deficiency is associated with a variety of chronic metabolic diseases. Limited evidence regarding vitamin D deficiency exists within the Chinese population. The present study aims to examine the association between serum vitamin D concentrations and cardiometabolic risk factors in the young and middle-aged, urban Chinese populationThe cross-sectional relationships between serum 25-hydroxyvitamin D [25(OH)D] concentrations and indices of adiposity and cardiometabolic risk factors (e.g., body mass index, waist circumference, fasting plasma glucose, etc.) were evaluated in 601 non-diabetic adults.Vitamin D deficiency or insufficiency was present in 66% of the tested population, and serum 25(OH)D levels were lower in patients who were overweight/obese or suffered metabolic syndrome when compared to individuals of healthy weight without metabolic syndrome (24.08 ± 8.08 vs 31.70 ± 11.77 ng/ml, 21.52 ± 6.9 vs 31.74 ± 10.21 ng/ml respectively). 25(OH)D was inversely associated with waist circumference, fasting glucose, fasting insulin, triglycerides and LDL-cholesterol, and it was positively associated with HDL-cholesterol in a multivariable-adjusted regression model.Vitamin D deficiency is common in the young and middle-aged, urban Chinese population, with high prevalence in overweight/obese individuals and patients with metabolic syndrome. Low vitamin D concentration was associated with indices of adiposity and cardiometabolic risk factors. Further studies are warranted to elucidate the cause-effect relation between vitamin D status, obesity and related metabolic disorders.

Background Lonicera japonica Thunb. is a plant used in traditional Chinese medicine known for its anti-inflammatory, anti-oxidative, anti-carcinogenic, and antiviral pharmacological properties. The major active secondary metabolites of this plant are chlorogenic acid (CGA) and luteoloside. While the biosynthetic pathways of these metabolites are relatively well known, the genetic information available for this species, especially the biosynthetic pathways of its active ingredients, is limited. Methodology/Principal Findings We obtained one million reads (average length of 400 bp) in a whole sequence run using a Roche/454 GS FLX titanium platform. Altogether, 85.69% of the unigenes covering the entire life cycle of the plant were annotated and 325 unigenes were assigned to secondary metabolic pathways. Moreover, 2039 unigenes were predicted as transcription factors. Nearly all of the possible enzymes involved in the biosynthesis of CGA and luteoloside were discovered in L. japonica. Three hydroxycinnamoyl transferase genes, including two hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase genes and one hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) gene featuring high similarity to known genes from other species, were cloned. The HCT gene was discovered for the first time in L. japonica. In addition, 188 candidate cytochrome P450 unigenes and 245 glycosyltransferase unigenes were found in the expressed sequence tag (EST) dataset. Conclusion This study provides a high quality EST database for L. japonica by 454 pyrosequencing. Based on the EST annotation, a set of putative genes involved in CGA and luteoloside biosynthetic pathways were discovered. The database serves as an important source of public information on genetic markers, gene expression, genomics, and functional genomics in L. japonica.

Panax ginseng Meyer is a traditional medicinal plant famous for its strong therapeutic effects and serves as an important herbal medicine. To understand and manipulate genes involved in secondary metabolic pathways including ginsenosides, transcriptome profiling of P. ginseng is essential.RNA-seq analysis of adventitious roots of two P. ginseng cultivars, Chunpoong (CP) and Cheongsun (CS), was performed using the Illumina HiSeq platform. After transcripts were assembled, expression profiling was performed.Assemblies were generated from ∼85 million and ∼77 million high-quality reads from CP and CS cultivars, respectively. A total of 35,527 and 27,716 transcripts were obtained from the CP and CS assemblies, respectively. Annotation of the transcriptomes showed that approximately 90% of the transcripts had significant matches in public databases. We identified several candidate genes involved in ginsenoside biosynthesis. In addition, a large number of transcripts (17%) with different gene ontology designations were uniquely detected in adventitious roots compared to normal ginseng roots.This study will provide a comprehensive insight into the transcriptome of ginseng adventitious roots, and a way for successful transcriptome analysis and profiling of resource plants with less genomic information. The transcriptome profiling data generated in this study are available in our newly created adventitious root transcriptome database (http://im-crop.snu.ac.kr/transdb/index.php) for public use.

Surface modification of Fe3O4 is an efficient strategy to improve its Fenton-like catalytic activity. In this study, the dispersible Fe3O4 cores were coated with SiO2 and C to prepare Fe3O4/SiO2/C nanospheres (FSCNS) for Fenton-like catalysis. FSCNS showed high catalytic activity in the pH range of 4–9. The dispersible formulation made FSCNS more efficient than aggregated Fe3O4/SiO2/C nanocomposites. Increasing the FSCNS amount, H2O2 concentration and temperature would accelerate the Fenton-like reaction. The radicals generated in the Fenton-like reaction was used for the decoloration of cationic dye methylene blue and showed good tolerance to radical scavenger t-butanol. After decoloration, FSCNS could be easily washed for regeneration. The implication to the applications of FSCNS in water treatment is discussed.

Bovine viral diarrhea caused by bovine viral diarrhea virus (BVDV) is an important disease in cattle, resulting in significant economic losses to the cattle industry worldwide. In order to develop an effective vaccine against BVDV infection, we constructed a dendritic cell (DC)-targeting oral probiotic vaccine (pPG-E2-DCpep/LC W56) using Lactobacillus casei as antigen delivery carrier to express BVDV glycoprotein E2 fused with DC-targeting peptide, and the immunogenicity of orally administered probiotic vaccine was evaluated in mice model. Our results showed that after immunization with the probiotic vaccine, significantly levels of antigen-specific sera IgG and mucosal sIgA antibodies (p < 0.05) with BVDV-neutralizing activity were induced in vivo. Challenge experiment showed that pPG-E2-DCpep/LC W56 can provide effective immune protection against BVDV, and BVDV could be effectively cleared from the intestine of immunized mice post-challenge. Moreover, the pPG-E2-DCpep/LC W56 could efficiently activate DCs in the intestinal Peyer's patches, and significantly levels of lymphoproliferative responses, Th1-associated IFN-γ, and Th2-associated IL-4 were observed in mice immunized with pPG-E2-DCpep/LC W56 (p < 0.01). Our results clearly demonstrate that the probiotic vaccine could efficiently induce anti-BVDV mucosal, humoral, and cellular immune responses via oral immunization, indicating a promising strategy for the development of oral vaccine against BVDV.

Abstract Background Sialic acids are widely distributed in animal tissues, and aberrantly expressed in a variety of cancer types. High expression of sialic acid contributes to tumor aggressiveness by promoting cell proliferation, migration, angiogenesis, and metastasis. Sialidases are responsible for removal of sialic acids from glycoproteins and glycolipids. Methods N-glycomics of bladder cancer cells were detected by MALDI-TOF mass spectrometry. Sialic acid modification in bladder cancer tissue was determined by lectin blot. The down-regulation of NEU1 in bladder cancer cells was determined by high resolution liquid chromatography mass spectrometry (HR LC-MS). The effects of sialidase NEU1 expression on proliferation and apoptosis of human bladder cancer cells were examined by western blot, RT-PCR, confocal imaging and flow cytometry. Moreover, the function of sialic acids on fibronectin-integrin α5β1 interaction were assayed by immunoprecipitation and ELISA. The importance of NEU1 in tumor formation in vivo was performed using BALB/c-nu mice. Expression of NEU1 in primary human bladder cancer tissue samples was estimated using bladder cancer tissue microarray. Results (1) Downregulation of NEU1 was primarily responsible for aberrant expression of sialic acids in bladder cancer cells. (2) Decreased NEU1 expression was correlated with bladder cancer progression. (3) NEU1 overexpression enhanced apoptosis and reduced proliferation of bladder cancer cells. (4) NEU1 disrupted FN-integrin α5β1 interaction and deactivated the Akt signaling pathway. (5) NEU1 significantly suppressed in vivo tumor formation in BALB/c-nu mice. Conclusions Our data showed that NEU1 inhibited cancer cell proliferation, induced apoptosis, and suppressed tumor formation both in vitro and in vivo, by disrupting interaction of FN and integrin β1 and inhibiting the Akt signaling pathway. Our observations indicate that NEU1 is an important modulator of the malignant properties of bladder cancer cells, and is a potential therapeutic target for prognosis and treatment of bladder cancer. Graphical abstract

The Alternaria alternata apple pathotype adversely affects apple (Malus domestica Borkh.) cultivation. However, the molecular mechanisms underlying enhanced resistance to this pathogen in apple remain poorly understood. We have previously reported that MdWRKY75 expression is upregulated by A. alternata infection in 'Sushuai' apples. In this study, we discovered that overexpression of MdWRKY75e increased the resistance of transgenic apple lines to A. alternata infection, whereas silencing this gene enhanced susceptibility to A. alternata infection. Furthermore, we found that MdWRKY75e directly binds to the MdLAC7 promoter to regulate the biosynthesis of laccase and increase the biosynthesis of lignin during A. alternata infection. Moreover, the thickening of the cell wall enhanced the mechanical defense capabilities of apple. In addition, we found that jasmonic acid remarkably induced MdWRKY75e expression, and its levels in transgenic apple lines were elevated. These results indicate that MdWRKY75e confers resistance to the A. alternata apple pathotype mainly via the jasmonic acid pathway and that pathogenesis-related genes and antioxidant-related enzyme activity are involved in the disease resistance of MdWRKY75e transgenic plants. In conclusion, our findings provide insights into the importance of MdWRKY75e for resistance to A. alternata infection in apples.

Virtual CSR co-creation activities have become effective strategic tools for enterprises to promote green consumption. The research objective of the current study is to find out whether the experience value acquired by users in the process of virtual CSR co-creation leads to pro-social behavior, namely, the willingness to purchase green products. This empirical study introduces a moderated mediation effect model with virtual CSR co-creation and self-construal as independent variables. Environmental involvement was introduced as a moderator in the research model, whereas the virtual community identity was introduced as a mediator. A scenario simulation experiment was conducted to explore the mechanism of the virtual CSR co-creation on customers’ green product purchase intention. The results indicate that the virtual CSR co-creation promoted the customers’ green product purchase intention. The virtual CSR co-creation and self-construal have an interactive effect on green product purchase intention. Environmental involvement plays a moderating role in the interactive effect. The virtual community identity partially mediates this interactive effect, thus impacting consumers’ green products purchase intentions indirectly. The research findings provide a new practical path for enterprises to develop target strategies to promote green consumption.

Not least because it is adaptable to a variety of geographies and climates, potato (Solanum tuberosum L.) is grown across much of the world. Pigmented potato tubers have been found to contain large quantities of flavonoids, which have various functional roles and act as antioxidants in the human diet. However, the effect of altitude on the biosynthesis and accumulation of flavonoids in potato tubers is poorly characterized. Here we carried out an integrated metabolomic and transcriptomic study in order to evaluate how cultivation at low (800 m), moderate (1800 m), and high (3600 m) altitude affects flavonoid biosynthesis in pigmented potato tubers. Both red and purple potato tubers grown at a high altitude contained the highest flavonoid content, and the most highly pigmented flesh, followed by those grown at a low altitude. Co-expression network analysis revealed three modules containing genes which were positively correlated with altitude-responsive flavonoid accumulation. The anthocyanin repressors StMYBATV and StMYB3 exhibited a significant positive relationship with altitude-responsive flavonoid accumulation. The repressive function of StMYB3 was further verified in tobacco flowers and potato tubers. The results presented here add to the growing body of knowledge regarding the response of flavonoid biosynthesis to environmental conditions, and should aid in efforts to develop novel varieties of pigmented potatoes for use across different geographies.

