PHLDA1 is a P53 target gene involved in P53-mediated cell apoptosis.

Pleckstrin homeolike domain, family A, member 1 (PHLDA1) is a multifunctional protein that plays diverse roles in A variety of biological processes, including cell death, and hence its altered expression has been found in different types of cancer. Although studies have shown a regulatory relationship between p53 and PHLDA1, the molecular mechanism is still unclear. Especially, the role of PHLDA1 in the process of apoptosis is still controversial. In this study, we found that the expression of PHLDA1 in human cervical cancer cell lines was correlated with the up-expression of p53 after treatment with apoptosis-inducing factors. Subsequently, the binding site and the binding effect of p53 on the promoter region of PHLDA1 were verified by our bioinformatics data analysis and luciferase reporter assay. Indeed, we used CRISPR-Cas9 to knockout the p53 gene in HeLa cells and further confirmed that p53 can bind to the promoter region of PHLDA1 gene, and then directly regulate the expression of PHLDA1 by recruiting P300 and CBP to change the acetylation and methylation levels in the promoter region. Finally, a series of gain-of-function experiments further confirmed that p53 re-expression in HeLap53-/- cell can up-regulate the reduction of PHLDA1 caused by p53 knockout, and affect cell apoptosis and proliferation. Our study is the first to explore the regulatory mechanism of p53 on PHLDA1 by using the p53 gene knockout cell model, which further proves that PHLDA1 is a target-gene in p53-mediated apoptosis, and reveals the important role of PHLDA1 in cell fate determination.

First report of Lily virus X infecting Paris polyphylla var. yunnanensis in Yunnnan, China.

Lily virus X (LVX) is a positive-sense ssRNA virus belonging to the genus Potexvirus in the family Alphaflexiviridae. LVX is known to infect plants of the genera Lilium and Tricyrtis in the family Liliacea. LVX was first reported in an asymptomatic lily (Lilium formosanum) from England (Stone, 1980), but has been shown to infect plants in the Netherlands (Chen et al. 2005), the United States (Jordan et al. 2008) and Japan (Nijo et al. 2018). To date, the complete genomes of two LVX isolates from the Netherlands and Japan have been reported. Paris polyphylla var. yunnanensis, known as Dianchonglou in China, is a perennial plant of the family Melanthiaceae (formerly belonging to the family Trillium). In China, its rhizome is commonly used as an antispasmodic agent for stroke and cancer treatment (Chang et al. 2017). From 2019 to 2022, leaf mottle and shrinkage which are typical symptoms of viral infections were observed on the leaves of P. polyphylla var. yunnanensis plants in Dianchonglou fields in Qujing, Yunnan. Disease incidence ranged from 19% to 45% across 5 fields (90 plants per field) in Qujing. To identify the possible viral pathogen(s) associated with the disease, the mirVanaTM miRNA isolation Kit was used to extract total RNA was from a mixed sample pool of 5 symptomatic leaf samples collected from the 5 fields. RNA sequencing library was constructed using TruSeqTM RNA sample preparation kit. Sequencing on the Illumina HiSeqTM 2500 platform (Illumina, USA) with 125-bp paired-end reads yielded 23,077,786 raw reads. 22,534,100 clean reads were obtained by removing reads of low quality and poly-N using Trimmomatic software (Bolger et al. 2014). By utilizing the paired-end splicing method in Trinity software (Grabherr et al. 2011) the the raw reads were De novo assembled into 184,596 contigs, of which 303 were related to viruses, including Paris mosaic necrosis virus (PMNV), Pear alphapartitivirus (PAPV), Dahlia mosaic virus (DMV), and Lily virus X (LVX). BLASTn analysis revealed that 12 contigs (lengths ranging from 344 nt to 5,981 nt, query cover 6% to 99%) were most similar (57.32% to 91.67% nt identities) to the genome sequences of LVX, suggesting a possible infection of LVX in the plants. To confirm the result, a full-length genomic sequence of LVX was obtained by reverse transcription polymerase chain reaction (RT-PCR) using specific primers designed based on the sequence of the assembled contigs. The PCR products were cloned into pGEM-T vector (Promega Corporation, USA) and sequenced using the Sanger method (Sangon Biotech, Shanghai, China). The obtained full-length genomic sequence of the LVX isolate (LVX-PP, accession number OM100017) was 5,981 nt in length. BLASTp analysis demonstrated that the putative Rep and CP of LVX-PP shared 76.27% to 81.05% and 80.81% to 81.82% aa sequence similarities with that of other LVX isolates, respectively. Maximum-likelihood phylogenetic trees inferred from the Rep and CP aa sequences showed that LVX-PP clustered closely with LVX isolates. The leaf samples were further analyzed using a lily virus X (LVX) ELISA kit (DEIAPV181, Creative Diagnostics, U.S.A.). Healthy P. polyphylla var. yunnanensis leaves were taken as a negative control and buffer solution as a blank control. The results showed a positive reaction for all five symptomatic plants (OD = 1.259 ± 0.007) relative to the negative (OD = 0.099) and blank (OD = 0.073) controls. These results indicate that LVX can infect P. polyphylla var. yunnanensis. To our knowledge, this is the first report that LVX has been detected in P. polyphylla var. yunnannensis. This study will serve as an important reference for the study of the host range of LVX. Further studies will be required to determine how LVX spreads between P. polyphylla var. yunnannensis and other host plants.

