Transcriptome-Wide Identification and Response Pattern Analysis of the Salix integra NAC Transcription Factor in Response to Pb Stress.

The NAC (NAM-ATAF1/2-CUC) transcription factor family is one of the largest plant-specific transcription factor families, playing an important role in plant growth and development and abiotic stress response. As a short-rotation woody plant, Salix integra (S. integra) has high lead (Pb) phytoremediation potential. To understand the role of NAC in S. integra Pb tolerance, 53 SiNAC transcripts were identified using third-generation and next-generation transcriptomic data from S. integra exposed to Pb stress, and a phylogenetic analysis revealed 11 subfamilies. A sequence alignment showed that multiple subfamilies represented by TIP and ATAF had a gene that produced more than one transcript under Pb stress, and different transcripts had different responses to Pb. By analyzing the expression profiles of SiNACs at 9 Pb stress time points, 41 of 53 SiNACs were found to be significantly responsive to Pb. Short time-series expression miner (STEM) analysis revealed that 41 SiNACs had two significant Pb positive response patterns (early and late), both containing 10 SiNACs. The SiNACs with the most significant Pb response were mainly from the ATAF and NAP subfamilies. Therefore, 4 and 3 SiNACs from the ATAF and NAP subfamilies, respectively, were selected as candidate Pb-responsive SiNACs for further structural and functional analysis. The RT-qPCR results of 7 transcripts also confirmed the different Pb response patterns of the ATAF and NAP subfamilies. SiNAC004 and SiNAC120, which were randomly selected from two subfamilies, were confirmed to be nuclear localization proteins by subcellular localization experiments. Functional prediction analysis of the associated transcripts of seven candidate SiNACs showed that the target pathways of ATAF subfamily SiNACs were "sulfur metabolism" and "glutathione metabolism", and the target pathways of NAP subfamily SiNACs were "ribosome" and "phenylpropanoid biosynthesis". This study not only identified two NAC subfamilies with different Pb response patterns but also identified Pb-responsive SiNACs that could provide a basis for subsequent gene function verification.

Small RNA and Degradome Sequencing Reveal Roles of miRNAs in the Petal Color Fading of Malus Crabapple.

The Malus crabapple is an important woody ornamental plant. The fading of petals during its development significantly affects their ornamental value. Petal color is related to anthocyanin content and miRNAs play an important role in the post-transcriptional regulation of anthocyanin synthesis. However, the mechanisms underlying miRNA regulation of petal fading have rarely been studied. Transcriptome and small RNA sequencing of petals from the blooming phases of Malus. 'Indian Summer' varieties S1 (small bud), S2 (initial-flowering), and S3 (late-flowering) allowed us to identify 230 known miRNAs and 17 novel miRNAs, including 52 differentially expressed miRNAs which targeted 494 genes and formed 823 miRNA-target pairs. Based on the target gene annotation results, miRNA-target pairs were screened that may be involved in the fading process of Malus crabapple petals through three different pathways: anthocyanin synthesis, transport, and degradation, involving mcr-miR858-MYB1\MYB5 and mcr-miR396-McCHI inhibiting anthocyanin synthesis; mcr-miR167, mcr-miR390, mcr-miR535, and mcr-miR858 inhibiting anthocyanin transport from the cytoplasm to the vacuole by targeting ABC transporter genes (ABCB, ABCC, ABCD, and ABCG); and mcr-miR398 targeting the superoxide dismutase genes (CZSOD2 and CCS) to accelerate anthocyanin degradation. These findings offer a novel approach to understanding the mechanism of petal fading and serve as a reference for other plants with floral fading.

Gene Structural Specificity and Expression of MADS-Box Gene Family in Camellia chekiangoleosa.