To assess the difference in survival between fertility-sparing surgery (FSS) and radical surgery and explore pregnancy outcomes after FSS in stage I malignant sex cord-stromal tumours (MSCSTs).We carried out a multicentre retrospective cohort study on patients who were diagnosed with MSCSTs and the tumour was confined to one ovary. The patients were divided into FSS and radical surgery groups. Inverse probability of treatment weighting (IPTW) was used to balance variables between the two groups. Kaplan-Meier analysis was used to compare the difference in disease-free survival (DFS). Univariate and multivariate Cox regression analysis was used to find risk factors of DFS. Univariate logistic regression analysis was used to assess risk factors of pregnancy.In total, 107 patients were included, of whom 54 (50.5%) women underwent FSS and 53 (49.5%) received radical surgery. After IPTW, a pseudo-population of 208 was determined and all of the covariates were well balanced. After a median follow-up time of 50 months (range 7-156 months), 10 patients experienced recurrence and two died. There was no significant difference in DFS between the two groups, both in unweighted (P = 0.969) or weighted cohorts (P = 0.792). In the weighted cohort, stage IC (P = 0.014), tumour diameter >8 cm (P = 0.003), incomplete staging surgery (P = 0.003) and no adjuvant chemotherapy (P < 0.001) were the four high-risk factors associated with a shorter DFS. Among 14 patients who had pregnancy desire, 11 (78.6%) women conceived successfully; the live birth rate was 76.9%. In univariate analysis, only adjuvant chemotherapy (P = 0.009) was associated with infertility.On the premise of complete staging surgery, FSS is safe and feasible in early stage MSCSTs with satisfactory reproductive outcomes.

American ginseng ( Panax quinquefolius L. ) has been used for a wide range of therapeutic purposes in China. The major bioactive phytochemicals responsible for this plant's pharmacological features are ginsenosides. Thus far, little is known regarding the genes involved in ginsenosides biosynthesis in this species. As a non‐model plant, information about its genomes is generally not available. In this study, we generated 6678 expressed sequence tags (ESTs) from the flower, leaf and root cDNA libraries of American ginseng. Assembly of ESTs resulted in 3349 unigenes including 534 contigs (with ESTs number ranging from 2 to 52) and 2815 singletons. By analyzing the predominant transcripts within specific tissues, a gene expression pattern was obtained in a tissue‐specific manner. They were assigned according to the functional classification of unigenes to broad ranges of Gene Ontology categories which include biological processes, cellular components and molecular functions. Based on blastx search results, 24 unigenes representing candidates related to ginsenosides biosynthesis were identified. Cloning and characterization of 3‐hydroxy‐3‐methylglutaryl‐coenzyme A reductase (HMGR, EC: 1.1.1.34), the rate‐limiting enzyme in mevalonic acid pathway, demonstrated that it belonged to the plant HMGR family and was highly expressed in leaves. Putative transcription factors were detected in 63 unigenes, including zinc finger, WRKY, homeobox and MADS‐box family proteins. Five hundred and eighty‐eight simple sequence repeat motifs were identified, of which, dimer was the most abundant motif. These data will provide useful information on transcript profiles, gene discovery, transcriptional regulation, flower biogenesis and marker‐assisted selections. The analysis and information from this study will greatly contribute to the improvement of this medicinal plant as well as of other species in the Araliaceae family, for the purpose of ensuring adequate drug resources.

Taxus species are highly valued as renewable resources for the production of Taxol. Despite the commercial and medicinal importance of Taxus, little genomic information is available for yew species, and Taxol biosynthesis still needs to be fully elucidated. In this study, 454 pyrosequencing technology was employed to produce an expressed sequence tag (EST) from the needles of Taxus cuspidata. In all, 81 148 high-quality reads from the needles of T. cuspidata were produced using Roche GS FLX Titanium. A total of 20,557 unique sequences were obtained, including 12 975 singletons and 7582 contigs. Approximately 14,095 unique sequences were annotated by a similarity search against five public databases. Gene ontology revealed 11,220 unique sequences that could be assigned to 45 vocabularies. In the Kyoto Encyclopedia of Genes and Genomes mapping, 2403 transcripts were established as associated with 3821 biochemical pathways. Enzymes in the plastidial 2-C-methyl-D-erythritol 4-phosphate pathway were well represented. Candidates of the putative genes of Taxol biosynthesis were revealed, including those in the remaining steps. In total, 291 transcripts were identified, representing putative homologues of transcription factors. Furthermore, 753 simple sequence repeat motifs, which are potential molecular markers for genetic application, were identified. These results provide the largest EST collections in TAXUS and will contribute to biosynthetic and biochemical studies that lead to drug improvement.

Fe2+-H2O2 Fenton system is the most applied system in advanced oxidation process for water remediation, but the drawbacks of acidic pH, sludge generation and poor regeneration are serious problems to overcome. Herein, we reported the Fe3O4/TiO2/reduced graphene oxide composites (RGO) as high-performance Fenton-like catalyst that could catalyze the decomposition of H2O2 in near neutral pH range of 5–9 for the decoloration of dye methylene blue (MB). The high catalytic activity of Fe3O4/TiO2/RGO was reflected by the fast absorbance decrease and chemical oxidation demand removal of MB solution within 20 min. The oxidation of MB was due to the radical generation in the Fe3O4/TiO2/RGO-H2O2 system, and the radicals could oxidize MB before the scavenging by the radical scavenger t-butanol. Higher Fe3O4/TiO2/RGO amount, H2O2 concentration and temperature accelerated the decoloration of MB. Fe3O4/TiO2/RGO could be easily regenerated by washing with water/ethanol mixture. The implication to the applications of Fe3O4/TiO2/RGO in water treatment is discussed.

Here, based on self-assembly of carbon quantum dots (CDs), an innovative method to prepare nanomaterials under the action of a metal catalyst was presented. CDs were synthesized by a one-step hydrothermal method with citric acid (CA) as the carbon source, ethylenediamine (EDA) as the passivator and FeSO4•7H2O as the pre-catalyst. In the experiment, it was found that the nano-carbon films with a graphene-like structure were formed on the surface of the solution. The structure of the films was studied by high-resolution transmission electron microscopy (HRTEM), Fourier transform infrared (FT-IR), etc. The results demonstrated that the films were formed by the self-assembly of CDs under the action of the gas-liquid interface template and the metal catalyst. Meanwhile, the electrochemical performance of the films was evaluated by linear cyclic voltammetry (CV) and galvanostatic charge discharge (GOD) tests. In addition, the bulk solution could be further reacted and self-assembled by reflux to form a bifunctional magnetic-fluorescent composite material. Characterizations such as X-ray diffractometer (XRD), fluorescence spectra (FL), vibrating sample magnetometer (VSM), etc. revealed that it was a composite of superparamagnetic γ-Fe2O3 and CDs. The results showed that self-assembly of CDs is a novel and effective method for preparing new carbon nanomaterials.

Potatoes consist of flavonoids that provide health benefits for human consumers. To learn more about how potato tuber flavonoid accumulation and flesh pigmentation are controlled, we analyzed the transcriptomic and metabolomic profile of potato tubers from three colored potato clones at three developmental phases using an integrated approach. From the 72 flavonoids identified in pigmented flesh, differential abundance was noted for anthocyanins, flavonols, and flavones. Weighted gene co-expression network analysis further allowed modules and candidate genes that positively or negatively regulate flavonoid biosynthesis to be identified. Furthermore, an R2R3-MYB repressor StMYB3 and an R3-MYB repressor StMYBATV involved in the modulation of anthocyanin biosynthesis during tuber development were identified. Both StMYB3 and StMYBATV could interact with the cofactor StbHLH1 and repress anthocyanin biosynthesis. Our results indicate a feedback regulatory mechanism of a coordinated MYB activator-repressor network on fine-tuning of potato tuber pigmentation during tuber development.

An association between neuroinflammation and cognitive decline has been established. The complement system regulates neuroinflammation. Dysregulation, impairment, or inadvertent activation of complement components contribute to preclinical Alzheimer's disease. The astrocyte-derived exosome (ADE) complement proteins, including C3b and C5b-9, may be predictive biomarkers of mild cognitive impairment conversion to Alzheimer's disease dementia. We hypothesized that complement proteins might be involved in cognitive impairment during obstructive sleep apnea (OSA). The aim of our study was to explore the correlation between the complement system and mild cognitive impairment (MCI) in patients with OSA.All participants with subjective snoring complaints from the Sleep Medicine Center underwent polysomnography. OSA was defined as apnea-hypopnea index ≥ 5 events/h. MCI was defined as the Montreal Cognitive Assessment < 26 and met the criteria: (1) a subjective cognitive impairment; (2) an objective impairment in 1 or more cognitive domains; (3) complex instrumental daily abilities can be slightly impaired but independent daily living abilities are maintained; and (4) no dementia. The ADEs were isolated immunochemically for enzyme-linked immunosorbent assay quantification of complement proteins, including C3b, C5b-9, and CD55. The participants who received continuous positive airway pressure were followed up and their complement protein levels were reassessed after 1 year of treatment.A total of 212 participants (66.98% males; mean age of 56.71 ± 10.10 years) were divided into the OSA+MCI group (n = 90), OSA-MCI group (n = 79), and controls (normal cognitive state without OSA) (n = 43). The ADE levels of C3b and C5b-9 in the OSA+MCI group were higher than those in the OSA-MCI and control groups. The C3b and C5b-9 were independently associated with cognitive impairment in patients with OSA. The relationship between apnea-hypopnea index and Montreal Cognitive Assessment scores was mediated by C3b and C5b-9. We found no linear correlation between the complement proteins and the severity of OSA. The complement proteins were negatively correlated with global cognitive performance and cognitive subdomains. The complement protein levels significantly decreased after continuous positive airway pressure treatment.Complement proteins were implicated in cognitive impairment in patients with OSA and may be promising biomarkers for predicting cognitive impairment in patients with OSA.Registry: Chinese Clinical Trial Registry; Name: Study on early diagnostic markers in patients with dementia and mild cognitive impairment; URL: https://www.chictr.org.cn/; Identifier: ChiCTR1900021544.Li M, Sun C, Xue S, et al. Complement proteins levels in serum astrocyte-derived exosomes are associated with cognitive impairment in obstructive sleep apnea. J Clin Sleep Med. 2023;19(4):727-739.

Huperzia serrata produces various types of lycopodium alkaloids, especially the huperzine A (HupA) that is a promising drug candidate for Alzheimer's disease. Despite the medicinal importance of H. serrata, little genomic or transcriptomic data are available from the public databases. A cDNA library was thus generated from RNA isolated from the leaves of H. serrata. A total of 4012 clones were randomly selected from the library, and 3451 high-quality expressed sequence tags (ESTs) were assembled to yield 1510 unique sequences with an average length of 712 bp. The majority (79.4%) of the unique sequences were assigned to the putative functions based on the BLAST searches against the public databases. The functions of these unique sequences covered a broad set of molecular functions, biological processes and biochemical pathways according to GO and KEGG assignments. The transcripts involved in the secondary metabolite biosynthesis of alkaloids, terpenoids and flavone/flavonoids, such as cytochrome P450, lysine decarboxylase (LDC), flavanone 3-hydroxylase, squalene synthetase and 2-oxoglutarate 3-dioxygenase, were well represented by 34 unique sequences in this EST dataset. The corresponding peptide sequence of the LDC contained the Pfam 03641 domain and was annotated as a putative LDC. The unique sequences encoding transcription factors, phytohormone biosynthetic enzymes and signaling components were also found in this EST collection. In addition, a total of 501 potential SSR-motif microsatellite loci were identified from the 393 H. serrata leaf unique sequences. This set of non-redundant ESTs and the molecular markers obtained in this study will establish valuable resources for a wide range of applications including gene discovery and identification, genetic mapping and analysis of genetic diversity, cultivar identification and marker-assisted selections in this important medicinal plant.

Ginkgo biloba is monotypic species native to China and has old, dioecious, medicinally important characteristics. The functional genes related to these characteristics have not been effectively explored due to a limited number of expressed sequence tags (ESTs) from Ginkgo . To discover novel functional genes efficiently and to understand the development of a living fossil tree, Ginkgo , we used massive parallel pyrosequencing on the Roche 454 GS FLX Titanium platform to generate 64 057 ESTs. The ESTs combined with the 21 590 Ginkgo ESTs in genbank were assembled into 22 304 unique putative transcripts, in which 13 922 novel unique putative transcripts were identified by 454 sequencing. After being assigned to putative functions with Gene Ontology terms, a detailed view of the Ginkgo biological systems was displayed, including characterization of unique putative transcripts with homology to known key enzymes and transcription factors involved in ginkgolide/bilobalide and flavonoid biosynthetic pathways, as well as unique putative transcripts related to development, response to disease and defence. The fact that three full‐length Ginkgo genes encoding key enzymes were found and cloned, suggests that high‐throughput sequencing technology is superior to traditional gene‐by‐gene approach in discovery of genes. Additionally, a total of 204 simple sequence repeat motifs were detected. Our study not only lays the foundations for transcriptome‐led studies in biosynthetic mechanisms, but also contributes significantly to the understanding of functional genomics and development in non‐model plants.