STAT3-Mediated Promoter-Enhancer Interaction Up-Regulates Inhibitor of DNA Binding 1 (ID1) to Promote Colon Cancer Progression.

BACKGROUND: High expression of inhibitor of DNA binding 1 (ID1) correlates with poor prognosis in colorectal cancer (CRC). Aberrant enhancer activation in regulating ID1 transcription is limited. METHODS: Immunohistochemistry (IHC), quantitative RT-PCR (RT-qPCR) and Western blotting (WB) were used to determine the expression of ID1. CRISPR-Cas9 was used to generate ID1 or enhancer E1 knockout cell lines. Dual-luciferase reporter assay, chromosome conformation capture assay and ChIP-qPCR were used to determine the active enhancers of ID1. Cell Counting Kit 8, colony-forming, transwell assays and tumorigenicity in nude mice were used to investigate the biological functions of ID1 and enhancer E1. RESULTS: Human CRC tissues and cell lines expressed a higher level of ID1 than normal controls. ID1 promoted CRC cell proliferation and colony formation. Enhancer E1 actively regulated ID1 promoter activity. Signal transducer and activator of transcription 3 (STAT3) bound to ID1 promoter and enhancer E1 to regulate their activity. The inhibitor of STAT3 Stattic attenuated ID1 promoter and enhancer E1 activity and the expression of ID1. Enhancer E1 knockout down-regulated ID1 expression level and cell proliferation in vitro and in vivo. CONCLUSIONS: Enhancer E1 is positively regulated by STAT3 and contributes to the regulation of ID1 to promote CRC cell progression and might be a potential target for anti-CRC drug studies.

First report of stem rot of Scrophularia ningpoensis caused by Fusarium commune in China.

Chinese figwort (Scrophularia ningpoensis Hemsl.) is an important annual herb and its dried root tubers are used as a traditional Chinese herbal medicine. In May 2021, a disease with stem rot symptoms on S. ningpoensis was observed at three randomly selected fields (~0.67 ha per field) in Nanchuan district (28.93°N, 107.27°E) of Chongqing, China. Disease incidence was estimated between 10% and 17% based on calculating the proportion of symptomatic plants. Initially, watery dark brown spots appeared on the epidermis of the stem. Then the spots expanded into spindle or strip shape, and the center of lesions were sunken, constricted and rotted finally (Figure 1A and Figure 1B). Leaves turned yellow and the plants wilted (Figure 1C). The infected parts of the stem broke easily and became brittle. The number of daughter buds used for reproduction was reduced by more than 24% and the production of root tubers decreased by more than 3%. Twelve stems with typical rot symptoms were sampled from the three fields for further investigation. Infested tissue fragments (4×4 mm) were surface sterilized with 75% ethanol for 30s and 2% sodium hypochlorite for 2 minutes in turn, finally, were rinsed 4 times with sterilized water. The disinfected tissue were air-dried and transferred onto potato dextrose agar (PDA) in the dark for 6 days at 25℃. The resulting fungal colonies were isolated by the single-spore isolation technique (Fang. 1998). Six different fungal colonies were isolated (X1-X6) and Koch's postulates were conducted to verify the pathogenicity of individual isolates. The stem surfaces of 8 months old plants were sterilized with 75% ethanol for 30 s, rinsed three times with sterilized water, and stabbed with a sterilized needle. Conidial from the fungal colonies grown on PDA plate were harvested by filtration through five layers of sterilized absorbent gauze. Conidial concentration was then adjusted to 106 conidia per mL. 10 µL of conidial suspension was sprayed on stems injured with a sterile syringe. For each isolate, 6 plants were inoculated. Stems inoculated with sterilized water were used as a blank control. All plants were all put in a growth chamber at 28℃ with 75 to 80% relative humidity under a 12 h photoperiod for 15 days. The pathogenicity test was repeated once. After 13 days, the stems inoculated with X3 showed the same rot symptoms as we observed in the fields (Figure 1D) whereas the control stems remained symptomless (Figure 1E). The fungus re-isolated from the plants showing 100% symptoms had a similar morphology than X3 as described below. At the same time, the stems inoculated with X1, X2, X4, X5 and X6 showed no sign of rot. After culturing on PDA for 9 days under 25℃ in dark, isolate X3 grew all over the dish with white or pale pink pigmentation in the center (Figure 1F). Macroconidia were produced on synthetic low nutrient agar (SNA) plates, which showed sickle or spindle, 3 septate, straight to slightly curved with a foot-shaped basal cell, ranging from 17.595~44.88 × 2.04~3.315 µm (n=30). Microconidia were oval, elliptical or reniform, 0 to 1 septate, 3.06~12.75 ×1.785~2.805 µm (n=30) in size (Figure 1G). Phialides of conidiophores were cylindrical, short and monophialides or polyphialides (Figure 1H). Chlamydospores were found terminal or cluster with round or oblong (Figure 1I). These morphological characteristics described as Fusarium commone (Skovgaard et al. 2003). For molecular identification, the ribosomal internal transcribed spacer (ITS), translation elongation factor 1-alpha (EF-1α), RNA polymerase II subunit 1 (RPB1), the largest subunit of RNA polymerase Ⅱ gene sequences (RPB2) and the mitochondrial small subunit rDNA (mtSSU) genes were amplified with primers V9G /ITS4 (Hoog et al. 1998; White et al. 1990), EF1-668F /EF1-1251R (Alves et al. 2008), Fa/G2R (O'Donnell et al. 2010), 5f2/7cr (Liu et al. 1999; O'Donnell et al. 2010) and NMS1/NMS2 (Li et al. 1994). The sequences of isolate X3 were deposited in GenBank (MZ571935 (ITS), MZ576201 (EF-1α), MZ882396 (RPB1), MZ882397 (RPB2) and MZ867716 (mtSSU)). All sequences were revealed more than 99.8% sequence identity with reported sequences of Fusarium commune (GenBank accession No: KY630717, JF740838, KU171680, KU171700 and MK439851). Based on the optimal nucleotide replacement model SYM of multi-gene series sequence matrix, the system development tree was constructed. Results showed the strain X3 and those of F. commune (Isolates numbers were NRRL 28387, MRC 2566, MRC 2564 and CZ3-5-6) were clustered into the same evolutionary branch with a post-mortem probability of 0.996 (Figure 2). According to the morphology, molecular identification and phylogenetic analysis based on the concatenated of EF-1α and RPB2 genes sequences, the isolated X3 was identified as F. commune. The ITS sequences of X1, X2, X4, X5 and X6 showed homology exceeding 97.1% to Fusarium tricinctum (MH931273), Plectosphaerella cucumerina (MH858371), Sordariomycetes sp. (JX179237), Whalleya microplace (EF026129) and Pestalotiopsis maculiformans (EU552147), respectively, suggested the five strains to be these species possibly. GeneBank accession number of X1, X2, X4, X5 and X6 was OM074010, OM074011, OM074013, OM074015 and OM074018, respectively. To our best knowledge, this is the first report of F. commune infecting S. ningpoensis in China. Stem rot caused by F. commune is a severe threat to Chinese figwort cultivation, and identification of this pathogen is important for effective disease management and control.