MADS-box genes encode transcription factors that affect plant growth and development. Camellia chekiangoleosa is an oil tree species with ornamental value, but there have been few molecular biological studies on the developmental regulation of this species. To explore their possible role in C. chekiangoleosa and lay a foundation for subsequent research, 89 MADS-box genes were identified across the whole genome of C. chekiangoleosa for the first time. These genes were present on all the chromosomes and were found to have expanded by tandem duplication and fragment duplication. Based on the results of a phylogenetic analysis, the 89 MADS-box genes could be divided into either type I (38) or type II (51). Both the number and proportion of the type II genes were significantly greater than those of Camellia sinensis and Arabidopsis thaliana, indicating that C. chekiangoleosa type II genes experienced a higher duplication rate or a lower loss rate. The results of both a sequence alignment and a conserved motif analysis suggest that the type II genes are more conserved, meaning that they may have originated and differentiated earlier than the type I genes did. At the same time, the presence of extra-long amino acid sequences may be an important feature of C. chekiangoleosa. Gene structure analysis revealed the number of introns of MADS-box genes: twenty-one type I genes had no introns, and 13 type I genes contained only 1~2 introns. The type II genes have far more introns and longer introns than the type I genes do. Some MIKCC genes have super large introns (≥15 kb), which are rare in other species. The super large introns of these MIKCC genes may indicate richer gene expression. Moreover, the results of a qPCR expression analysis of the roots, flowers, leaves and seeds of C. chekiangoleosa showed that the MADS-box genes were expressed in all those tissues. Overall, compared with that of the type I genes, the expression of the type II genes was significantly higher. The CchMADS31 and CchMADS58 genes (type II) were highly expressed specifically in the flowers, which may in turn regulate the size of the flower meristem and petals. CchMADS55 was expressed specifically in the seeds, which might affect seed development. This study provides additional information for the functional characterization of the MADS-box gene family and lays an important foundation for in-depth study of related genes, such as those involved in the development of the reproductive organs of C. chekiangoleosa.

Identification and Expression Analysis of the Populus trichocarpa GASA-Gene Family.

The gibberellic acid-stimulated Arabidopsis (GASA) gene family plays an important regulatory role in the growth and development of plants. In this study, we identified 19 GASA genes using bioinformatics-based methods in Populus trichocarpa, and these PtGASA genes could be divided into three categories based on their phylogenetic relationships. Based on an analysis of the structure and motifs of these genes, it was concluded that PtGASA class II members are more conserved than class I and class III members are, and the results of collinearity analysis showed that members of class II are collinearly related in poplar. Expression analysis of Populus trichocarpa roots, stems, and leaves showed that most of the PtGASA genes are expressed at higher levels in the stems or roots than in the leaves; a similar expression pattern was found in Vitis vinifera, indicating that the GASA-family members mainly play a role in the morphogenesis of poplar. Considering the phenomenon of gene amplification, we found that the higher the similarity of homologous genes was, the more similar the expression patterns. This study represents the first whole-genome identification and expression-profile analysis of the GASA-gene family in poplar, a model species, laying a foundation for functional studies of poplar GASA genes and serving as a reference for related research on other woody plant species.

Genome-Wide Identification of the Ginkgo (Ginkgo biloba L.) LBD Transcription Factor Gene and Characterization of Its Expression.

Lateral organ boundaries domain (LBD) proteins are plant-specific transcription factors involved in various transcriptional regulation processes. We identified a total of 37 GbLBD genes in ginkgo, and based on gene structure and phylogenetic analysis, the GbLBD gene family was classified into class I (33, with the largest number of Id genes (16)) and class II (4). The ginkgo LBD gene was also analyzed regarding its chromosomal distributions, gene duplications, promoters, and introns/exons. In addition, gene expression profiling and real-time quantitative PCR analysis showed that the expression of 14 GbLBD genes differed in six different tissues and three developmental stages. The GbLBD gene of class II were highly expressed relative to the class I gene in all tissues and developmental stages, while class Id gene were generally at low levels or were not expressed, especially in seed developmental stages. The expression pattern analysis of cold/drought treatment and IAA/ABA hormone treatment showed that abiotic stress treatment could significantly induce the expression of GbLBD gene, of which class II genes played a key role in stress treatment. Our study provides a solid foundation for further evolutionary and functional analysis of the ginkgo LBD gene family.