Cabbage (Brassica oleracea L. var. capitata), an important vegetable crop in the Brassicaceae family, is economically important worldwide. In the process of hybrid seed production, Ogura cytoplasmic male sterility (OguCMS), controlled by the mitochondrial gene orf138, has been extensively used for cabbage hybrid production with complete and stable male sterility. To identify the critical genes and pathways involved in the sterility and to better understand the underlying molecular mechanisms, the anther of OguCMS line R2P2CMS and the fertile line R2P2 were used for RNA-seq and iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) proteome analysis. RNA-seq analysis generated 13,037,109 to 13,066,594 SE50-clean reads, from the sterile and fertile lines, which were assembled into 36,890 unigenes. Among them, 1,323 differentially expressed genes (DEGs) were identified, consisting of 307 up- and 1016 down-regulated genes. For ITRAQ analysis, a total of 7,147 unique proteins were identified, and 833 were differentially expressed including 538 up- and 295 down-regulated proteins. These were mainly annotated to the ribosome, spliceosome and mRNA surveillance pathways. Combined transcriptomic and proteomic analyses identified 22 and 70 genes with the same and opposite expression profiles, respectively. Using KEGG analysis of DEGs, gibberellin mediated signaling pathways regulating tapetum programmed cell death and four different pathways involved in sporopollenin synthesis were identified. Secretion and translocation of the sporopollenin precursors were identified, and the key genes participating in these pathways were all significantly down-regulated in R2P2CMS. Light and transmission electron (TE) microscopy revealed fat abnormal tapetum rather than vacuolization and degradation at the tetrad and microspore stages of the OguCMS line. This resulted in the failed deposition of sporopollenin on the pollen resulting in sterility. This study provides a comprehensive understanding of the mechanism underlying OguCMS in cabbage.

Graphene nanomaterials are emerging environmental pollutants and their toxicity to plants requires careful investigation in environmental matrixes. Actually, the transportation of graphene in hydroponic systems is completely different to that in soil, which might affect the interaction between graphene and plants. In this study, we compared the toxicity of graphene oxide (GO) to naked oats (Avena sativa L.) in hydroponic and soil cultures. Serious toxicity of GO was only observed in hydroponic culture. GO induced the inhibition of biomass gain, seedling length and photosynthesis of naked oats. The root structure was disturbed by GO and oxidative stress was aroused in the root. In contrast, the soil (vermiculite) interacted strongly with GO and restricted the transportation of GO in soil. This reduced the contact between GO and the roots and largely alleviated its toxicity. Our results collectively suggested that environmental biosafety evaluation should consider the impact of environmental behaviors of nanomaterials to better reflect the real bioeffect of nanomaterials.

RNA editing is a widespread, post-transcriptional molecular phenomenon that diversifies hereditary information across various organisms. However, little is known about genome-scale RNA editing in fungi. In this study, we screened for fungal RNA editing sites at the genomic level in Ganoderma lucidum, a valuable medicinal fungus. On the basis of our pipeline that predicted the editing sites from genomic and transcriptomic data, a total of 8906 possible RNA-editing sites were identified within the G. lucidum genome, including the exon and intron sequences and the 5'-/3'-untranslated regions of 2991 genes and the intergenic regions. The major editing types included C-to-U, A-to-G, G-to-A, and U-to-C conversions. Four putative RNA-editing enzymes were identified, including three adenosine deaminases acting on transfer RNA and a deoxycytidylate deaminase. The genes containing RNA-editing sites were functionally classified by the Kyoto Encyclopedia of Genes and Genomes enrichment and gene ontology analysis. The key functional groupings enriched for RNA-editing sites included laccase genes involved in lignin degradation, key enzymes involved in triterpenoid biosynthesis, and transcription factors. A total of 97 putative editing sites were randomly selected and validated by using PCR and Sanger sequencing. We presented an accurate and large-scale identification of RNA-editing events in G. lucidum, providing global and quantitative cataloging of RNA editing in the fungal genome. This study will shed light on the role of transcriptional plasticity in the growth and development of G. lucidum, as well as its adaptation to the environment and the regulation of valuable secondary metabolite pathways.

The nitrogen-doped carbon dots (N-CQDs) were synthesized by citric acid as a raw material and propylene diamine as a passivation agent. Structure, optical properties and biocompatibility of N-CQDs were analyzed. It was found that the N-CQDs possessed concentration-dependent, multicolor photoluminescence and low toxicity. As demonstrated in the imaging of bioluminescence, by adjusting the concentration of N-CQDs, the cell imaging effect can be adjusted. The internalized N-CQDs were concentrated in the nucleus. A novel tool for studying the nuclear changes during the cell cycle was developed.

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.

To predict the incidence of radiation-induced hypothyroidism (RHT) in nasopharyngeal carcinoma (NPC) patients, dosiomics features based prediction models were established.A total of 145 NPC patients treated with radiotherapy from January 2012 to January 2015 were included. Dosiomics features of the dose distribution within thyroid gland were extracted. The minimal-redundancy-maximal-relevance (mRMR) criterion was used to rank the extracted features and selected the most relevant features. Machine learning (ML) algorithms including logistic regression (LR), support vector machine (SVM), random forest (RF), and k-nearest neighbor (KNN) were utilized to establish prediction models, respectively. Nested sampling and hyper-tuning methods were adopted to train and validate the prediction models. The dosiomics-based (DO) prediction models were evaluated through comparing with the dose-volume factor-based (DV) models in terms of the area under the receiver operating characteristic (ROC) curve (AUC). The demographics factors (age and gender) were included in both DO model and DV model.Age, V45 and 37 dosiomics features exhibited significant correlations with RHT in univariate analysis. For prediction performance, DO prediction models exhibited better results with the best AUC value 0.7 while DV prediction models 0.61. In DO prediction models, the AUC values displayed a trend from ascending to descending with the increasing of selected features. The highest AUC value was achieved when the number of selected features was 3. In DV prediction model, similar trend was not observed.This study established a prediction model based on the dosiomics features with better performance than conventional dose-volume factors, leading to early predict the possible RHT among NPC patients who had received radiotherapy and take precaution measures for NPC patients.

Abstract Brassica parachinensis is a popular leafy vegetable. It is able to accumulate high concentrations of cadmium (Cd), but the molecular mechanism of Cd accumulation is unknown. This study investigated the function and regulatory mechanism of the Cd-responsive metal ion transporter gene BrpHMA2. BrpHMA2 was induced by Cd stress and specifically expressed in vascular tissues, and the protein was localized in the plasma membrane. Heterologous expression of BrpHMA2 enhanced Cd accumulation and Cd sensitivity in transgenic Arabidopsis and yeast. After Cd stress, the transcription factors BrpNAC895 and BrpABI449, which may recognize the abscisic acid-responsive elements in the BrpHMA2 promoter, were also differentially expressed. The transcriptional regulation of BrpHMA2 was further investigated using the chromatin immunoprecipitation–quantitative PCR (ChIP–qPCR) assay, the electrophoretic mobility shift assay (EMSA), and luciferase (LUC) reporter activity analysis employing the transient expression system of B. parachinensis protoplasts and tobacco leaves and the Escherichia coli expression system. By binding to the promoter, BrpNAC895 induced the transcription of BrpHMA2. BrpABI449 might bind to the BrpHMA2 promoter or interact with BrpNAC895 to interfere with the action of BrpNAC895. The findings suggest that BrpHMA2 is a membrane-based afflux-type Cd transporter involved in Cd2+ uptake and long-distance transport in plants. BrpNAC895 and BrpABI449, which function as the transcription activator and repressor, respectively, coregulate BrpHMA2 expression.

DELLA proteins are essential components of GA signal transduction. MhGAI1 was isolated from the tea crabapple (Malus hupehensis Redh. var. pingyiensis), and it was found to encode a DELLA protein. Mhgai1 is a GA-insensitive allele that was artificially generated via a bridge-PCR approach. Ectopic expression of Mhgai1 reduced plant stature, decreased spontaneous fruit-set-ratio and enhanced drought-tolerance in transgenic tomatoes. In addition, we examined the long-distance movement of Mhgai1 mRNAs by grafting experiments and SqRT-PCR analysis. It was found that the wild-type scions accumulated Mhgai1 transcripts trafficked from the transgenic rootstocks and therefore exhibited dwarf phenotypes. Furthermore, transgenic tomato plants produced more soluble solids, sugars and organic acids compared to wild-type tomatoes, suggesting an involvement of GA signaling in the regulation of fruit quality. Despite noticeable accumulation in the leaves and stems of WT scions, Mhgai1 transcripts were undetectable in flowers and fruit. Therefore, fruit quality was less influenced by the grafting of WT scions onto transgenic rootstocks than they were by the ectopic expression of Mhgai1 in transgenic rootstocks. Taken together, MhGAI1, which functions as a repressor in the GA signaling pathway, and its GA-insensitive allele, Mhgai1, could turn out to be useful targets for the genetic improvement of dwarfing rootstocks in apples.

MicroRNAs (miRNAs) are a family of non-coding RNAs approximately 21 nucleotides in length that play pivotal roles at the post-transcriptional level in animals, plants and viruses. These molecules silence their target genes by degrading transcription or suppressing translation. Studies have shown that miRNAs are involved in biological responses to a variety of biotic and abiotic stresses. Identification of these molecules and their targets can aid the understanding of regulatory processes. Recently, prediction methods based on machine learning have been widely used for miRNA prediction. However, most of these methods were designed for mammalian miRNA prediction, and few are available for predicting miRNAs in the pre-miRNAs of specific plant species. Although the complete Solanum lycopersicum genome has been published, only 77 Solanum lycopersicum miRNAs have been identified, far less than the estimated number. Therefore, it is essential to develop a prediction method based on machine learning to identify new plant miRNAs.A novel classification model based on a support vector machine (SVM) was trained to identify real and pseudo plant pre-miRNAs together with their miRNAs. An initial set of 152 novel features related to sequential structures was used to train the model. By applying feature selection, we obtained the best subset of 47 features for use with the Back Support Vector Machine-Recursive Feature Elimination (B-SVM-RFE) method for the classification of plant pre-miRNAs. Using this method, 63 features were obtained for plant miRNA classification. We then developed an integrated classification model, miPlantPreMat, which comprises MiPlantPre and MiPlantMat, to identify plant pre-miRNAs and their miRNAs. This model achieved approximately 90% accuracy using plant datasets from nine plant species, including Arabidopsis thaliana, Glycine max, Oryza sativa, Physcomitrella patens, Medicago truncatula, Sorghum bicolor, Arabidopsis lyrata, Zea mays and Solanum lycopersicum. Using miPlantPreMat, 522 Solanum lycopersicum miRNAs were identified in the Solanum lycopersicum genome sequence.We developed an integrated classification model, miPlantPreMat, based on structure-sequence features and SVM. MiPlantPreMat was used to identify both plant pre-miRNAs and the corresponding mature miRNAs. An improved feature selection method was proposed, resulting in high classification accuracy, sensitivity and specificity.

The impact of pregnancy on the clinical course of acute hepatitis B (AHB) is still largely unclear, mainly because most studies have not included matched controls. This study was conducted to investigate the clinical features and outcome of AHB in pregnancy using matched controls.Consecutive AHB inpatients who were admitted to Jinan Infectious Disease Hospital, Jinan, between January 2006 and December 2010 were evaluated and followed. Demographic data, clinical manifestations, and results of laboratory tests were compared between pregnant patients and age and sex matched non-pregnant patients at admission, discharge, and final follow-up.A total of 618 AHB inpatients were identified during the study period. 22 pregnant patients and 87 age and sex matched non-pregnant patients were enrolled in this study. Prodromal fever was less common (0% vs. 20.7%, P=0.02), serum alanine aminotransferase levels were significantly lower, and HBsAg>250 IU/mL rate and serum bilirubin levels were significantly higher in pregnant patients than in non-pregnant patients. After a mean (range) of 7(5.2-8.3) months follow-up, 18.2% pregnant patients and 4.6% non-pregnant patients were still HBsAg positive (P=0.03). For pregnant patients, the relative risk (95% confidence interval) of HBsAg positive at the end of follow-up was 4.6 (1.1-20.2). The median (95% confidence interval) days of HBsAg seroclearance form disease onset in pregnant and non-pregnant patients were 145.0 (110.5-179.5) and 80.0 (62.6-97.4), respectively.The HBsAg loss and seroconversion were delayed and lower in pregnant patients. Pregnancy might be a possible risk of chronicity following acute HBV infection.

