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.

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.

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.

Metabolomic analysis reveals dynamic changes in secondary metabolites of Sophora japonica L. during flower maturation.

Sophora japonica L. is widely consumed in China because of its medicinal and nutritional value. Its quality is greatly affected by the accumulation of metabolites, which varies with the stage of flower development. However, changes in the characteristics of the secondary metabolites during flower maturity remain unclear. Ultra-high-performance liquid chromatography coupled with electrospray ionization-triple quadrupole-linear ion trap mass spectrometry (UPLC-ESI-QTRAP-MS/MS) revealed dynamic changes in the secondary metabolites of S. japonica during the five flower-maturity stages. We monitored 331 metabolites and screened 164. The differential metabolites showed seven trends during flower maturation, with flavonoids and phenolic acids having the most varied expressions. Flower buds (S2-S3) are rich in flavonoids and are thus suitable for use in high-quality medicine or industrial extraction. Our study provides an empirical basis for the informed harvesting of S. japonica based on its mode of utilization.

Corrigendum: PHLDA1 Suppresses TLR4-Triggered Proinflammatory Cytokine Production by Interaction With Tollip.

[This corrects the article DOI: 10.3389/fimmu.2022.731500.].

PHLDA1 Suppresses TLR4-Triggered Proinflammatory Cytokine Production by Interaction With Tollip.

Pleckstrin homology-like domain, family A, member 1 (PHLDA1) has been reported to be expressed in many mammalian tissues and cells. However, the functions and exact mechanisms of PHLDA1 remain unclear. In this study, we found that PHLDA1 expression was significantly altered in macrophages after exposure to lipopolysaccharide (LPS) in vitro, suggesting that PHLDA1 may be involved in the regulation of TLR4 signaling pathway activated by LPS. PHLDA1 attenuated the production of LPS-stimulated proinflammatory cytokines (TNF-α, IL-6, and IL-1ß). Further research showed that the phosphorylation levels of some important signal molecules in TLR4/MyD88-mediated MAPK and NF-κB signaling pathways were reduced by PHLDA1, which in turn impaired the transcription factors NF-κB and AP1 nuclear translocation and their responsive element activities. Furthermore, we found that PHLDA1 repressed LPS-induced proinflammatory cytokine production via binding to Tollip which restrained TLR4 signaling pathway. A mouse model of endotoxemia was established to confirm the above similar results. In brief, our findings demonstrate that PHLDA1 is a negative regulator of LPS-induced proinflammatory cytokine production by Tollip, suggesting that PHLDA1 plays an anti-inflammatory role through inhibiting the TLR4/MyD88 signaling pathway with the help of Tollip. PHLDA1 may be a novel therapeutic target in treating endotoxemia.

Purine-Induced IFN-γ Promotes Uric Acid Production by Upregulating Xanthine Oxidoreductase Expression.

Objective: Limiting purine intake, inhibiting xanthine oxidoreductase (XOR) and inhibiting urate reabsorption in proximal tubule by uricosuric drugs, to reduce serum uric acid (UA) levels, are recognized treatments for gout. However, the mechanism of increased how XOR expression and activity in hyperuricemia and gout remains unclear. This study aims to explore whether exogenous purines are responsible for increased XOR expression and activity. Methods: HepG2 and Bel-7402 human hepatoma cells were stimulated with exogenous purine, or were exposed to conditioned growth medium of purine-stimulated Jurkat cells, followed by measurement of XOR expression and UA production to determine the effect of lymphocyte-secreted cytokines on XOR expression in hepatocytes. The expression of STAT1, IRF1 and CBP and their binding on the XDH promoter were detected by western blotting and ChIP-qPCR. The level of DNA methylation was determined by bisulfite sequencing PCR. Blood samples from 117 hyperuricemia patients and 119 healthy individuals were collected to analyze the correlation between purine, UA and IFN-γ concentrations. Results: Excess of purine was metabolized to UA in hepatocyte metabolism by XOR that was induced by IFN-γ secreted in the conditioned growth medium of Jurkat cells in response to exogenous purine, but it did not directly induce XOR expression. IFN-γ upregulated XOR expression due to the enhanced binding of STAT1 to IRF1 to further recruit CBP to the XDH promoter. Clinical data showed positive correlation of serum IFN-γ with both purine and UA, and associated risk of hyperuricemia. Conclusion: Purine not only acts as a metabolic substrate of XOR for UA production, but it induces inflammation through IFN-γ secretion that stimulates UA production through elevation of XOR expression.

A 15-Gene Signature and Prognostic Nomogram for Predicting Overall Survival in Non-Distant Metastatic Oral Tongue Squamous Cell Carcinoma.