Comparison among three methods for obtaining chloroplast genome sequences from the conifer Pinus massoniana.

The chloroplast genome (CPG) is a powerful tool for phylogenetic studies. Many CPGs have been determined using NGS. However, the large nuclear-genome and difficult CPG-DNA separation in conifers limit their application in related research. In this study, three methods (PCR + Sanger, PCR + HiSeq, cpDNA+HiSeq) for obtaining the CPGs of Pinus massoniana were compared for sequence accuracy, time and cost. PCR + Sanger obtained the most accurate CPGs with advantages in cost (3.08$/kb) and time (2-3 days); PCR + HiSeq generated some DNA fragments with low depth, and the SNPs false-positive-rate (0.44) and sequencing error-rate (0.0265) of this method were higher than those of the cpDNA+HiSeq. Moreover, the cost (~6.17$/kb) and time (4-5 weeks) would significantly increase when HiSeq sequencing were outsourced to sequencing service company. Thus, for the study of intraspecific and interspecies variation in CPGs, CPG sequences can be obtained by comprehensive methods to bridge the method shortcomings. Scuh as sequence accuracy, cost and time.

Overexpression of GbF3'5'H1 Provides a Potential to Improve the Content of Epicatechin and Gallocatechin.

The flavonoids in Ginkgo biloba L. (ginkgo) have important medicinal uses due to their antioxidant, antitumor, and blood circulation-promoting effects. However, the genetic mechanisms underlying flavonoid biosynthesis in ginkgo remain elusive. Flavonoid 3', 5'-hydroxylase (F3'5'H) is an important enzyme in flavonoid synthesis. We detected a novel differentially expressed GbF3'5'H1 gene homologous to the F3'5'H enzyme involved in the flavonoid synthesis pathway through transcriptome sequencing. In this study, we characterized this gene, performed an expression analysis, and heterologously overexpressed GbF3'5'H1 in Populus. Our results showed that GbF3'5'H1 is abundant in the leaf and highly expressed during April. We also found four metabolites closely related to flavonoid biosynthesis. Importantly, the contents of 4',5-dihydroxy-7-glucosyloxyflavanone, epicatechin, and gallocatechin were significantly higher in transgenic plants than in nontransgenic plants. Our findings revealed that the GbF3'5'H1 gene functions in the biosynthesis of flavonoid-related metabolites, suggesting that GbF3'5'H1 represents a prime candidate for future studies (e.g., gene-editing) aiming to optimize ginkgo flavonoid production, especially that of flavan-3-ols.

Roles of the SPL gene family and miR156 in the salt stress responses of tamarisk (Tamarix chinensis).

BACKGROUND: Accumulating evidences show that SPLs are crucial regulators of plant abiotic stress tolerance and the highly conserved module miR156/SPL appears to balance plant growth and stress responses. The halophyte Tamarix chinensis is highly resistant to salt tress. SPLs of T. chinensis (TcSPLs) and theirs roles in salt stress responses remain elusive. RESULTS: In this study, we conducted a systematic analysis of the TcSPLs gene family including 12 members belonging to 7 groups. The physicochemical properties and conserved motifs showed divergence among groups and similarity in each group. The microRNA response elements (MREs) are conserved in location and sequence, with the exception of first MRE within TcSPL5. The miR156-targeted SPLs are identified by dual-luciferase reporter assay of MRE-miR156 interaction. The digital expression gene profiles cluster suggested potential different functions of miR156-targeted SPLs vs non-targeted SPLs in response to salt stress. The expression patterns analysis of miR156-targeted SPLs with a reverse expression trend to TcmiR156 suggested 1 h (salt stress time) could be a critical time point of post-transcription regulation in salt stress responses. CONCLUSIONS: Our work demonstrated the post-transcription regulation of miR156-targeted TcSPLs and transcription regulation of non-targeted TcSPLs in salt stress responses, and would be helpful to expound the miR156/SPL-mediated molecular mechanisms underlying T. chinensis salt stress tolerance.