First report of Diaporthe eres causing root rot of Coptis chinensis Franchet.

Chongqing coptis (Coptis chinensis Franchet) industry produces more than 60% of the Chinese coptis crop, and has been exported to many countries and regions. Since 2008, root rot has become a serious and widespread disease on coptis plants in Shizhu county with an average incidence of 40%, and yield losses up to 67%. Symptomatic coptis plants showed stunted growth, with the fibrous roots and main roots having brown or black, rotten, necrotic lesions. To our knowledge, Fusarium solani, F. carminascens, F. oxysporum and F. tricinctum have been previously reported as pathogens of coptis root rot (Luo et al. 2014; Cheng et al. 2020; Wu et al. 2020), but non Fusarium pathogens has not been reported yet. In order to identify new pathogens, 33 diseased roots were collected from Shizhu (30°18'N, 108°30'E) in October 2019. Small samples (0.5 cm in length) were cut from the border between diseased and healthy tissue, and then put on PDA after surface sterilization. Cultures were incubated at 25°C in dark until fungal colonies were observed. After subculturing for 3 times, 3 out of 21 isolates yielded a similar type of fungal colony. White, aerial, fluffy mycelium were formed and reached 8.3 cm diameter within 7 days, and dark pigmentation developed in the centre. Colonies turned to gray with age, and abundant dark brown pycnidia and black stromata were formed at maturity. Alpha conidia were aseptate, hyaline, fusiform to ellipsoidal, often biguttulate, measuring (6.0-8.5)×(2.0-3.0) µm. Beta conidia were aseptate, hyaline, linear to hooked, measuring (18-30)×(1.0-1.5) µm (Figure S1). For further identification, a multigene phylogenetic analysis was carried out. The internal transcribed spacer (ITS), translation elongation factor 1ɑ (tef1-ɑ), histone H3 (his3), calmodulin (cal), and ß-tubulin (tub2) gene regions were amplified with ITS1/ITS4, EF1-728F/EF1-986R, CYLH3F/H3-1b, CAL228F/CAL737R, T1/Bt2b (White et al. 1990; Glass and Donaldson 1995; Carbone and Kohn 1999; Crous et al. 2004). GenBank accession numbers of isolate H13 were MT463391 for the ITS region, MT975573 for tef1-ɑ, MT975574 for his3, MT975575 for cal, and MT975576 for tub2. BLAST results showed the ITS, tef1-ɑ, his3, cal and tub2 sequences revealed 99.82% (553/554 base pairs), 100% (347/347 base pairs), 100% (474/474 base pairs), 99.39% (486/489 base pairs), and 99.14% (803/810 base pairs) homology respectively with those of Diaporthe eres (MN816416.1, KU557616.1, KC343564.1, KU557595.1, and KY569366.1). Thus, H13 were identified as D. eres based on its morphological and molecular characteristics. Pathogenicity of D. eres in coptis was investigated using the H13 isolate (1 of the 3 isolates). The roots of 10 healthy 2-year-old coptis plants were individually inoculated with 5 ml of a 106 conidia/mL conidial suspension and sterilized water was used to mock inoculate. Thirty days after inoculation, most of the inoculated coptis roots showed dark brown and rotten root, similar to those observed in the field, whereas mock inoculated roots showed healthy. D. eres was recovered from symptomatic roots and identified based on morphology. To our knowledge, this is the first report of D. eres causing root rot of coptis not only in China but anywhere in the world.