BACKGROUND: Oral tongue squamous cell carcinoma (OTSCC) is a devastating tumor with poor prognosis. There is an urgent need for reliable biomarkers to help predict prognosis and guide treatment for OTSCC. In the current study, we aimed to develop a robust multi-gene signature and prognostic nomogram to predict the prognosis of patients with non-distant metastatic OTSCC. METHODS: OTSCC-related differentially-expressed genes were screened from The Cancer Genome Atlas (TCGA) database. Univariate Cox regression based on 1,000 bootstrap replicates, LASSO regression and stepwise multivariate Cox regression were utilized to develop a novel multi-mRNA signature for predicting overall survival in OTSCC. The concordance index, area under receiver operating characteristic (ROC AUC) and calibration curve were employed to assess the prediction capacity of the novel multi-gene model. In addition, a prognostic nomogram was constructed to facilitate the clinical use of the fitted model. The Kaplan-Meier with log-rank test was employed to assess differences in overall survival. RESULTS: We successfully established a novel 15-mRNA prognostic model for predicting overall survival of non-distant metastatic OTSCC, involving ADTRP, ITGA3, RFC4, CCDC96, CYP2J2, NELL2, SPHK1, SPAG16, HBEGF, S100A9, EGFL6, ADGRG6, PDE4D, ABCA4, and CTTN. The prediction ability of this 15-gene signature was independent of other clinicopathological factors, with an HR of 11.5 (95% CI: 4.70-28.3). Moreover, internal validation by bootstrap analysis yielded a C-index of 0.849, with a 3-year AUC of 0.907 and 5-year AUC of 0.944, which implied excellent prediction accuracy of the fitted model. In addition, external validation by using the GEO dataset (GSE41116) yielded a C-index of 0.804, with a 3-year AUC of 0.868 and 5-year AUC of 0.855, which also indicated good prediction ability of the 15-gene model. Finally, a prognostic nomogram integrating risk group, grade, T stage and N stage was established. CONCLUSION: Our results demonstrate our 15-gene signature was independently associated with overall survival in non-distant metastatic OTSCC. Moreover, the prognostic nomogram integrating the 15-gene signature and clinicopathological factors has potential to be developed as a prognostic tool.

[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 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.

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.

Berberine inhibits cancer cells growth by suppressing fatty acid synthesis and biogenesis of extracellular vesicles.

AIMS: Berberine is an isoquinoline alkaloid extracted from the root, rhizome and stem bark of Coptidis Rhizoma. Previous studies have revealed the anti-tumor potential of berberine against various types of cancer cells. However, the underlying mechanisms are not yet fully understood. In this study, we focused on the effects of berberine on fatty acid synthesis and extracellular vesicles formation in cancer cells, and revealed the internal mechanism of berberine inhibition on cancer cell proliferation. MATERIALS AND METHODS: Anti-proliferative activity of berberine was determined by cell counting and microscope observation and cell cycle analysis. Activities of AMPK and ACC, expression of extracellular vesicles markers were detected by western blotting. 13C labeling metabolic flux analysis was used for determination of de novo synthesis of fatty acids. The excreted extracellular vesicles in culture mediums were separated by both polyethylene glycol enrichment of extracellular vesicles and differential centrifugation separation. KEY FINDINGS: Among our early experiments, 5-10 µmol/L berberine exhibited the substantial anti-proliferative effect against human colon cancer cell line HCT116, cervical cancer cell line HeLa and other cancer cells. It was also revealed that, through activating AMPK, berberine inhibited ACC activity then suppressed intracellular fatty acid synthesis, finally decreased the biogenesis of extracellular vesicles. Moreover, supplement with citrate acid, palmitic acid, as well as exogenous extracellular vesicles, could rescue the inhibitory effect of berberine on cell proliferation, suggesting that inhibited ACC activity, suppressed fatty acid synthesis and decreased extracellular vesicles production were important mechanisms account for berberine inhibiting cancer cell proliferation. SIGNIFICANCE: Our study indicates that berberine suppresses cancer cell proliferation through inhibiting the synthesis of fatty acids and decreasing biogenesis and secretion of extracellular vesicles, suggests that berberine is a promising candidate for the development of new therapies for cancer.

The Efficacy of Psychological Capital Intervention (PCI) for Depression From the Perspective of Positive Psychology: A Pilot Study.