Transcriptome analysis and identification of genes related to terpenoid biosynthesis in Cinnamomum camphora.

BACKGROUND: Cinnamomum camphora has been cultivated as an economically important tree for its medicinal and aromatic properties. Selective breeding has produced Cinnamomum plants for special uses, including spice strains with characteristic flavors and aromas and high-potency medicinal cultivars. The molecular biology underlying terpenoid biosynthesis is still unexplored. RESULTS: Gas chromatography-mass spectrometry was used to analyze the differences in contents and compositions of essential oil terpenoids in linalool- and borneol-type chemotypes of C. camphora. The data revealed that the essential oils consist primarily of monoterpenes with only very minor quantities of sesquiterpenes and diterpenes and that the essential oil differs in different chemotypes of C. camphora, with higher yields of (-)-borneol from the borneol-type than from the linalool-type. To study the terpenoid biosynthesis of signature compounds of the major monoterpenes, we performed RNA sequencing to profile the leaf transcriptomes of the two chemotypes of C. camphora. A total of 23.76 Gb clean data was generated from two chemotypes and assembled into 156,184 unigenes. The total length, average length, N50 and GC content of unigenes were 155,645,929 bp, 997 bp, 1430 bp, and 46.5%, respectively. Among them, 76,421 unigenes were annotated by publicly available databases, of which 67 candidate unigenes were identified to be involved in terpenoid biosynthesis in C. camphora. A total of 2863 unigenes were identified to be differentially expression between borneol-type and linalool-type, including 1714 up-regulated and 1149 down-regulated unigenes. Most genes encoding proteins involved in terpenoid precursor MVA and MEP pathways were expressed in similar levels in both chemotypes of C. camphora. In addition, 10 and 17 DEGs were significantly enriched in the terpene synthase activity and oxidoreductase activity terms of their directed acyclic graphs (DAG), respectively. Three monoterpene synthase genes, TPS14-like1, TPS14-like2 and TPS14-like3 were up-regulated in the borneol-type compared to the linalool-type, and their expression levels were further verified using quantitative real-time PCR. CONCLUSIONS: This study provides a global overview of gene expression patterns related to terpenoid biosynthesis in C. camphora, and could contribute to a better understanding of the differential accumulation of terpenoids in different C. camphora chemotypes.

Nivolumab versus Everolimus in Advanced Renal-Cell Carcinoma.

BACKGROUND: Nivolumab, a programmed death 1 (PD-1) checkpoint inhibitor, was associated with encouraging overall survival in uncontrolled studies involving previously treated patients with advanced renal-cell carcinoma. This randomized, open-label, phase 3 study compared nivolumab with everolimus in patients with renal-cell carcinoma who had received previous treatment. METHODS: A total of 821 patients with advanced clear-cell renal-cell carcinoma for which they had received previous treatment with one or two regimens of antiangiogenic therapy were randomly assigned (in a 1:1 ratio) to receive 3 mg of nivolumab per kilogram of body weight intravenously every 2 weeks or a 10-mg everolimus tablet orally once daily. The primary end point was overall survival. The secondary end points included the objective response rate and safety. RESULTS: The median overall survival was 25.0 months (95% confidence interval [CI], 21.8 to not estimable) with nivolumab and 19.6 months (95% CI, 17.6 to 23.1) with everolimus. The hazard ratio for death with nivolumab versus everolimus was 0.73 (98.5% CI, 0.57 to 0.93; P=0.002), which met the prespecified criterion for superiority (P≤0.0148). The objective response rate was greater with nivolumab than with everolimus (25% vs. 5%; odds ratio, 5.98 [95% CI, 3.68 to 9.72]; P<0.001). The median progression-free survival was 4.6 months (95% CI, 3.7 to 5.4) with nivolumab and 4.4 months (95% CI, 3.7 to 5.5) with everolimus (hazard ratio, 0.88; 95% CI, 0.75 to 1.03; P=0.11). Grade 3 or 4 treatment-related adverse events occurred in 19% of the patients receiving nivolumab and in 37% of the patients receiving everolimus; the most common event with nivolumab was fatigue (in 2% of the patients), and the most common event with everolimus was anemia (in 8%). CONCLUSIONS: Among patients with previously treated advanced renal-cell carcinoma, overall survival was longer and fewer grade 3 or 4 adverse events occurred with nivolumab than with everolimus. (Funded by Bristol-Myers Squibb; CheckMate 025 ClinicalTrials.gov number, NCT01668784.).