[Effect of drying processing methods on different specifications of Sophorae Flos based on comprehensive statistical analysis].

To investigate the effects of six common drying methods on the quality of different specifications of Sophorae Flos, in order to select their suitable drying methods. According to appearance and morphology, Sophorae Flos was divided into the following three specifications: flower bud type(HL), half-open type(BK) and blooming type(SK). All specifications of samples were treated with shade-drying method(25 ℃, natural temperature), sun-drying method, hot-air-drying method(60, 105 ℃), and drying method(60 ℃) after steaming. The contents of total flavonoids, rutin, narcissus, quercetin, isorhamnetin, and Fe~(3+) reducing ability, DPPH free radical scavenging ability, ABTS free radical scavenging ability and fluorescence recovery after photobleaching(FRAP) were detected by UV, HPLC and colorimetry, respectively. Principal component analysis(PCA), cluster analysis(CA) and correlation analysis were used to comprehensively evaluate the quality of samples. According to the results, there were significant differences in the effect of drying methods on different specifications of samples. The drying method(60 ℃) after steaming was suitable for HL and BK, while the hot-air-drying method(60 ℃) was suitable for SK. When the fresh medicinal materials could not be treated in time, they should be spread out in a cool and ventilated place. Under high and low temperature conditions, the quality of three specifications of Sophorae Flos would be reduced. The hot-air-drying method(105 ℃) and shade-drying method(25 ℃) were not suitable for the treatment of fresh flowers and flower buds of Sophora japonicus. There were obviously differences of chemical compositions and antioxidant activities among the three specifications of samples. Therefore, the specifications of medicinal materials should be controlled to ensure the uniform quality. The study provided the abundant data reference for the selection of appropriate drying methods for the three specifications of Sophorae Flos, and useful exploration for the classification and processing of medicinal materials of flowers.

First Report of Root Rot Caused by Fusarium avenaceum on Coptis chinensis Franchet in Chongqing, China.

Coptis chinensis Franchet, is a perennial herb used as a traditional Chinese medicine. Annual production of Coptis is about 3000 tons in Shizhu, Chongqing. In recent years, root rot has become a serious and widespread disease on Coptis in Shizhu with an average incidence of 40%, and yield losses up to 67%. Infected plants were easy to pull from the soil, and most of the fibrous roots and main roots were brown or black compared to healthy roots that were yellow. Severely infected plants were wilted and necrotic. In October 2019, 33 diseased roots were collected from Shizhu (30°18'N, 108°30'E), and small samples (0.5 cm in length) were cut from the border between diseased and healthy tissue, successively sterilized with 75% ethanol and 2% sodium hypochlorite, rinsed 3 times with sterilized water, dried on sterilized filter paper, and transferred onto PDA, and incubated at 25°C for 7 days in dark. Eighteen distinct fungal isolates (H1-H18) were isolated and Koch's postulates were conducted to verify the pathogenicity of individual isolates. The rhizosphere soil of healthy 2-year-old Coptis plants was inoculated by pouring 5 mL of conidial suspension (106 conidia/mL) scraped from a culture of each isolate on PDA. Sterilized water was used to mock inoculate. For each isolate, 6 plants were inoculated. After 20 days, the roots of all plants inoculated with H15 or H18 were dark brown and rotten, while mock inoculated plants were healthy. The isolates H15 and H18 were re-isolated from symptomatic plants. Isolate H15 formed abundant white mycelium on PDA and produced rose pigment in the agar. Conidia were long and slender, straight to slightly curved, with 1-3 septate. The apical cells were tapering and bent, and the foot cells were distinctly notched. Conidiogenous cells were monophialides and polyphialides. No chlamydospores were observed (Figure S1). Isolate H18 formed white sparse mycelium on PDA and produced no pigment in the agar. Conidia were relatively wide, straight and stout, with 3-5 septate. The apical cells were blunt and rounded, and the foot cells were barely notched. Conidiogenous cells were long monophialides. Chlamydospores were formed intercalary in the hyphae (Figure S2). For further identification, the internal transcribed spacer (ITS), ß-tubulin, translation elongation factor 1ɑ (EF1ɑ) and RNA polymerase second largest subunit (RPB2) gene regions were amplified with ITS1/ITS4, Bt2a/Bt2b, EF1/EF2 and 5f2/7cr (White et al. 1990; Glass and Donaldson, 1995; O'Donnell et al. 2010). GenBank accession numbers of H15 and H18 were MT463390 and MT463389 for the ITS region, MT465656 and MT465654 for ß-tubulin, MT653321 and MT465651 for EF1ɑ, and MT653323 and MT653322 for RPB2. BLAST results showed the ITS, ß-tubulin, EF1ɑ, and RPB2 sequences revealed 100% (533/533 base pairs), 100% (265/265 base pairs), 98% (622/632 base pairs), and 99% (936/947 base pairs) homology respectively with those of Fusarium avenaceum (MN186746.1, MH791368.1, KU238140.1, and MK185027.1), and 100% (537/537 base pairs), 100% (227/227 base pairs), 100% (688/688 base pairs), and 99.03% (918/927 base pairs) with F. solani in GenBank (MH857319.1, MN692929.1, KP674211.1, and MH300549.1), respectively. Thus, H15 and H18 were identified as F. avenaceum and F. solani based on its morphological and molecular characteristics. To our knowledge, F. solani has been previously reported as a pathogen on Coptis (Luo et al. 2014), and this is the first report of root rot on Coptis caused by F. avenaceum in the world. Identification of the pathogens is important for effective disease management and control.