Integrin alpha M (ITGAM; CD11b) is a component of the macrophage-1 antigen complex, which mediates leukocyte adhesion, migration and phagocytosis as part of the immune system. We previously identified a missense polymorphism, rs1143679 (R77H), strongly associated with systemic lupus erythematosus (SLE). However, the molecular mechanisms of this variant are incompletely understood. A meta-analysis of published and novel data on 28 439 individuals with European, African, Hispanic and Asian ancestries reinforces genetic association between rs1143679 and SLE [Pmeta = 3.60 × 10−90, odds ratio (OR) = 1.76]. Since rs1143679 is in the most active region of chromatin regulation and transcription factor binding in ITGAM, we quantitated ITGAM RNA and surface protein levels in monocytes from patients with each rs1143679 genotype. We observed that transcript levels significantly decreased for the risk allele (‘A’) relative to the non-risk allele (‘G’), in a dose-dependent fashion: (‘AA’ < ‘AG’ < ‘GG’). CD11b protein levels in patients' monocytes were directly correlated with RNA levels. Strikingly, heterozygous individuals express much lower (average 10- to 15-fold reduction) amounts of the ‘A’ transcript than ‘G’ transcript. We found that the non-risk sequence surrounding rs1143679 exhibits transcriptional enhancer activity in vivo and binds to Ku70/80, NFKB1 and EBF1 in vitro, functions that are significantly reduced with the risk allele. Mutant CD11b protein shows significantly reduced binding to fibrinogen and vitronectin, relative to non-risk, both in purified protein and in cellular models. This two-pronged contribution (nucleic acid- and protein-level) of the rs1143679 risk allele to decreasing ITGAM activity provides insight into the molecular mechanisms of its potent association with SLE.

Abstract Background Apple is one of the most popular fruit crops world-wide and its skin color is an important quality consideration essential for commercial value. However, the strategy on genetic breeding for red skin apple and the genetic basis of skin color differentiation is very limited and still largely unknown. Results Here, we reported a bud sport mutant of Fuji apple with red skin color and enhanced anthocyanins accumulation. Quantitative SWATH-MS (sequential window acquisition of all theoretical spectra-mass spectrometry) proteomics investigations revealed proteome changes in the apple red skin bud mutation and a total of 451 differentially expressed proteins were identified in apple skin. The mutant showed significantly increased expression levels of photosynthesis-related proteins, stress-related proteins as well as anthocyanins biosynthesis pathway. On the other hand, substantial downregulation of mitogen-activated protein kinase 4 (MAPK4) and mevalonate kinase (MVK) were detected, indicating a promising role for the red skin color development in the mutant. Furthermore, we also hypothesize that a post-transcriptional regulation of the skin color formation occurs in the mutant through the advanced SWATH-MS analysis. Conclusion Our work provides important information on the application of proteomic methods for analysing proteomes changes in Fuji apple and highlights a clade of regulatory proteins potentially contributing for the molecular breeding of fruit skin color.

Cell and gene therapies are being rapidly developed to treat solid tumors. Some have been approved, while the most of them are still under clinical investigation. Adoptive cell therapy (ACT) takes advantage of immune cells to eliminate tumors. ACT can be broadly classified as non-genetically and genetically engineered cell products or classified based on the cell type, such as T cell, natural killer (NK) cell, dendritic cell (DC), cytokine-induced killer (CIK), tumor-infiltrating lymphocyte (TIL), and mesenchymal stromal cell (MSC) (Stroncek et al., 2012Stroncek D.F. Berger C. Cheever M.A. Childs R.W. Dudley M.E. Flynn P. Gattinoni L. Heath J.R. Kalos M. Marincola F.M. et al.New directions in cellular therapy of cancer: a summary of the summit on cellular therapy for cancer.J. Transl. Med. 2012; 10: 48Crossref PubMed Scopus (39) Google Scholar). Both non-genetically and genetically engineered ACTs are either available or being actively investigated in clinical settings, including T cells (CAR-T, TCR-T, and γδ T cells as well as gene-edited T cells, respectively) (Stadtmauer et al., 2020Stadtmauer E.A. Fraietta J.A. Davis M.M. Cohen A.D. Weber K.L. Lancaster E. Mangan P.A. Kulikovskaya I. Gupta M. Chen F. et al.CRISPR-engineered T cells in patients with refractory cancer.Science. 2020; 367: eaba7365Crossref PubMed Scopus (328) Google Scholar), TILs, DCs, and macrophages. Genetically engineered cells also belong to the category of gene therapy, using extraneous genetic products (DNA or RNA) to treat diseases such as tumor. The gene introduction system can include viral and non-viral vector products. Oncolytic viruses (OVs) are a promising gene therapy because of their tumor lysis effect, their tumor selectivity, and their desirable immunogenic properties. Cell and gene therapies have shown great potential for treating solid tumors. Many have already been approved. From January 1, 2010, to December 31, 2019, 491 clinical trials on a total of 178 cell and gene therapies were carried out worldwide (INFORMA database). The number of trials increased by 16.1% on average annually and almost doubled in 2017 from the previous year. Phase 1 and 2 trials accounted for >90% of new trials each year (Figure S1A). These trials covered 26 solid tumor types, with melanoma, non-small cell lung cancer (NSCLC), and ovarian cancer as the top three most frequently tested (Figure S1B). In total, 318 ACT clinical trials have been carried out during the last decade, spanning all treatment stages from neoadjuvant to the last line treatment (Figure S1C) and mainly targeting advanced solid tumors (88.1%, Figure S1D). Cancer vaccines were the most popular in all treatment stages. In addition, 374 gene therapy (including genetically engineered ACT) clinical trials were registered in INFORMA. Although China was ranked second place by number of cell and gene therapy clinical trials, there was a large gap between China and the top country, the United States (39 versus 120 trials, Figure S1E). There were 14 and 5 investigational CAR-T and TCR-T trials in China, respectively, versus 22 and 16 in the rest of the world. The landscape of targets in those trials was also different between China and the other countries. The top three CAR-T targets were glypican-3, IL13Rα2/CEA/MUC1, and claudin 18.2/AFP in China and mesothelin, NKG2D, and IL13Rα2/CEA/MUC1 outside of China. The top three TCR-T targets were NY-ESO-1, HBV, and HPV E6 in China and NY-ESO-1, individualized tumor-specific neoantigen, and MAGE A4 outside of China. The higher representation of HBV and HPV E6 in China reflected higher incidences of HBV-related liver cancer and HPV-related cervical cancer in the Chinese population. In China, most trials were carried out in Shanghai, Guangzhou, and Beijing, followed by Shenzhen, Hangzhou, and Nanjing. Most cell and gene therapies didn’t move beyond phase 1 or 2 trials because of many remaining difficulties. First, few tumor-specific antigens have so far been identified on the cell surface in solid tumors (Leko and Rosenberg, 2020Leko V. Rosenberg S.A. Identifying and targeting human tumor antigens for T cell-based immunotherapy of solid tumors.Cancer Cell. 2020; 38: 454-472Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Second, solid tumors usually display substantial heterogeneity of antigens. Third, transferred T cells have limited tumor infiltration. Fourth, immunosuppressive tumor microenvironment is a common feature in late-stage cancer patients, leading to T cell exhaustion (Weigelin et al., 2011Weigelin B. Krause M. Friedl P. Cytotoxic T lymphocyte migration and effector function in the tumor microenvironment.Immunol. Lett. 2011; 138: 19-21Crossref PubMed Scopus (34) Google Scholar). Finally, clinical trials with biomarkers were limited, accounting for only 19.2% of ACT trials (61/318) and 14.7% gene therapy trials (55/374). In addition, operational challenges exist. For example, how can the preparation procedures of cell and gene therapies be simplified? How can more effective training be provided to all parties who are involved in cell and gene therapy trials? Various strategies have been developed to address those issues. For instance, better CAR design principles were utilized, and additional factors were included to promote antitumor efficacy, improve tumor infiltration, and overcome the immunosuppressive microenvironment (Hong et al., 2020Hong M. Clubb J.D. Chen Y.Y. Engineering CAR-T cells for next-generation cancer therapy.Cancer Cell. 2020; 38: 473-488Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Furthermore, different therapies can be combined in the clinics to improve efficacy. Finally, a new manufacturing process called Generation-2 has been developed for TIL generation, with only 22 days of completion instead of the original 6-week process. The Generation-2 TIL product for melanoma and cervical cancer might soon become commercialized in the United States. In conclusion, cell and gene therapies have been actively developed to treat solid tumors, and related clinical trials covering almost all solid tumor types increased quickly especially after 2017. Cell and gene therapies still face many challenges in solid tumors, and innovative solutions are required to move these therapies to the clinics. This work was supported by Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (Platform Improvement of Clinical Trial Capability 2020-I2M-2-007 ), Chinese Academy of Medical Sciences (grant 2019XK320068 ), and Beijing Municipal Science and Technology Commission ( International Pharmaceutical Clinical Research and Development Platform 2015 ). Q.-J.L. is a co-founder and stockholder for TCRCure Biopharma. T.D. is the founder of Chengdu MedGenCell Co Ltd., Chengdu, Sichuan, China. Download .pdf (.36 MB) Help with pdf files Document S1. Figure S1

The success of quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) to quantify gene expression depends on the stability of the reference genes used for data normalization. To date, systematic screening for reference genes in persimmon (Diospyros kaki Thunb) has never been reported. In this study, 13 candidate reference genes were cloned from 'Nantongxiaofangshi' using information available in the transcriptome database. Their expression stability was assessed by geNorm and NormFinder algorithms under abiotic stress and hormone stimulation. Our results showed that the most suitable reference genes across all samples were UBC and GAPDH, and not the commonly used persimmon reference gene ACT. In addition, UBC combined with RPII or TUA were found to be appropriate for the "abiotic stress" group and α-TUB combined with PP2A were found to be appropriate for the "hormone stimuli" group. For further validation, the transcript level of the DkDREB2C homologue under heat stress was studied with the selected genes (CYP, GAPDH, TUA, UBC, α-TUB, and EF1-α). The results suggested that it is necessary to choose appropriate reference genes according to the test materials or experimental conditions. Our study will be useful for future studies on gene expression in persimmon.

Quantitative real-time reverse transcription PCR (qRT-PCR) requires a stable internal control to avoid misinterpretation of data or errors for gene expression normalization. However, there are still no validated reference genes for stable internal control in Poria cocos (Schw.) Wolf (Fuling). This study aims to validate the reference genes of P. cocos. This study firstly collected the 14 candidate reference genes by BLASTP from the genome of P. cocos for qRT-PCR analysis to determine the expression levels of 14 housekeeping genes (GAPDH, MAPK, β-Act, RPB2, RPB1-1, RPB1-2, his3-1, his3-2, APT, SAMDC, RP, β-Tub, EIF, and CYP) under different temperatures and in response to different plant hormones (indole-3-acetic acid, abscisic acid, 6-benzylaminopurine, methyl jasmonate, and gibberellic acid), and the threshold cycle (Ct) values. The results were analyzed by four programs (i.e., geNorm, NormFinder, BestKeeper, and RefFinder) for evaluating the candidate reference genes. SAMDC, his3-2, RP, RPB2, and his3-1 were recommended as reference genes for treating P. cocos with indole-3-acetic acid, abscisic acid, 6-benzylaminopurine, methyl jasmonate, and gibberellic acid, respectively. Under different temperatures RPB2 was the most stable reference gene. CYP was the most stable gene for all 90 samples by RefFinder. SAMDC, his3-2, RP, RPB2, and his3-1 were evaluated to be suitable reference genes for P. cocos following different treatments. RPB2 was the most stable reference gene under different temperatures and CYP was the most stable gene in the mycelia under all six evaluated conditions.