Complete Chloroplast Genome of Pinus massoniana (Pinaceae): Gene Rearrangements, Loss of ndh Genes, and Short Inverted Repeats Contraction, Expansion.

The chloroplast genome (CPG) of Pinus massoniana belonging to the genus Pinus (Pinaceae), which is a primary source of turpentine, was sequenced and analyzed in terms of gene rearrangements, ndh genes loss, and the contraction and expansion of short inverted repeats (IRs). P. massoniana CPG has a typical quadripartite structure that includes large single copy (LSC) (65,563 bp), small single copy (SSC) (53,230 bp) and two IRs (IRa and IRb, 485 bp). The 108 unique genes were identified, including 73 protein-coding genes, 31 tRNAs, and 4 rRNAs. Most of the 81 simple sequence repeats (SSRs) identified in CPG were mononucleotides motifs of A/T types and located in non-coding regions. Comparisons with related species revealed an inversion (21,556 bp) in the LSC region; P. massoniana CPG lacks all 11 intact ndh genes (four ndh genes lost completely; the five remained truncated as pseudogenes; and the other two ndh genes remain as pseudogenes because of short insertions or deletions). A pair of short IRs was found instead of large IRs, and size variations among pine species were observed, which resulted from short insertions or deletions and non-synchronized variations between "IRa" and "IRb". The results of phylogenetic analyses based on whole CPG sequences of 16 conifers indicated that the whole CPG sequences could be used as a powerful tool in phylogenetic analyses.

Anti-IP-10 antibody (BMS-936557) for ulcerative colitis: a phase II randomised study.

OBJECTIVE: Interferon-γ-inducible protein-10 (IP-10 or CXCL10) plays a role in inflammatory cell migration and epithelial cell survival and migration. It is expressed in higher levels in the colonic tissue and plasma of patients with ulcerative colitis (UC). This phase II study assessed the efficacy and safety of BMS-936557, a fully human, monoclonal antibody to IP-10, in the treatment of moderately-to-severely active UC. DESIGN: In this 8-week, phase II, double-blind, multicentre, randomised study, patients with active UC received placebo or BMS-936557 (10 mg/kg) intravenously every other week. The primary endpoint was the rate of clinical response at Day 57; clinical remission and mucosal healing rates were secondary endpoints. Post hoc analyses evaluated the drug exposure-response relationship and histological improvement. RESULTS: 109 patients were included (BMS-936557: n=55; placebo: n=54). Prespecified primary and secondary endpoints were not met; clinical response rate at Day 57 was 52.7% versus 35.2% for BMS-936557 versus placebo (p=0.083), and clinical remission and mucosal healing rates were 18.2% versus 16.7% (p=1.00) and 41.8% versus 35.2% (p=0.556), respectively. However, higher BMS-936557 steady-state trough concentration (Cminss) was associated with increased clinical response (87.5% vs 37.0% (p<0.001) for patients with Cminss 108-235 µg/ml vs placebo) and histological improvements (73.0% vs 41.0%; p=0.004). Infections occurred in 7 (12.7%) BMS-936557-treated patients and 3 (5.8%) placebo-treated patients. 2 (3.6%) BMS-936557 patients discontinued due to adverse events. CONCLUSIONS: Anti-IP-10 antibody, BMS-936557, is a potentially effective therapy for moderately-to-severely active UC. Higher drug exposure correlated with increasing clinical response and histological improvement. Further dose-response studies are warranted. CLINICAL TRIAL REGISTRATION NUMBER: ClinicalTrials.gov NCT00656890.