A novel lncRNA NR4A1AS up-regulates orphan nuclear receptor NR4A1 expression by blocking UPF1-mediated mRNA destabilization in colorectal cancer.

Long non-coding RNAs (lncRNAs) play important roles in tumorigenesis and cancer progression. The orphan nuclear receptor subfamily 4 group A member 1 (NR4A1) acts as an oncogene, and is involved in colorectal cancer (CRC) development. However, the mechanism through which lncRNA regulates NR4A1 expression remains unknown. We aimed to identify lncRNAs that regulate NR4A1 and assess their underlying mechanisms in CRC. We first identified an antisense lncRNA of NR4A1 that was up-regulated in CRC tissues and cells with rapid amplification of cDNA ends (RACE), and designated it as NR4A1AS. Spearman correlation analysis showed that NR4A1AS was positively correlated with NR4A1 mRNA levels in 37 CRC tissues. Mechanistically, NR4A1AS stabilized NR4A1 mRNA by forming RNA-RNA complexes via partial base-pairing and up-regulated NR4A1 expression in CRC cells. RNA immunoprecipitation (RIP) assays revealed that knockdown of NR4A1AS expression by siRNA enhanced up-frameshift 1 (UPF1) recruitment to NR4A1 mRNA, thereby decreasing NR4A1 mRNA stability. Moreover, depletion of NR4A1AS was found to mimic the effect of NR4A1 knockdown, specifically by suppressing cell proliferation, migration and invasion, and inducing apoptosis and cell cycle arrest. Accordingly, restoring NR4A1 expression ameliorated the effects of NR4A1AS knockdown on tumor growth and metastasis of CRC cells in vitro and in vivo Thus, we conclude that NR4A1AS up-regulates NR4A1 expression by forming RNA-RNA complexes and blocking UPF1-mediated mRNA destabilization, and it functions in tumor growth and metastasis of CRC cells at least partly through regulating NR4A1, suggesting that NR4A1AS might be as a potential target for RNA-based anti-CRC drug studies.

Variations in the Components and Antioxidant and Tyrosinase Inhibitory Activities of Styphnolobium japonicum (L.) Schott Extract during Flower Maturity Stages.

Styphnolobium japonicum (L.) Schott is widely cultivated in China, and its flowers and flower buds (FFB-SJ) are commonly used as traditional Chinese medicine. This work aimed to assess variations in the chemical components and antioxidant and tyrosinase inhibitory activities of S. japonicum extract during five flower maturity stages (ES1-ES5). The results showed that the contents of total flavonoids, rutin, and narcissin were highest at ES1, whereas the contents of quercetin and isorhamnetin were highest at ES3. ES1 presented considerable antioxidant activities in terms of reducing power (RP) and 1,1-diphenyl-2-picrylhydrazyl radical (DPPH. ) and hydroxyl radical (. OH) scavenging capacity, whereas ES3 showed excellent tyrosinase inhibitory activity and 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS.+ )- and O2 .- -scavenging capacity. Rutin and quercetin are the main bioactive components of FFB-SJ with antioxidant and tyrosinase inhibition, and the immature flower buds of S. japonicum (S2 and S3) with excellent biological activities and relatively high extract yields were the best for product development.

[Transcriptome analysis reveals candidate genes involved in flavonols biosyhthesis in Sophora japonica].

In order to study the pathways of biosynthesis of flavonoids in Sophora japonica, 113 797 unigenes were obtained by Trinity software, with an average length of 803 bp, of which 72 752 unigenes were obtained from the database by high-throughput sequencing, and a total of 38 891 SSR loci were searched. Through the metabolic pathway analysis, we found that there were 135 unigenes involved in the biosynthesis of flavonoids and 959 unigene involved in other secondary metabolic pathways. Further analysis of genes involved in rutin biosynthesis revealed that 24 were associated with CHS, 52 were associated with FLS, and 11 were associated with UFGT. The obtained data of S. japonica transcriptome lays the foundation for studying the pathways of biosynthesis of flavonoids in S. japonica and provides theoretical basis for the formation of the quality of S. japonica.

[Research for dynamic changes of growth and alkaloids of Coptis chinensis].