Comprehensive Genomic Profiling may be informative for novel treatment strategies and to improve outcomes for patients with rare tumors. This study aims to discover opportunities for use of targeted therapies already approved for routine use in patients with rare tumors. Solid tumors with an incidence lower than 2.5/100,000 per year was defined as rare tumors in China after comprehensive analysis based on epidemiological data and current availability of standardized treatment. Genomic data of rare tumors from the public database cBioPortal were compared with that of the Chinese population for targetable genomic alterations (TGAs). TGAs were defined as mutations of ALK, ATM, BRAF, BRCA1, BRCA2, CDKN2A, EGFR, ERBB2, FGFR1,2,3, KIT, MET, NF1, NTRK1,2,3, PIK3CA, PTEN, RET, and ROS1 with level 1 to 4 of evidence according to the OncoKB knowledge database. Genomic data of 4,901 patients covering 63 subtypes of rare tumor from cBioPortal were used as the western cohort. The Chinese cohort was comprised of next generation sequencing (NGS) data of 1,312 patients from across China covering 67 subtypes. Forty-one subtypes were common between the two cohorts. The accumulative prevalence of TGAs was 20.40% (1000/4901) in cBioPortal cohort, and 53.43% (701/1312) in Chinese cohort (p < 0.001). Among those 41 overlapping subtypes, it was still significantly higher in Chinese cohort compared with cBioPortal cohort (54.1%% vs. 26.1%, p < 0.001). Generally, targetable mutations in BRAF, BRCA2, CDKN2A, EGFR, ERBB2, KIT, MET, NF1, ROS1 were ≥3 times more frequent in Chinese cohort compared with that of the cBioPortal cohort. Cancer of unknown primary tumor type, gastrointestinal stromal tumor, gallbladder cancer, intrahepatic cholangiocarcinoma, and sarcomatoid carcinoma of the lung were the top 5 tumor types with the highest number of TGAs per tumor. The incidence of TGAs in rare tumors was substantial worldwide and was even higher in our Chinese rare tumor population. Comprehensive genomic profiling may offer novel treatment paradigms to address the limited options for patients with rare tumors.

Immuno-oncology therapies have revolutionised the standard of care for many types of cancer. The advent of immune checkpoint inhibitors and chimeric antigen receptor T (CAR-T) cell immunotherapy in the past decade marks a new era of modern immunotherapy treatment for oncology. 1 Tang J Shalabi A Hubbard-Lucey VM Comprehensive analysis of the clinical immuno-oncology landscape. Ann Oncol. 2018; 29: 84-91 Summary Full Text Full Text PDF PubMed Scopus (260) Google Scholar The emergence of these immunotherapies, along with policies made by the Chinese government to strengthen medical innovations and drug research and development (R&D), 2 Li N Huang HY Wu DW et al. Changes in clinical trials of cancer drugs in mainland China over the decade 2009-18: a systematic review. Lancet Oncol. 2019; 20: e619-e626 Summary Full Text Full Text PDF PubMed Scopus (20) Google Scholar means that the immuno-oncology drug pipeline for oncology treatment has strengthened in China over the past 5 years. 3 Yu JX Hubbard-Lucey VM Tang J Immuno-oncology drug development goes global. Nat Rev Drug Discov. 2019; 18: 899-900 Crossref PubMed Scopus (80) Google Scholar The developments in immunotherapies for oncology in China led us to explore the data on the outlook and development of these drugs in this country, which can inform future targets for industry, policy makers, and other stakeholders.

Although PD-L1 expression is a crucial predictive biomarker for immunotherapy, it can be influenced by many factors.A total of 248 Chinese patients with lung adenocarcinoma was retrospectively identified. Data for clinical features, gene alternations, signaling pathways and immune signatures was analyzed among negative expression group (TPS < 1%, n = 124), intermediate expression group (1% ≤ TPS < 50%, n = 93), and high expression group (TPS ≥ 50%, n = 38). Clinical outcomes among different expression groups were also evaluated from public database.Firstly, high tumor mutation burden was significantly associated with high PD-L1 expression in these Chinese patients with lung adenocarcinoma. In addition, gene alternations including TP53, PRKDC, KMT2D, TET1 and SETD2 apparently occurred in high PD-L1 expression group. Moreover, pathway analysis showed that mutations involving in DDR pathway, TP53 pathway, cell-cycle pathway and NOTCH pathway were obviously varied among three PD-L1 expression groups. Besides, most of patients in high PD-L1 expression group from TCGA database were determined as high-grade immune subtypes (C2-C4), showing significant higher proportions of IFN-gamma, CD8+ T-cells, NK cells, NK CD56 dim cells, Th1 cells, Th2 cells (P < 0.0001). Moreover, SETD2 mutation slightly correlated with overall survival from MSKCC cohort (HR 1.92 [95%CI 0.90-4.10], P = 0.085), and the percentage of IFN-gamma was significantly higher in SETD2 mutant group than in wild-type group (P < 0.01).This study illustrated in-depth genomic correlates of PD-L1 expression in Chinese lung adenocarcinoma patients and relevant immune signatures from public database, which might interpret more potential molecular mechanisms for immunotherapy in NSCLC.

A plasma sprayed Cu11.85Al3.2Mn0.1Ti coating on Ti6Al4V alloy was processed with in–situ laser remelting to improve its tribological performance. The effects of laser remelting on the structural evolution and high–temperature tribological properties of plasma sprayed coating were investigated using a wear tester, and the wear mechanism was also discussed in detail. The results show that the pores, layered structures and un–melted particles of plasma sprayed coating are eliminated by laser remelting, and its microstructure and hardness are further improved by in–situ laser remelting. The coefficient of friction of laser remelted coating is reduced by 26% compared with the plasma sprayed coating, and the corresponding wear rate is also decreased by 70%, showing that the superior friction reduction and wear resistance.

CuAlMnTi-xTiC composite coatings were prepared on Ti6Al4V alloy by laser cladding to enhance its tribological properties. The effects of TiC mass fraction on the friction–wear properties of CuAlMnTi–xTiC composite coatings at high temperature were investigated using an ball–on–disc tester. The results show that the grains of CuAlMnTi–xTiC composite coatings are refined by the addition of TiC, and the metallurgical bonding is formed between the coating and the substrate. The hardness of CuAlMnTi–15%TiC composite coating is higher 1.98 times than that of CuAlMnTi coating, in which the TiC as wear–resistant framework prevents micro–cutting to enhance wear resistance. The average coefficients of friction of CuAlMnTi–0%TiC, –5%TiC, –10%TiC and –15% TiC composite coatings in the stable wear period are 0.465, 0.491, 0.514, and 0.587, respectively, and the corresponding wear rates are 116.8, 111.3, 107.8, and 95.4 μm3•N–1•mm–1, respectively, showing that the wear rate is decreased with the increase of TiC mass fraction. The wear mechanism is transferred from adhesive wear to abrasive wear and slight oxidation wear by the addition of TiC, which is attributed to the improvement of hardness by the refined grain strengthening effect of TiC.

The best way to increase patient survival rate is to identify patients who are likely to progress to muscle-invasive or metastatic disease upfront and treat them more aggressively. The human cell lines HCV29 (normal bladder epithelia), KK47 (low grade nonmuscle invasive bladder cancer, NMIBC), and YTS1 (metastatic bladder cancer) have been widely used in studies of molecular mechanisms and cell signaling during bladder cancer (BC) progression. However, little attention has been paid to global quantitative proteome analysis of these three cell lines. We labeled HCV29, KK47, and YTS1 cells by the SILAC method using three stable isotopes each of arginine and lysine. Labeled proteins were analyzed by 2D ultrahigh-resolution liquid chromatography LTQ Orbitrap mass spectrometry. Among 3721 unique identified and annotated proteins in KK47 and YTS1 cells, 36 were significantly upregulated and 74 were significantly downregulated with >95% confidence. Differential expression of these proteins was confirmed by western blotting, quantitative RT-PCR, and cell staining with specific antibodies. Gene ontology (GO) term and pathway analysis indicated that the differentially regulated proteins were involved in DNA replication and molecular transport, cell growth and proliferation, cellular movement, immune cell trafficking, and cell death and survival. These proteins and the advanced proteome techniques described here will be useful for further elucidation of molecular mechanisms in BC and other types of cancer.

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.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.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.

Immune checkpoint blockade has led to a significant improvement of patient survival in metastatic colorectal cancer (CRC) with DNA mismatch repair-deficiency (dMMR)/microsatellite instability-high (MSI-H). However, not all these patients are sensitive to monoimmunotherapy. We firstly presented a case series of advanced dMMR/MSI-H CRCs treating with PD-1 inhibitor-based chemoradioimmunotherapy (CRIT).We assessed the short-term efficacy and safety of CRIT in advanced dMMR/MSI-H CRCs, and also did next-generation sequencing (NGS) assays.Our analysis included five advanced dMMR/MSI-H CRCs who have received toripalimab-based CRIT. Toripalimab was given 240mg every three weeks, and the radiation dose was 45-50 gray in 25 fractions. Chemotherapy regimens consisted of CAPOX in three patients, capecitabine in one patient, and mFOLFOX6 in one patient. Initially, two patients displayed complete response (CR), and three patients achieved partial response (PR) on imaging findings. Afterwards, one PR patient was confirmed pathological complete response after surgery, leading to three CR cases in total. Hematological toxicity was the most common adverse effect, and only two patients developed mild immune-related adverse effects besides. All the treatment-related adverse events were under control. Based on the NGS results, the median intratumor heterogeneity was 0.19 (range 0-0.957), which was less in CR patients than PR patients (P = 0.019). Genetic mutations at DNA damage repair genes and the JAK1 gene were also observed.For advanced dMMR/MSI-H CRC, anti-PD-1 based CRIT is effective and safe. Further studies are required to better clarify the potential role and mechanism of CRIT as a viable therapeutic strategy in this population.

Nuclear factor I (NFI) family is emerging found playing oncogenic or tumor-suppressive potential in cancers. However, the function and underlying mechanisms of NFIC, in the progression of Lung Squamous Cell Carcinoma (LUSC) remain unclear. Therefore, this study aims to probe into the function of NFIC in the development of LUSC. In the present study, we reported that NFIC was low expressed in human LUSC tissues and cell lines. NFIC inhibited LUSC cell proliferation and promoted cell apoptosis in vitro and in vivo. Moreover, NFIC also inhibited LUSC cell migration and invasion. Furthermore, we found that there were binding sites between lncRNA cancer susceptibility candidate 2 (CASC2) and NFIC, whose relationship was confirmed by the luciferase reporter assay. The expression of CASC2 and the expression of NFIC were positively correlated, and the function of CASC2 overexpression is similar to that of NFIC overexpression, which suggested that CASC2 may play a key role in LUSC development. Our study provided a new perspective for NFIC acting as an antioncogene in LUSC tumorigenesis, and NFIC and CASC2 may serve as novel potential targets for the treatment of LUSC.

Gynostemma is an important medicinal and food plant of the Cucurbitaceae family. The phylogenetic position of the genus Gynostemma in the Cucurbitaceae family has been determined by morphology and phylogenetics, but the evolutionary relationships within the genus Gynostemma remain to be explored. The chloroplast genomes of seven species of the genus Gynostemma were sequenced and annotated, of which the genomes of Gynostemma simplicifolium, Gynostemma guangxiense and Gynostemma laxum were sequenced and annotated for the first time. The chloroplast genomes ranged from 157,419 bp (Gynostemma compressum) to 157,840 bp (G. simplicifolium) in length, including 133 identical genes: 87 protein-coding genes, 37 tRNA genes, eight rRNA genes and one pseudogene. Phylogenetic analysis showed that the genus Gynostemma is divided into three primary taxonomic clusters, which differs from the traditional morphological classification of the genus Gynostemma into the subgenus Gynostemma and Trirostellum. The highly variable regions of atpH-atpL, rpl32-trnL, and ccsA-ndhD, the repeat unilts of AAG/CTT and ATC/ATG in simple sequence repeats (SSRs) and the length of overlapping regions between rps19 and inverted repeats(IRb) and between ycf1 and small single-copy (SSC) were found to be consistent with the phylogeny. Observations of fruit morphology of the genus Gynostemma revealed that transitional state species have independent morphological characteristics, such as oblate fruit and inferior ovaries. In conclusion, both molecular and morphological results showed consistency with those of phylogenetic analysis.