PMAT: an efficient plant mitogenome assembly toolkit using low-coverage HiFi sequencing data.

Complete mitochondrial genomes (mitogenomes) of plants are valuable resources for nucleocytoplasmic interactions, plant evolution, and plant cytoplasmic male sterile line breeding. However, the complete assembly of plant mitogenomes is challenging due to frequent recombination events and horizontal gene transfers. Previous studies have adopted Illumina, PacBio, and Nanopore sequencing data to assemble plant mitogenomes, but the poor assembly completeness, low sequencing accuracy, and high cost limit the sampling capacity. Here, we present an efficient assembly toolkit (PMAT) for de novo assembly of plant mitogenomes using low-coverage HiFi sequencing data. PMAT has been applied to the de novo assembly of 13 broadly representative plant mitogenomes, outperforming existing organelle genome assemblers in terms of assembly accuracy and completeness. By evaluating the assembly of plant mitogenomes from different sequencing data, it was confirmed that PMAT only requires 1× HiFi sequencing data to obtain a complete plant mitogenome. The source code for PMAT is available at https://github.com/bichangwei/PMAT. The developed PMAT toolkit will indeed accelerate the understanding of evolutionary variation and breeding application of plant mitogenomes.

A phase II, randomized, double-blind, placebo-controlled study evaluating the efficacy and safety of MDX-1100, a fully human anti-CXCL10 monoclonal antibody, in combination with methotrexate in patients with rheumatoid arthritis.

OBJECTIVE: CXCL10 (also known as interferon-γ-inducible 10-kd protein [IP-10]) is a chemokine that potentially plays a role in the immunopathogenesis of rheumatoid arthritis (RA). We undertook this phase II study to evaluate the efficacy and safety of MDX-1100, a fully human, anti-CXCL10 (anti-IP-10) monoclonal antibody, in RA patients whose disease responded inadequately to methotrexate (MTX). METHODS: Patients with active RA receiving stable doses of MTX (10-25 mg weekly) were randomized to receive intravenous doses of 10 mg/kg MDX-1100 (n = 35) or placebo (n = 35) every other week. The primary end point was the proportion of patients meeting the American College of Rheumatology 20% improvement criteria (achieving an ACR20 response) on day 85, and patients were followed up for safety to day 141. RESULTS: The ACR20 response rate was significantly higher among MDX-1100-treated patients than among placebo-treated patients (54% versus 17%; P = 0.0024). Statistically significant differences in the ACR20 response rate between treatments were observed starting on day 43 (P < 0.05). The ACR50 and ACR70 response rates on day 85 did not differ between the groups. Overall, 51.4% of MDX-1100-treated patients and 30.3% of placebo-treated patients experienced at least 1 adverse event (AE). No study drug-related serious AEs were reported. CONCLUSION: MDX-1100 was well tolerated and demonstrated clinical efficacy in RA patients whose disease responded inadequately to MTX. This is the first study to demonstrate clinical efficacy of a chemokine inhibitor in RA and supports the notion of a potential role of IP-10 in the immunopathogenesis of RA.

GbFLSa overexpression negatively regulates proanthocyanin biosynthesis.