With Coptis chinensis in high-yielding soil as the object,the growth regularity of plant and dynamic change of alkaloid content was studied. The plant growth model of C. chinensis was constructed. The plant height equation was y=3.030 9+0.732 6x-0.009 6x²,the number of leaves equation was y=111.882 6-2 234.881 7/x+15 218.960 8/x²-31 740.960 8/x³,the leaf area equation was y=-217.136 1+30.552 2x-0.359 0x²,the roots talk biomass equation was y=-2.748 8+0.210 3x+0.006 4x²,the number of rootstalk equation was y=-1.246 0+0.192 6x+0.000 8x²,the fibrous root biomass equation was y=-4.973 5+0.589 4x -0.002 6x². The results indicated that the number of leaves and leaf area were increasing continuously after seedling transplanting,the leaf area of 3-year-old C. chinensis reached a maximum value of 425.83 cm²/plant,after declining.The number of leave of 5-year-old C. chinensis reached a maximum value of 70.91. With the increasing of years of growth, the number of rootstalk and rootstalk biomass of C. chinensis was increasing continuously. The biomass growth of 3-year-old and 4-year-old rootstalk was the fastest in the whole development stage of C. chinensis,the annual increase of more than 300%. The change curve of rootstalk number, rootstalk biomass and fibrous root biomass in the whole growth stage was a s-type.The dry matter partition of leafwas the highest in 1-year-old C. chinensis, and then gradually decreased,the change trend of dry matter partition of rootstalk was just the opposite, the dry matter partition of fibrous root increases with the increase of the growing year, reaching the maximum value in 3-year-old, then gradually lower trend. The root-shootratio of 1-year-old C. chinensis was the smallest, then gradually increases, the growth center gradually shifted to the roots from stems and leaves, The weight of underground part of 3-year-old C. chinensis exceeded the aboveground part, the 5-year-old C. chinensis root-shoot ratio reached the maximum value of 1.91:1.With the increasing of years of growth, the contents of coptisine, berberine, epiberberine and palmatine in rootstalk was increasing continuously. The jatrorrhizine content in 2-year-old C. chinensis was significantly lower than that in other years, the content was no significant change after that. The columbamine content reached a maximum value in 3-year-old C. chinensis,then the decreased gradually. The content of magnoflorine gradually increased and reached maximum value in 5-year-old C. chinensis.

[Research on bacteria microecology in root rot rhizosphere soil of Coptis chinensis produced in Shizhu city].

Illumina Hiseq 2500 high-throughput sequencing platform was used to study the bacteria richness and diversity, the soil enzyme activities, nutrients in unplanted soil, root-rot and healthy rhizophere soil of Coptis chinensis for deeply discussing the mechanism of the root-rot of C. chinensis. The high-throughput sequencing result showed that the artificial cultivation effected the bacteria community richness and diversity. The bacteria community richness in healthy and diseased rhizosphere soil showed significant lower than that of in unplanted soil (P<0.05) and declined bacteria diversity. The bacteria community richness in root-rot rhizosphere soil increased significantly than that of health and unplanted soil and the diversity was lower significant than that of unplanted soil (P<0.05). The results of soil nutrients and enzyme activities detected that the pH value, available phosphorus and urease activity decreased and the sucrase activity increased significantly (P<0.05). The content of organic carbon and alkaline hydrolysis nitrogen the catalase and urease activity in root rot soil samples was significantly lower than that of healthy soil samples (P<0.05). However, the contents of available phosphorus and available potassium were significantly in root-rot sample higher than that of healthy soil samples (P<0.05). Comprehensive analysis showed that the artificial cultivation declined the bacteria community richness and diversity. The bacteria community richness decreased significantly and the decreased diversity may be the cause of the root-rot. Meanwhile, the decrease of carbon and the catalase activity may be another cause of the root-rot in C. chinensis produced in Shizhu city, Chongqing province.

[Transcriptome characterization for Scrophularia ningpoensis based on high-throughput sequencing technology and related genes for synthesis of terpenoid compounds].

To investigate the profile of gene function and search for SSR, a new technology of high-throughput Solexa/Illumina sequencing was used to generate the root transcriptome of Scrophularia ningpoensis, and 65 602 036 raw reads were obtained. Based on the bioinformatics analysis and Trinity, 73 983 unigenes were obtained with an average length of 823 bp. The comparison of sequence homology in database showed that 56 389 unigenes had different degrees of homology. A total of 520 metabolic pathways related genes and 191 relDODO transcription factors were identified by the Swiss-Prot, GO, KEGG and COG.The 11 659 SSRs were found by MISA and the highest frequency was AG/CT. In this study, we obtained numerous SSRs to provide references for the study of functional gene cloning and genetic diversity of S. ningpoensis. The key genes involved in the secondary metabolism are the basis for the study of biosynthesis and regulatory mechanism of the secondary metabolites.

[Identification and phylogenetic analysis of endophytic fungi isolated from Scrophularia ningpoensis].