Herba Epimedii (Epimedium) leaves are rich in prenylated flavonol glycosides (PFGs) with high medicinal value. However, the dynamics and regulatory network of PFG biosynthesis remain largely unclear. Here, we combined metabolite profiling (targeted to PFGs) and a high-temporal-resolution transcriptome to elucidate PFGs' regulatory network in Epimedium pubescens and identified key candidate structural genes and transcription factors (TFs) involved in PFG accumulation. Chemical profile analysis revealed that PFG content was quite different between buds and leaves and displayed a continuous decline with leaf development. The structural genes are the determinant reasons, and they are strictly regulated by TFs under temporal cues. We further constructed seven time-ordered gene co-expression networks (TO-GCNs) of PFG biosynthesis genes (including EpPAL2, EpC4H, EpCHS2, EpCHI2, EpF3H, EpFLS3, and EpPT8), and three flavonol biosynthesis routines were then predicted. The TFs involved in TO-GCNs were further confirmed by WGCNA analysis. Fourteen hub genes, comprising 5 MYBs, 1 bHLH, 1 WD40, 2 bZIPs, 1 BES1, 1 C2H2, 1 Trihelix, 1 HD-ZIP, and 1 GATA were identified as candidate key TFs. The results were further validated by TF binding site (TFBS) analysis and qRT-PCR. Overall, these findings provide valuable information for understanding the molecular regulatory mechanism of PFGs biosynthesis, enriching the gene resources, which will guide further research on PFG accumulation in Epimedium.

Global poultry production is still severely affected by H9N2 avian influenza virus (AIV), and the development of a novel universal AIV vaccine is still urgently needed. Neuraminidase (NA) has recently been shown to be an efficient conserved protective antigen. In this study, we fused the extracellular region of the NA gene with a ferritin cassette (pYL281), which resulted in self-assembled 24-mer nanoparticles with the NA protein displayed outside the nanoparticles. In addition, a chicken dendritic cell-targeting nanobody-phage74 was also inserted ahead of the NA protein to yield pYL294. Incubation with chicken bone marrow-derived dendritic cells (chBMDCs) showed that the DC-targeting nanoparticles purified from the pYL294 strain significantly increased the maturation of chBMDCs, as shown by increased levels of CCL5, CCR7, CD83 and CD86 compared with nontargeting proteins. Then, a chicken study was performed using Salmonella oral administration together with intranasal boost with purified proteins. Compared with the other groups, oral immunization with Salmonella harboring pYL294 followed by intranasal boost with purified DC-targeting nanoparticles dramatically increased the humoral IgY and mucosal IgA antibody response, as well as increased the cellular immune response, as shown by elevated splenic lymphocyte proliferation and intracellular mRNA levels of IL-4 and IFN-γ. Finally, sequential immunization with DC-targeting nanoparticles showed increased protection against G57 subtype H9N2 virus challenge compared with other groups, as shown by significantly decreased virus RNA copy numbers in oropharyngeal washes (Days 3, 5 and 7 post challenge) and cloacal washes (Day 7), significantly decreased lung virus titers on Day 5 post challenge and increased body weight gains during the challenge.

Lignocellulosic materials are composed of cellulose, hemicellulose and lignin and are one of the most abundant biopolymers in marine environments. The extent of the involvement of marine microorganisms in lignin degradation and their contribution to the oceanic carbon cycle remains elusive. In this study, a novel lignin-degrading bacterial strain, LCG003, was isolated from intertidal seawater in Lu Chao Harbor, East China Sea. Phylogenetically, strain LCG003 was affiliated with the genus Aliiglaciecola within the family Alteromonadaceae. Metabolically, strain LCG003 contains various extracellular (signal-fused) glycoside hydrolase genes and carbohydrate transporter genes and can grow with various carbohydrates as the sole carbon source, including glucose, fructose, sucrose, rhamnose, maltose, stachyose and cellulose. Moreover, strain LCG003 contains many genes of amino acid and oligopeptide transporters and extracellular peptidases and can grow with peptone as the sole carbon and nitrogen source, indicating a proteolytic lifestyle. Notably, strain LCG003 contains a gene of dyp-type peroxidase and strain-specific genes involved in the degradation of 4-hydroxy-benzoate and vanillate. We further confirmed that it can decolorize aniline blue and grow with lignin as the sole carbon source. Our results indicate that the Aliiglaciecola species can depolymerize and mineralize lignocellulosic materials and potentially play an important role in the marine carbon cycle.

Diabetic nephropathy (DN) is a common complication of diabetes mellitus and cell death is a key issue in DN. Ferroptosis is a recently discovered type of iron-dependent cell death and different from other kinds of cell death including apoptosis and necrosis. However, ferroptosis has not been described in the context of DN. This study was to explore the role of ferroptosis in the DN pathophysiology and to explore the efficacy of ferroptosis inhibitor SRS 16-86 on DN. The STZ injection was used to establish the DM and DN animal models. We detected the levels of iron, reactive oxygen species, and ferroptosis-specific markers in a rat DN model to investigate whether there was ferroptosis in the process of DN. The hematoxylin-eosin staining, blood biochemistry, urine biochemistry and the of function kidney were used to evaluate the efficacy of ferroptosis inhibitor-SRS 16-86 in repairing DN. We found that SRS 16-86 could improve the recovery of renal function after DN by improving the antiferroptosis factors glutathione peroxidase 4, glutathione, and system Xc-light chain and could lower the lipid peroxidation marker and 4-hydroxynonenal. SRS 16-86 treatment may improve the structure of renal organization after DN. Inflammatory cytokines-interleukin 1β and tumor necrosis factor α, and intercellular adhesion molecule 1 were decreased significantly following SRS 16-86 treatment after DN. Results indicate that there is a strong connection between ferroptosis and the pathological mechanism of DN. The validity of SRS 16-86, a ferroptosis inhibitor in DN repair, supports its potential as a new therapeutic target for DN.

Abstract This meta‐analysis systematically evaluates the impact of the enhanced recovery after surgery (ERAS) concept on surgical site wound infections and postoperative complications in gastric cancer patients. A comprehensive computerized search was conducted in PubMed, Embase, Google Scholar, Cochrane Library, China National Knowledge Infrastructure and Wanfang databases for randomized controlled trials (RCTs), from database inception to November 2023, exploring the application of the ERAS concept in the perioperative period of gastric cancer surgery. Two researchers independently screened the literature, extracted data, and conducted quality assessments based on inclusion and exclusion criteria. Data analysis was performed using Stata 17.0 software. A total of 24 RCTs involving 2050 gastric cancer patients were included. The analysis revealed a significantly lower incidence of wound infections (OR = 0.23, 95% CI: 0.14–0.40, p &lt; 0.001) and postoperative complications (OR = 0.20, 95% CI: 0.15–0.27, p &lt; 0.001) in the ERAS group compared to the standard care group. This study demonstrates that the application of the ERAS concept during the perioperative period in gastric cancer surgery can effectively reduce the occurrence of wound infections and complications, thereby facilitating postoperative recovery.

The C3HC4 RING finger gene (RING-HC) family is a zinc finger protein crucial to plant growth. However, there have been no studies on the RING-HC gene family in potato. In this study, 77 putative StRING-HCs were identified in the potato genome and grouped into three clusters based on phylogenetic relationships, the chromosome distribution, gene structure, conserved motif, gene duplication events, and synteny relationships, and cis-acting elements were systematically analyzed. By analyzing RNA-seq data of potato cultivars, the candidate StRING-HC genes that might participate in tissue development, abiotic stress, especially drought stress, and anthocyanin biosynthesis were further determined. Finally, a StRING-HC gene (Soltu.DM.09G017280 annotated as StRNF4-like), which was highly expressed in pigmented potato tubers was focused on. StRNF4-like localized in the nucleus, and Y2H assays showed that it could interact with the anthocyanin-regulating transcription factors (TFs) StbHLH1 of potato tubers, which is localized in the nucleus and membrane. Transient assays showed that StRNF4-like repressed anthocyanin accumulation in the leaves of Nicotiana tabacum and Nicotiana benthamiana by directly suppressing the activity of the dihydroflavonol reductase (DFR) promoter activated by StAN1 and StbHLH1. The results suggest that StRNF4-like might repress anthocyanin accumulation in potato tubers by interacting with StbHLH1. Our comprehensive analysis of the potato StRING-HCs family contributes valuable knowledge to the understanding of their functions in potato development, abiotic stress, hormone signaling, and anthocyanin biosynthesis.

Abstract Tanshinones and phenolic acids are two major classes of bioactive compounds in Salvia miltiorrhiza. Revealing the regulatory mechanism of their biosynthesis is crucial for quality improvement of S. miltiorrhiza medicinal materials. Here we demonstrated that Smi-miR858a–Smi-miR858c, a miRNA family previously known to regulate flavonoid biosynthesis, also played critical regulatory roles in tanshinone and phenolic acid biosynthesis in S. miltiorrhiza. Overexpression of Smi-miR858a in S. miltiorrhiza plants caused significant growth retardation and tanshinone and phenolic acid reduction. Computational prediction and degradome and RNA-seq analyses revealed that Smi-miR858a could directly cleave the transcripts of SmMYB6, SmMYB97, SmMYB111, and SmMYB112. Yeast one-hybrid and transient transcriptional activity assays showed that Smi-miR858a-regulated SmMYBs, such as SmMYB6 and SmMYB112, could activate the expression of SmPAL1 and SmTAT1 involved in phenolic acid biosynthesis and SmCPS1 and SmKSL1 associated with tanshinone biosynthesis. In addition to directly activating the genes involved in bioactive compound biosynthesis pathways, SmMYB6, SmMYB97, and SmMYB112 could also activate SmAOC2, SmAOS4, and SmJMT2 involved in the biosynthesis of methyl jasmonate, a significant elicitor of plant secondary metabolism. The results suggest the existence of dual signaling pathways for the regulation of Smi-miR858a in bioactive compound biosynthesis in S. miltiorrhiza.

Abstract Using user-generated content (UGC) is of utmost importance for e-commerce platforms to extract valuable commercial information. In this paper, we propose an explainable multimodal learning approach named the visual–semantic embedding model with a self-attention mechanism for multimodal interaction (VSEM-SAMMI) to predict user-generated image (UGI) helpfulness and product sales. Focusing on SHEIN (i.e. a fast-fashion retailer), we collect the images posted by consumers, along with product and portrait characteristics. Moreover, we use VSEM-SAMMI, which adopts a self-attention mechanism to enforce attention weights between image and text, to extract features from UGI then use machine learning algorithms to predict UGI helpfulness and product sales. We explain features using a caption generation model and test the predictive power of embeddings and portrait characteristics. The results indicate that when predicting commercial information, embeddings are more informative than product and portrait characteristics. Combining VSEM-SAMMI with light gradient boosting (LightGBM) yields a mean squared error (MSE) of 0.208 for UGI helpfulness prediction and 0.184 for product sales prediction. Our study offers valuable insights for e-commerce platforms, enhances feature extraction from UGI through image–text joint embeddings for UGI helpfulness and product sales prediction, and pioneers a caption generation model for interpreting image embeddings in the e-commerce domain.

<h2>Abstract</h2><h3>Purpose</h3> To establish a deep learning-based model to predict radiotherapy-induced temporal lobe injury (TLI). <h3>Materials and methods</h3> Spatial features of dose distribution within the temporal lobe were extracted using both the three-dimensional convolution (C3D) network and the dosiomics method. The Minimal Redundancy-Maximal-Relevance (mRMR) method was employed to rank the extracted features and select the most relevant ones. Four machine learning (ML) classifiers, including logistic regression (LR), k-nearest neighbors (kNN), support vector machines (SVM) and random forest (RF), were used to establish prediction models. Nested sampling and hyperparameter tuning methods were applied to train and validate the prediction models. For comparison, a prediction model base on the conventional D_0.5cc of the temporal lobe obtained from dose volume (DV) histogram was established. The area under the receiver operating characteristic (ROC) curve (AUC) was utilized to compare the predictive performance of the different models. <h3>Results</h3> A total of 127 nasopharyngeal carcinoma (NPC) patients were included in the study. In the model based on C3D deep learning features, the highest AUC value of 0.843 was achieved with 5 features. For the dosiomics features model, the highest AUC value of 0.715 was attained with 1 feature. Both of these models demonstrated superior performance compared to the prediction model based on DV parameters, which yielded an AUC of 0.695. <h3>Conclusion</h3> The prediction model utilizing C3D deep learning features outperformed models based on dosiomics features or traditional parameters in predicting the onset of TLI. This approach holds promise for predicting radiation-induced toxicities and guide individualized radiotherapy.