Flavonoids are important secondary metabolites with extensive pharmacological functions. Ginkgo biloba L. (ginkgo) has attracted extensive attention because of its high flavonoid medicinal value. However, little is understood about ginkgo flavonol biosynthesis. Herein, we cloned the full-length gingko GbFLSa gene (1314 bp), which encodes a 363 amino acid protein that has a typical 2-oxoglutarate (2OG)-Fe(II) oxygenase region. Recombinant GbFLSa protein with a molecular mass of 41 kDa was expressed in Escherichia coli BL21(DE3). The protein was localized to the cytoplasm. Moreover, proanthocyanins, including catechin, epicatechin, epigallocatechin and gallocatechin, were significantly less abundant in transgenic poplar than in nontransgenic (CK) plants. In addition, dihydroflavonol 4-reductase, anthocyanidin synthase and leucoanthocyanidin reductase expression levels were significantly lower than those of their CK counterparts. GbFLSa thus encodes a functional protein that might negatively regulate proanthocyanin biosynthesis. This study helps elucidate the role of GbFLSa in plant metabolism and the potential molecular mechanism of flavonoid biosynthesis.

Protoplast isolation and transient transformation system for Ginkgo biloba L.

Ginkgo biloba L. has a unique evolutionary status. Owing to its high medicinal and ornamental value, ginkgo has also recently become a research hotspot. However, the large genome and long juvenile period, as well as the lack of an effective genetic transformation system, have hindered gaining a full understanding of the comprehensive functions of ginkgo genes. At present, heterologous expression of genes in model plants is the primary method used in ginkgo-related research; however, these distant plant model relatives limit reliable interpretation of the results for direct applications in ginkgo breeding. To overcome these limitations, in this study, an efficient isolation and transient expression system for ginkgo protoplasts was established. A large number of intact and homogeneous ginkgo mesophyll protoplasts were isolated using 2% cellulase and 0.25% pectinase in 0.4 M mannitol. The activity of these protoplasts remained above 90% even after 24 h. Furthermore, when the concentration of the polyethylene glycol 4000 solution was 30%-40% (w/v), the transformation efficiency of the protoplasts reached 40%. Finally, the reliability of the system was verified using subcellular localization, transient overexpression, and protein interaction experiments with ginkgo genes, thereby providing a technical platform for the identification and analysis of ginkgo gene functions. The proposed method partially compensates for the limitations associated with the lack of a genetic transformation system and provides technical support to expand research on elucidating the functions of ginkgo genes.

Novel mechanisms for the synthesis of important secondary metabolites in Ginkgo biloba seed revealed by multi-omics data.

Although the detailed biosynthetic mechanism is still unclear, the unique secondary metabolites of Ginkgo biloba, including ginkgolic acids (GAs) and terpene trilactones, have attracted increasing attention for their potent medicinal, physiological and biochemical properties. In particular, GAs have shown great potential in the fields of antibacterial and insecticidal activities, making it urgent to elucidate their biosynthetic mechanism. In this study, we systematically revealed the landscape of metabolic-transcriptional regulation across continuous growth stages of G. biloba seeds (GBS) based on multi-omics mining and experimental verification, and successfully identified all major types of GAs and terpene trilactones along with more than a thousand kinds of other metabolites. The phenological changes and the essential gene families associated with these unique metabolites were analyzed in detail, and several potential regulatory factors were successfully identified based on co-expression association analysis. In addition, we unexpectedly found the close relationship between large introns and the biosynthesis of these secondary metabolites. These genes with large introns related to the synthesis of secondary metabolites showed higher gene expression and expression stability in different tissues or growth stages. Our results may provide a new perspective for the study of the regulatory mechanism of these unique secondary metabolites in GBS.

Assessment of the Impact of Alternative Fixatives on HER2 Detection in Breast Cancer and Gastric Cancer Tumor Specimens.