The endophytic fungi from root, main stem, branch and leaf of Scrophularia ningpoensis were isolated and identified from Wulong and Chongqing, and the population diversity analysis and phylogenetic analysis were followed. The result indicated that, as to population diversity index, S. ningpoensis from Wulong： leaf>main stem=branch>root, branch from Chongqing>branch from Wulong. Fifty-eight endophytic fungi were obtained, most of which were the pathogens of the plant. Colletotrichum was the prevailing genus, of which C. gloeosporioides and C. boninense were the prevailing strains. Leaf and seedlings might be the main path of infection. Endophytic fungi and pathogen might convert to each other, influenced by such factors as environment, genotype et al.

Histone acetylation regulates orphan nuclear receptor NR4A1 expression in hypercholesterolaemia.

Hypercholesterolaemia and inflammation are correlated with atherogenesis. Orphan nuclear receptor NR4A1, as a key regulator of inflammation, is closely associated with lipid levels in vivo. However, the mechanism by which lipids regulate NR4A1 expression remains unknown. We aimed to elucidate the underlying mechanism of NR4A1 expression in monocytes during hypercholesterolaemia, and reveal the potential role of NR4A1 in hypercholesterolaemia-induced circulating inflammation. Circulating leucocytes were collected from blood samples of 139 patients with hypercholesterolaemia and 139 sex- and age-matched healthy subjects. We found that there was a low-grade inflammatory state and higher expression of NR4A1 in patients. Both total cholesterol and low-density lipoprotein cholesterol levels in plasma were positively correlated with NR4A1 mRNA level. ChIP revealed that acetylation of histone H3 was enriched in the NR4A1 promoter region in patients. Human mononuclear cell lines THP-1 and U937 were treated with cholesterol. Supporting our clinical observations, cholesterol enhanced p300 acetyltransferase and decreased HDAC7 (histone deacetylase 7) recruitment to the NR4A1 promoter region, resulting in histone H3 hyperacetylation and further contributing to NR4A1 up-regulation in monocytes. Moreover, cytosporone B, an NR4A1 agonist, completely reversed cholesterol-induced IL-6 (interleukin 6) and MCP-1 (monocyte chemoattractant protein 1) expression to below basal levels, and knockdown of NR4A1 expression by siRNA not only mimicked, but also exaggerated the effects of cholesterol on inflammatory biomarker up-regulation. Thus we conclude that histone acetylation contributes to the regulation of NR4A1 expression in hypercholesterolaemia, and that NR4A1 expression reduces hypercholesterolaemia-induced inflammation.

AS1DHRS4, a head-to-head natural antisense transcript, silences the DHRS4 gene cluster in cis and trans.

The human genome, like other mammalian genomes, encodes numerous natural antisense transcripts (NATs) that have been classified into head-to-head, tail-to-tail, or fully overlapped categories in reference to their sense transcripts. Evidence for NAT-mediated epigenetic silencing of sense transcription remains scanty. The DHRS4 gene encodes a metabolic enzyme and forms a gene cluster with its two immediately downstream homologous genes, DHRS4L2 and DHRS4L1, generated by gene duplication. We identified a head-to-head NAT of DHRS4, designated AS1DHRS4, which markedly regulates the expression of these three genes in the DHRS4 gene cluster. By pairing with ongoing sense transcripts, AS1DHRS4 not only mediates deacetylation of histone H3 and demethylation of H3K4 in cis for the DHRS4 gene, but also interacts physically in trans with the epigenetic modifiers H3K9- and H3K27-specific histone methyltransferases G9a and EZH2, targeting the promoters of the downstream DHRS4L2 and DHRS4L1 genes to induce local repressive H3K9me2 and H3K27me3 histone modifications. Furthermore, AS1DHRS4 induces DNA methylation in the promoter regions of DHRS4L2 by recruiting DNA methyltransferases. This study demonstrates that AS1DHRS4, as a long noncoding RNA, simultaneously controls the chromatin state of each gene within the DHRS4 gene cluster in a discriminative manner. This finding provides an example of transcriptional control over the multiple and highly homologous genes in a tight gene cluster, and may help explain the role of antisense RNAs in the regulation of duplicated genes as the result of genomic evolution.

AMP-activated protein kinase is required for cell survival and growth in HeLa-S3 cells in vivo.

Activation of the AMP-dependent protein kinase (AMPK) is linked to cancer cell survival in a variety of cancer cell lines, particularly under conditions of stress. As a potent activator of AMPK, metformin has become a hot topic of discussion for its effect on cancer cell. Here, we report that AMPK activated by metformin promotes HeLa-S3 cell survival and growth in vivo. Our results show that metformin inhibited cell proliferation in MCF-7 cells, but not in LKB1-deficient HeLa-S3 cells. Re-expression of LKB-1 in HeLa-S3 cells restored the growth inhibitory effect of metformin, indicating a requirement for LKB-1 in metformin-induced growth inhibition. Moreover, AMPK activation exerted a protective effect in HeLa-S3 cells by relieving ER stress, modulating ER Ca(2+) storage, and finally contributing to cellular adaptation and resistance to apoptosis. Our findings identify a link between AMPK activation and cell survival in HeLa-S3 cells, which demonstrates a beneficial effect of AMPK activated by metformin in cancer cell, and suggests a discrete re-evaluation on the role of metformin/AMPK activation on tumor cell growth, proliferation, and on clinical application in cancer therapy.