Verticillium wilt is a disastrous disease causing significant yield losses of many crops. Isolation of verticillium wilt resistance gene is a fundamental work for controlling this disease through genetic engineering. In this report, we describe the cloning and characterization of a Ve like gene (StVe) from Solanum torvum Swartz. The nucleotide sequence of StVe is 3640 bp long with an open reading frame of 3414 bp encoding a protein precursor of 1138 aa. Sharing high homologies to tomato verticillium wilt disease resistance genes Ve1 and Ve2, the leucine rich (15.89%) protein StVe has a calculated molecular weight of 126.48kDa with an isoelectric point of 5.62. It possesses a hydrophobic N-terminal signal peptide of 20 aa and 38 predicted leucine-rich repeats containing 32 potential N-glycosylation sites (28 being significant). Fifty-seven predicted phosphorylation sites (36 for S, 8 for T and 13 for Y) distribute in StVe protein. A PEST-like sequence and a mammalian endocytosis signals YCVF are found within the C-terminal region. The C terminus of StVe concludes with the residues KKF similar to the KKX motif that confers endoplasmic reticulum localization in plants as well as mammals and yeast. The sequence analysis of the StVe gene implies that the StVe is a potential verticillium wilt disease resistance gene encoding a cell surface-like receptor protein.

To investigate the profile of gene expression in Salvia miltiorrhiza and elucidate its functional gene, 454 GS FLX platform and Titanium regent were used to produce a substantial expressed sequence tags (ESTs) dataset from the root of S. miltiorrhiza. A total of 46 722 ESTs with an average read length of 414 bp were generated. 454 ESTs were combined with the S. miltiorrhiza ESTs from GenBank. These ESTs were assembled into 18 235 unigenes. Of these unigenes, 454 sequencing identified 13 980 novel unigenes. 73% of these unigenes (13 308) were annotated using BLAST searches (E-value < or = 1e-5) against the SwissProt, KEGG TAIR, Nr and Nt databases. Twenty-seven unigenes (encoding 15 enzymes) were found to be involved in tanshinones biosynthesis, and 29 unigenes (encoding 11 enzymes) involved in phenolic acids biosynthesis. Seventy putative genes were found to encode cytochromes P450 and 577 putative transcription factor genes. Data presented in this study will constitute an important resource for the scientific community that is interested in the molecular genetics and functional genomics of S. miltiorrhiza.

Radiation-induced lung injury is a well-described complication of nuclear accidents, marrow-transplant pretreatment and thoracic radiotherapy. The mechanism is complex and no special therapy for it is available at present. To study radiation pulmonary injury following heavy ion radiotherapy for thoracic tumors, Kunming mice were randomly divided into 4 groups: normal control and 2, 4 and 6 Gy irradiation groups which underwent whole-body exposure to 235 MeV/u (12)C(6+) administered at the Heavy Ion Research Facility in Lanzhou (HIRFL). The pathological changes were observed by hematoxylin and eosin staining and the hydroxyproline (HP) content was assessed by spectrophotometry at months 1, 2, 3, 4, 5 and 6 after radiation exposure. In addition, the expression of tumor necrosis factor (TNF)-α and transforming growth factor (TGF)-β in the lung tissues was measured. The results showed that, compared with the control group, the lung tissue HP content was increased following irradiation but did not statistically significantly change after 4 months in the 4- and 6-Gy-treated groups. However, in the 2-Gy-treated group, the HP content was markedly increased between months 1 and 4 and decreased after month 4. The extent of the lung injury was significantly increased by the higher radiation dosages but was relieved in the 2 Gy group as the time since irradiation increased. The results also revealed that the levels of TNF-α were upregulated and reached a maximum at month 2, but decreased noticeably 2 months later in the experimental groups. The expression of TGF-β increased markedly in month 4 and was altered little in the 4- and 6-Gy-treated groups but decreased sharply in the 2 Gy irradiation group after month 4. These findings suggest that heavy ion radiotherapy for chest tumors causes lung injury to a certain extent, while there is likely to be little injury to lungs treated with <2 Gy, which provides scientific evidence for the use of heavy ion therapy for thoracic tumors.

Abstract Background Little genomic or trancriptomic information on Ganoderma lucidum ( Lingzhi ) is known. This study aims to discover the transcripts involved in secondary metabolite biosynthesis and developmental regulation of G. lucidum using an expressed sequence tag (EST) library. Methods A cDNA library was constructed from the G . lucidum fruiting body. Its high-quality ESTs were assembled into unique sequences with contigs and singletons. The unique sequences were annotated according to sequence similarities to genes or proteins available in public databases. The detection of simple sequence repeats (SSRs) was preformed by online analysis. Results A total of 1,023 clones were randomly selected from the G . lucidum library and sequenced, yielding 879 high-quality ESTs. These ESTs showed similarities to a diverse range of genes. The sequences encoding squalene epoxidase (SE) and farnesyl-diphosphate synthase (FPS) were identified in this EST collection. Several candidate genes, such as hydrophobin , MOB2 , profilin and PHO84 were detected for the first time in G . lucidum . Thirteen (13) potential SSR-motif microsatellite loci were also identified. Conclusion The present study demonstrates a successful application of EST analysis in the discovery of transcripts involved in the secondary metabolite biosynthesis and the developmental regulation of G. lucidum .

The products of the FIS genes play important regulatory roles in diverse developmental processes, especially in seed formation after fertilization. In this study, a FIS-class gene MhFIE was isolated from apple. It encoded a predicted protein highly similar to polycomb group (PcG) protein FERTILIZATION-INDEPENDENT ENDOSPERM (FIE). MhFIE functioned as an Arabidopsis FIE homologue, as indicated by functional complementation experiment using Arabidopsis fie mutant. In addition, BiFC assay showed that MhFIE protein interacted with AtCLF. Furthermore, transgenic Arabidopsis ectopically expressing MhFIE produced less APETALA3 (AtAP3) and AGAMOUS (AtAG) transcripts than WT control, and therefore exhibited abnormal flower, seed development. These results suggested that polycomb complex including FIE and CLF proteins played an important role in reproductive development by regulating the expression of its downstream genes. In addition, it was found that MhFIE constitutively expressed in various tissues tested. Its expression levels were lower in apomictic apple species than the sexual reproductive species, suggested it was possibly involved into apomixis in apple. Furthermore, the hybrids of tea crabapple generated MhFIE transcripts at different levels. The parthenogenesis capacity was negatively correlated with MhFIE expression level in these hybrids. These results suggested that MhFIE was involved into the regulation of flower development and apomixis in apple.

Giant cell tumour of the distal radius is a locally aggressive lesion. In this study, we performed a wrist arthrodesis reconstruction with an ipsilateral double barrel segmental ulnar bone graft combined with a modified Sauve-Kapandji procedure for a giant cell tumour of the distal radius. From January 2007 to September 2013, we followed eight patients for a mean duration of 36 months. One patient developed a recurrence and was treated by amputation; the other seven patients achieved radiological union in about 8 months. There was no wrist instability, deformation or dislocation; the mean range of motion of the forearm achieved 75° of supination and 70° of pronation. The patients could recover reasonable grip strength. This new operative procedure can excise the tumour with a low rate of recurrence, fewer functional deficits and fewer complications than reported for other procedures.IV, therapeutic.

Lung cancer is a series of gene-driven disease. EGFR, ALK, and ROS1 are 3 major driver genes that play an important role in lung cancer development and precision management. Additionally, rare genetic alterations continue to be discovered and may become novel targets for therapy. The RET gene is one of such rare genetic alteration of non-small cell lung cancer (NSCLC). In this report, we present a RET-positive case that benefited from cabozantinib treatment.A 50-year-old male patient was diagnosed with lung adenocarcinoma 2 years ago, at that time he received palliative surgery of pulmonary carcinoma and completed 4 cycles of chemotherapy with gemcitabine and cisplatin. Six months later, he was hospitalized in our cancer center due to the disease recurrence, presenting with pleural metastasis.Gene alteration was examined using the intraoperative specimen by PCR method, and KIF5B/RET gene fusion was detected. Therefore, the patient was diagnosed with late-stage lung adenocarcinoma with RET gene mutation.The patient received treatment with cabozantinib from June 2017.Cabozantinib was administered (140 mg orally, once daily) for approximate 9 months, and his disease achieved stable disease (SD). During that period, there were no severe adverse events (AE), except for a grade II rash (CTCAE 4.0).We found that the RET fusion gene is a novel driver molecular of lung adenocarcinoma in patients without common mutations in such genes as EGFR, ALK, and ROS1. This case report supports a rationale for the treatment of lung adenocarcinoma patients with a RET fusion and provides alternative treatment options for these types of NSCLC patients.

A biosimilar is a biological agent highly similar to an approved reference biological drug in terms of efficacy, safety, and purity, that often has a relatively lower price. 1 European Medicines AgencyGuideline on similar biological medicinal products. https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-similar-biological-medicinal-products-rev1_en.pdfDate: 2015 Date accessed: August 16, 2019 Google Scholar The availability of biosimilars offers health systems a chance to strike a balance between the drive to control costs and the need to improve access—deemed fundamental to achieving the Sustainable Development Goals, in particular target 3.8: achieving universal health coverage. 2 Lyman GH Zon R Harvey RD Rationale, opportunities, and reality of biosimilar medications. N Engl J Med. 2018; 379: 694-695 Crossref PubMed Scopus (12) Google Scholar The remarkable expansion in the development of biosimilars over the last decade has led to a focus on gaining a complete picture of all approved anticancer biosimilars available worldwide, and the disparities in the availability among different countries. Understanding global anticancer biosimilar availability will provide essential data and insight into the possible causes of global health inequality and could support the development of strategies to address it.

Alternate wetting and drying (AWD) irrigation has been widely used as an efficient rice production method to obtain better yield without continuous flooding (CF) of the paddy field. However, how this practice affects gene expression to regulate rice physiology and morphology is largely unknown. In this study, we used two rice varieties, Nipponbare, a lowland rice cultivar, and Gaoshan 1, an upland cultivar, and found that root dry weight (RDW) and root oxidation activity (ROA) in both cultivars substantially increased in response to AWD. We then analyzed the differences in transcriptome profiles of their roots irrigated in AWD vs. CF conditions. AWD responsive genes are mainly involved in lignin biosynthetic pathway and phytohormone signal transduction pathway and belong mainly to bHLH, bZIP, NAC, WRKY, and HSF transcription factor families. We discussed how these differentially expressed genes may contribute to the morphological adaptations observed in roots exposed to AWD. This analysis also provides useful information to explain the similarities and differences in adaptation to AWD irrigation between the two rice ecotypes.

Wolfiporia cocos (F. A. Wolf) has been praised as a food delicacy and medicine for centuries in China. Here, we present the genome and transcriptome of the Chinese strain CGMCC5.78 of W. cocos. High-confidence functional prediction was made for 9277 genes among the 10,908 total predicted gene models in the W. cocos genome. Up to 2838 differentially expressed genes (DEGs) were identified to be related to sclerotial development by comparing the transcriptomes of mycelial and sclerotial tissues. These DEGs are involved in mating processes, differentiation of fruiting body tissues, and metabolic pathways. A number of genes encoding enzymes and regulatory factors related to polysaccharide and triterpenoid production were strikingly regulated. A potential triterpenoid gene cluster including the signature lanosterol synthase (LSS) gene and its modified components were annotated. In addition, five nonribosomal peptide synthase (NRPS)-like gene clusters, eight polyketide synthase (PKS) gene clusters, and 15 terpene gene clusters were discovered in the genome. The differential expression of the velevt family proteins, transcription factors, carbohydrate-active enzymes, and signaling components indicated their essential roles in the regulation of fungal development and secondary metabolism in W. cocos. These genomic and transcriptomic resources will be valuable for further investigations of the molecular mechanisms controlling sclerotial formation and for its improved medicinal applications.