The type of fixative used for preserving tumor specimens can significantly impact the performance of the immunohistochemistry and in situ hybridization assays used for assessing human epidermal growth factor receptor 2 (HER2) status. This study reports the prevalence of the use of alternative fixatives other than the guideline-recommended 10% neutral buffered formalin (NBF) during HER2 testing in a real-world setting. The effects of alternative fixatives [20% NBF and 10% unbuffered formalin (UBF) fixatives] on HER2 testing of breast cancer (BC) and gastric cancer (GC) cell lines and tissues are also assessed. Overall, 117,636 tumor samples received at a central laboratory from >8000 clinical trial sites across 60 countries were reviewed to determine the prevalence of alternative fixative usage. To investigate the impact of alternative fixatives, 27 cell lines (21 BC and 6 GC) and 76 tumor tissue samples (50 BC and 26 GC) were fixed in 10% NBF, 20% NBF, or 10% UBF, and evaluated for HER2 status by immunohistochemistry and in situ hybridization. Real-world data showed that 9195 (7.8%) tumor samples were preserved using an alternative fixative. In cell lines, overall percentage agreement, negative percentage agreement, and positive percentage agreement among the 3 fixatives were 100%. In tumor tissues, the agreement among 10% NBF, 20% NBF, and 10% UBF ranged between 94.7% and 96.6% for negative percentage agreement and 90.9% for overall percentage agreement compared with a range of 58.3% to 66.7% for positive percentage agreement. These results suggest that alternative fixatives may have the potential to convert HER2 status in tissues from positive to negative.

A phase II, double-blind, randomised, placebo-controlled study of BMS945429 (ALD518) in patients with rheumatoid arthritis with an inadequate response to methotrexate.

BACKGROUND: Interleukin 6 (IL-6) plays a key role in the inflammatory cascade in rheumatoid arthritis. BMS945429 is a humanised, monoclonal antibody that potently binds IL-6. OBJECTIVE: To conduct aphase II study to determine the efficacy and safety of BMS945429 in patients with active rheumatoid arthritis and an inadequate response to methotrexate. METHODS: Patients were randomised 1:1:1:1 to BMS945429 (80, 160 or 320 mg; administered intravenously) or placebo plus methotrexate during this 16-week, double-blind trial. The primary efficacy end point was the proportion of patients with a 20% improvement in American College of Rheumatology responses (ACR20) at week 12. Additional end points included ACR50 and ACR70 responses and 28-joint Disease Activity Scores (DAS28). RESULTS: Of 127 randomised and treated patients, 116 completed the trial. ACR20 responders at week 12 were 81% (80 mg; p<0.0001 vs placebo), 71% (160 mg; p=0.0005 vs placebo), 82% (320 mg; p<0.0001 vs placebo) and 27% (placebo), respectively. By week 16, 14% (80 mg), 28% (160 mg) and 44% (320 mg) of BMS945429 patients were in DAS28 remission (DAS28 score <2.6). Statistically significant and clinically meaningful improvements in health-related quality of life (HRQoL) were reported in all active treatment groups. Administration of BMS945429 was associated with increases in liver enzymes and in serum cholesterol. There were no serious infections, infusion reactions or apparent immunogenicity. CONCLUSIONS: In this phase II study, BMS945429 was associated with rapid and significant improvements in disease activity and HRQoL in patients with active rheumatoid arthritis and an inadequate response to methotrexate.

Development of 198 novel EST-derived microsatellites in Eucalyptus (Myrtaceae).

PREMISE OF THE STUDY: Expressed sequence tag (EST)-derived microsatellites were identified in Eucalyptus through screening the GenBank database. The loci were sequence-verified and explored for polymorphism among 20 genotypes. METHODS AND RESULTS: In total, 198 novel microsatellites were developed from 8262 unigenes, with the identity of 73.6-100% to the original sequences and presence of the expected repeat motifs. One hundred and eighty-four markers proved to be polymorphic among 10 E. urophylla and 10 E. tereticornis genotypes, with the number of alleles per locus, observed heterozygosity, and polymorphic information content being 2-17 (mean: 7.11), 0-1.0 (mean: 0.4511), and 0.0940-0.9131 (mean: 0.6571), respectively. CONCLUSIONS: These markers will be useful for germplasm characterization, genome mapping, and gene tagging for economic traits in the two species examined and may have potential for genetic applications in Eucalyptus.