[Growth and developmental rhythm of Scrophularia ningpoensisin southwest middle mountain area of China].

Plant samples were collected and investigated periodically. According to the growth of different parts and the characteristics of dry substance accumulation of Scrophularia ningpoensis, the development of S. ningpoensis could be divided into four stages: seeding stage, stem and leaf growth stage, expanding period of root tubers, and dry substance accumulation stage of root tuber. Leaf numbers of S. ningpoensis grew gradually from one at first to 370 at the final stage, main stem leaf were 50 pieces. Leaf size increasesed with the fastest growth at the stem and leaf growth stage, average daily increase amount was 225 cm2. By the middle of August, leaf size reached to 16,270 cm2. Leaf area indexrose sharply in the seeding stage, and remained above 8 among stem and leaf growth stage and expanding period of root tubers, and rapidly reduced to zero in the stage of dry substance accumulation of root tuber. Leaf area ratio has a tendency of obvious dropping. The net assimilation rate had a small change ranges, two small peak were seeding stage and dry substance accumulation of root tuber. The value of specific leaf area was higher in seeding stage, and in the earlier stage of dry substance accumulation of root tuber. Relative growth rate changed with large ranges, higher in seeding stage, rapid decrease in stem and leaf growth stage, rose in expanding period of root tubers, and declined again in the stage of dry substance accumulation of root tuber. Crop growth rate was higher in the first and last stages, and smaller in interim stage. The growth parameters of S. ningpoensis such as relative growth rate, net assimilation rate, leaf area index, leaf area ratio, specific leaf area, crop growth rate changed along with the growth. The rule of dry matter accumulation was as follows: the dry matter increased slowly during the seeding stage and speeded up in the middle and late stages, and in dry substance accumulation of root tuber increased slower, the growth of dry matter all appeared an "S" curve, and accorded with logistic equation. Cultivation technologies of S. ningpoensis and the relevant management methods could be established according to the growth of different parts of S. ningpoensis and the characteristics of dry substance accumulation in different stage.

Isolation and characterization of Aschersonia placenta from citrus orchards and its pathogenicity towards Dialeurodes citri (Ashmead).

For selecting potential biological agents to control Dialeurodes citri, a major insect pest in China's citrus orchards, 75 native Aschersonia isolates were selected from infected whiteflies in citrus orchards and were evaluated for virulence to third instar nymphs of D. citri at a concentration of 1 × 10(6)conidia/ml. The mortality rates of D. citri varied from 0% to 72.70%. Among the 75 Aschersonia isolates, three (HB10, HB12 and ZJ9) were the most virulent and pathogenic to D. citri and caused more than 69% mortality. The 50% lethal concentration (LC(50)) values of them were 1.86 × 10(6), 3.40 × 10(6) and 6.81 × 10(6)conidia/ml, respectively. The three isolates also produced conidia abundantly on potato dextrose agar medium (3.95 × 10(6), 6.19 × 10(6) and 7.34 × 10(6)conidia/cm(2), respectively). They were identified to be Aschersonia placenta based on their morphological analysis. Phylogenetic analysis showed that these three highly virulent Aschersonia isolates belong to a strongly supported clade that includes two other A. placenta isolates obtained from Vietnam and Thailand. In summary, these results indicate the possibility of developing the three most virulent A. placenta isolates as microbiological control agents against citrus whitefly.

Multi-Omics Analysis Reveals the Resistance Mechanism and the Pathogens Causing Root Rot of Coptis chinensis.

Coptis chinensis is a traditional Chinese medicinal herb used for more than 2,000 years. Root rot in C. chinensis can cause brown discoloration (necrosis) in the fibrous roots and rhizomes, leading to plants wilting and dying. However, little information exists about the resistance mechanism and the potential pathogens of the root rot of C. chinensis plants. As a result, in order to investigate the relationship between the underlying molecular processes and the pathogenesis of root rot, transcriptome and microbiome analyses were performed on healthy and diseased C. chinensis rhizomes. This study found that root rot can lead to the significant reduction of medicinal components of Coptis, including thaliotrine, columbamine, epiberberin, coptisine, palmatine chloride, and berberine, affecting its efficacy quality. In the present study, Diaporthe eres, Fusarium avenaceum, and Fusarium solani were identified as the main pathogens causing root rot in C. chinensis. At the same time, the genes in phenylpropanoid biosynthesis, plant hormone signal transduction, plant-pathogen interaction, and alkaloid synthesis pathways were involved in the regulation of root rot resistance and medicinal component synthesis. In addition, harmful pathogens (D. eres, F. avenaceum and F. solani) also induce the expression of related genes in C. chinensis root tissues to reduce active medicinal ingredients. These results provide insights into the root rot tolerance study and pave the way for process disease resistance breeding and quality production of C. chinensis. IMPORTANCE Root rot disease significantly reduces the medicinal quality of Coptis chinensis. In the present study, results found that the C. chinensis fibrous and taproot have different tactics in response to rot pathogen infection. Diaporthe eres, Fusarium avenaceum, and Fusarium solani were isolated and identified to cause different degrees of C. chinensis root rot. These results are helpful for researchers to further explore the mechanism of resistance to rhizoma Coptis root rot.