To facilitate functional genomic analysis and molecular breeding of velvet ash (Fraxinus velutina Torr), the de novo sequencing was carried out by Illumina sequencing technology. The cDNA samples were prepared from eleven different tissues of velvet ash and sequenced by using the Illumina genome analyzer. Subsequently, de novo assemebly, functional annotation databases, and the screening of expressed sequence tag-simple sequence repeats (EST-SSRs) were performed by comparing with corresponding databases using BLASTx and software tools. We obtained 51 698 unigenes with an average length of 661 bp and an N50 length of 980 bp. Among all these unigenes, 41 267 (79.8 %) were annotated in the NCBI non-redundant protein database and 25 236 (48.8 %) were annotated in the Swiss-Prot database. A total of 31 546 (61.0 %) and 13 281 (25.7 %) unigenes were successfully categorized to 59 and 25 functional groups, respectively, by gene ontology categories and clusters of orthologous group categories. A total of 22 323 (43.2 %) unigenes were assigned to 128 pathways using the Kyoto encyclopedia of genes and genomes pathway database. Additionally, 3 249 EST-SSRs markers were detected in 51 698 unigenes from velvet ash. Based on 3 249 EST-SSRs markers, 1 800 primer pairs were successfully designed using Primer 3. In the 50 randomly selected primers, 48 successfully amplified fragments, and 42 showed polymorphisms. We completed a successful application of the Illumina platform to de novo transcriptome assembly of velvet ash, which has the potential to be used for discovering novel genes and further characterization of gene expression profiles.

Introduction National Early Warning Score (NEWS) was launched in 2012 by the Royal College of Physicians in UK with an aim to improve the assessment of critical patients and timely detection of clinical deterioration. Objective To assess the performance of NEWS in emergency intensive care unit (EICU) patients in Beijing, PROC. Design prospective cohort study. Setting EICU in a university hospital. Methods The inclusion criteria were patients who stayed in the EICU beds under Department of Emergency Medicine, Xuanwu Hospital of Capital Medical University. Data of patients on admission were collected and calculated NEWS. Main outcome measure was death within 24 hours. The ability to predict mortality was assessed by area under the receiver operating characteristic curve (AUROC) analysis. Results Data on 540 consecutive EICU patients were collected from 1st January, 2013 to 31st March, 2013. Scores of 7 or more were associated with increased risk of death (OR=16.8; 95% CI 6.6-42.9). The AUROC for death within 24 h of admission was 0.85 (95% CI 0.79-0.90). Conclusions NEWS is applicable and feasible for EICU patients in Beijing. This study shows that the prediction power of NEWS for death within 24 hours of acutely ill patients attending Xuan Wu Hospital is comparable to that reported for the United Kingdom patients. (Hong Kong j.emerg.med. 2015;22:137-144)

Epithelial-to-mesenchymal transition (EMT) is an essential biological process involved in embryonic development, cancer progression, and metastatic diseases. EMT has often been used as a model for elucidating the mechanisms that underlie bladder cancer progression. However, no study to date has addressed the quantitative global variation of proteins in EMT using normal and non-malignant bladder cells. We treated normal bladder epithelial HCV29 cells and low grade nonmuscle invasive bladder cancer KK47 cells with transforming growth factor-beta (TGF-β) to establish an EMT model, and studied non-treated and treated HCV29 and KK47 cells by the stable isotope labeling amino acids in cell culture (SILAC) method. Labeled proteins were analyzed by 2D ultrahigh-resolution liquid chromatography/LTQ Orbitrap mass spectrometry. Among a total of 2994 unique identified and annotated proteins in HCV29 and KK47 cells undergoing EMT, 48 and 56 proteins, respectively, were significantly upregulated, and 106 and 24 proteins were significantly downregulated. Gene ontology (GO) term analysis and pathways analysis indicated that the differentially regulated proteins were involved mainly in enhancement of DNA maintenance and inhibition of cell-cell adhesion. Proteomes were compared for bladder cell EMT vs. bladder cancer cells, revealing 16 proteins that displayed similar changes in the two situations. Studies are in progress to further characterize these 16 proteins and their biological functions in EMT.

Cisplatin (CP), one of the most commonly used antitumor drugs in clinic, could induce reproductive and genetic toxicity. Traditional Chinese medicine believed that this side effect might be caused by the deficiency of both qi and blood. Panax notoginseng (Burk.) F. H. Chen (PN) is a traditional precious Chinese medicine for nourishing blood and hemostasis, which had the synergistic antitumor and reducing toxicity effects. However, the protective effect and mechanism of PN on CP-induced reproductive and genetic toxicity were still unknown. Aim of the study: This study was designed to illuminate the possible protective effect and mechanism of PN on CP-induced reproductive and genetic toxicity. Network pharmacology was first applied to analyze the potential components and targets of PN against CP-induced reproductive and genetic toxicity. Then, the results of network pharmacology were validated in a mouse model of reproductive and genotoxicity induced by CP. Body weight, testis weight, epididymis weight, sperm count, sperm viability and sperm morphology were used to assess protective effects of PN on CP-induced reproductive toxicity. Tail moment in peripheral blood cells and micronucleus in bone marrow cells were used to assess protective effects of PN on CP-induced genetic toxicity. Finally, possible protective targets obtained from network pharmacology, including 8-hydroxy-2-deoxyguanosine (8-OHdG), malondialdehyde (MDA), total superoxide dismutase (T-SOD) and glutathione peroxidase (GSH-Px), were experimentally validated by ELISA. One hundred and nineteen components of PN and sixty-eight targets of reproductive/genetic toxicity were acquired and constituted as the component-target network. Network pharmacology analysis showed alleviating oxidative stress might play important role in therapeutic mechanism of PN. In verified experiments, PN significantly improved the decline of body weight, testis weight and epididymis weight, increased sperm count and viability, decreased abnormal sperm morphology rate induced by CP in mice. Moreover, PN also significantly decreased the tail moment in peripheral blood cells and micronucleus formation rate in bone marrow cells in CP-induced mice. Finally, not only the decrease of T-SOD and GSH-Px level but also the increase of 8-OHdG and MDA level in serum were restored under PN treatment. Current study found that PN could improve CP-induced reproductive and genetic toxicity, which were probably attributed to alleviating oxidative stress. This finding provided the new perspective for understanding the therapeutic effect of PN on CP-induced reproductive and genetic toxicity and facilitating the clinical use of PN.

Fritillaria cirrhosa D. Don is an endangered species in the Liliaceae family, the bulb of which is the primary plant source for the Chinese traditional medicine “Chuan-beimu”, having activities that relieve coughs and eliminates phlegm. The major pharmacologically active constituents of F. cirrhosa are steroidal alkaloids. Two thousand one hundred and fifty-eight high-quality expressed sequence tags (ESTs) were generated from a cDNA library of F. cirrhosa bulbs and assembled to total 1343 unique transcripts. After removing ribosomal RNA sequences, 1330 putative protein coding sequences were obtained; among these, 765 (57.5%) had at least one significant match to the Swiss-Prot protein database via a BLASTX similarity search. The 1330 unique transcripts were further functionally classified for gene discovery purposes using the gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) databases. More than ten transcripts that are likely involved in the biosynthesis of F. cirrhosa alkaloids and corresponding regulatory activities were discovered in this EST dataset, including HMGR, FPSs, CYP450s and aminotransferases. This report describes the first example of EST analysis from the Fritillaria genus and lays the foundation for further cloning and identification of candidate genes related to steroidal alkaloid biosynthesis in F. cirrhosa.   Key words: Fritillaria cirrhosa, expressed sequence tags, steroidal alkaloids.

Scutellaria baicalensis is a well-known medicinal plant that produces biologically active flavonoids, such as baicalin, baicalein, and wogonin. Pharmacological studies have shown that these compounds have anti-inflammatory, anti-bacterial, and anti-cancer activities. Therefore, it is of great significance to investigate the genetic information of S. baicalensis, particularly the genes related to the biosynthetic pathways of these compounds. Here, we constructed the full-length transcriptome of S. baicalensis using a hybrid sequencing strategy and acquired 338,136 full-length sequences, accounting for 93.3% of the total reads. After the removal of redundancy and correction with Illumina short reads, 75,785 nonredundant transcripts were generated, among which approximately 98% were annotated with significant hits in the protein databases, and 11,135 sequences were classified as lncRNAs. Differentially expressed gene (DEG) analysis showed that most of the genes related to flavonoid biosynthesis were highly expressed in the roots, consistent with previous reports that the flavonoids were mainly synthesized and accumulated in the roots of S. baicalensis. By constructing unique transcription models, a total of 44,071 alternative splicing (AS) events were identified, with intron retention (IR) accounting for the highest proportion (44.5%). A total of 94 AS events were present in five key genes related to flavonoid biosynthesis, suggesting that AS may play important roles in the regulation of flavonoid biosynthesis in S. baicalensis. This study provided a large number of highly accurate full-length transcripts, which represents a valuable genetic resource for further research of the molecular biology of S. baicalensis, such as the development, breeding, and biosynthesis of active ingredients.

Background and Purpose: The availability of oncology biosimilars is deemed as a fundamental strategy to achieve sustainable health care. However, there is scarce systematic evidence on economic effectiveness of cancer biosimilars. We aimed to synthesize evidence from pharmacoeconomic evaluation of oncology biosimilars globally, provide essential data and methodological reference for involved stakeholders. Materials and Methods: This systematic review was conducted in PubMed, embase, the Cochrane library, CRD, ISPOR and NICE utill December 31, 2019. Information on basic characteristics, evaluation methodology and results were extracted. Quality of included studies was assessed using the Consolidated Health Economic Evaluation Reporting Standards Checklist. Results: For 17 studies identified (13 from Europe and four from United States), the overall quality was generally acceptable. A total of seven biological molecules involved with filgrastim, EPOETIN α , and trastuzumab leading the three. The mostly common evaluation perspective was payer, but the time horizon varied greatly. There were ten studies which adopted cost minimization analysis to evaluate efficiency while seven studies adopted budget impact analysis to address affordability, with cost ratio and cost saving being its corresponding primary endpoint. Although the comparability of included studies was limited and specific results were largely affected by uptake and price discount rates of the oncology biosimilar, the comprehensive results consistently favored its promotion. Conclusion: Globally, the economic evaluation of cancer biosimilars is in its initial phase. However, limited evidence from developed countries consistently supported both cost-effectiveness of efficiency and affordability of oncology biosimilars, while they were largely affected by uptake and price discount rate.

The phytohormone abscisic acid (ABA) plays a central role in mediating plant responses to environmental stresses. As a part of the stress response, the biosynthesis of certain secondary metabolites can be triggered by ABA. Salvia miltiorrhiza is a well-known medicinal plant that produces many biologically active components, such as tanshinones and salvianolic acid B. In this study, for the first time, we investigated S. miltiorrhiza transcriptome variations in response to ABA treatment by using Oxford Nanopore Technology (ONT). More than 76 Gb of clean ONT reads were generated from twelve cDNA libraries, and a total of 22,233 genes were detected. The expression pattern of approximately 50% of these genes was changed upon ABA induction, including most genes involved in ABA biosynthesis and ABA signal transduction, which suggests that in plants, multiple ABA signaling-induced cellular processes respond in a coordinated fashion to environmental stress. In addition, in S. miltiorrhiza leaves, ABA can change alternative splicing outputs mainly by reducing intron retention (IR) events through posttranscriptional splicing, which can quickly enhance protein biosynthesis to help plants cope with environmental stresses. Interestingly, ABA can upregulate the expression of genes involved in salvianolic acid biosynthesis but exerts a lesser effect on tanshinone biosynthesis, which indicates the different roles of these two classes of metabolites in response to environmental stresses. A better understanding of the ABA regulatory mechanism and ABA-triggered secondary metabolism will boost our ability to improve herb quality through molecular-assisted breeding, abiotic stress management and chemical manipulation with ABA-based bioactive agents.