Extensive psychological interventions primarily target the negative symptoms of depression and the deficits in positive resources have been systematically neglected. So far, little attention has been devoted to psychological capital (PsyCap) intervention from the perspective of developing positive resources. The aim of the present pilot study was to evaluate the efficacy of psychological capital intervention (PCI) for depression in a randomized controlled trial. A total of 56 patients were randomized to either care as usual (CAU) for normal medication or psychological capital intervention (PCI) group, where the normal medication was supplemented with the PCI. Participants were assessed at pre- and post-treatment, as well as 6-month follow-up, on measures of depressive symptoms and PsyCap. The PCI group displayed significantly larger improvements in PsyCap and larger reductions in depression symptoms from pre- to post treatment compared to control group. Improvements were sustained over the 6-month follow-up period. Targeting the positive resources intervention in the PCI may be effective against the treatment of depression.

Androgen Receptor-Activated Enhancers Simultaneously Regulate Oncogene TMPRSS2 and lncRNA PRCAT38 in Prostate Cancer.

Prostate cancer is a common carcinoma in males, the development of which involves the androgen receptor (AR) as a key regulator. AR transactivation induces the high expression of androgen-regulated genes, including transmembrane protease serine 2 (TMPRSS2) and long noncoding RNA prostate cancer-associated transcript 38 (PRCAT38). PRCAT38 and TMPRSS2 are both located on chromosome 21, separated by a series of enhancers. PRCAT38 is a prostate-specific long noncoding RNA that is highly expressed in cancer tissue as compared to normal tissue. Here, we show chromatin looping by enhancers E1 and E2 with the promoters for PRCAT38 and TMPRSS2, indicating the co-regulation of PRCAT38 and TMPRSS2 by the same enhancers. The knockout of enhancer E1 or E2 simultaneously impaired the transcription of PRCAT38 and TMPRSS2 and inhibited cell growth and migration. Moreover, the loop formation and enhancer activity were mediated by AR/FOXA1 binding and the activity of acetyltransferase p300. Our findings demonstrate the utilization of shared enhancers in the joint regulation of two oncogenes in prostate cancer cells.

MiR-506 Suppresses Colorectal Cancer Development by Inhibiting Orphan Nuclear Receptor NR4A1 Expression.

NR4A1 acts as an oncogene and plays an important role in colorectal cancer development and progression, but little is known about the regulatory mechanism of NR4A1 expression. MicroRNA (miRNA) is involved in the progression of various tumors, affecting proliferation, apoptosis or migration. We aimed to elucidate whether miRNA regulates NR4A1 expression and determine its underlying significance in colorectal cancer. By using the TargetScan database, we identified a miR-506 binding site in the NR4A1 3'-UTR. Examination of colorectal cancer tissues and cells revealed that NR4A1 mRNA and protein were up-regulated, while miR-506 expression was down-regulated. Spearman correlation analysis revealed that expression of NR4A1 mRNA was negatively correlated with miR-506 levels in colorectal cancer tissue. Further studies indicated that miR-506 decreased NR4A1 expression through directly targeting the NR4A1 mRNA 3'-UTR. Functional experiments showed that rescue of NR4A1 expression in cells reversed the inhibitory effects of miR-506 on proliferation, migration and invasion of colorectal cancer cells. In conclusion, miR-506 acts as a tumor suppressor and inhibits proliferation, migration and invasion in colorectal cancer cells partly through decreasing NR4A1 expression.

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.

Aberrant activation of CYR61 enhancers in colorectal cancer development.

BACKGROUND: High expression of secreted matricellular protein cysteine-rich 61 (CYR61) correlates with poor prognosis in colorectal cancer (CRC). Aberrant enhancer activation has been shown to correlate with expression of key genes involved in cancer progression. However, such mechanisms in CYR61 transcription regulation remain unexplored. METHODS: Expression of CYR61 was determined by immunohistochemistry (IHC), quantitative real-time PCR (qRT-PCR) and western blotting (WB) in CRC patients paraffin specimens and colon cell lines. ChIP-seq data of enhancer-characteristic histone modifications, in CRC tissues from the Gene Expression Omnibus (GEO) database, were reanalyzed to search for putative enhancers of CYR61. Dual-luciferase reporter assay was used to detected enhancer activity. Physical interactions between putative enhancers and CYR61 promoter were detected by chromosome conformation capture (3C) assay. Histone modification and transcription factors (TFs) enrichment were detected by ChIP-qPCR. Additionally, biological function of enhancers was investigated by transwell migration assays. RESULTS: CRC tissues and cell lines expressed higher level of CYR61 than normal colon mucosa. Three putative enhancers located downstream of CYR61 were found in CRC tissues by ChIP-seq data reanalysis. Consistent with the ChIP-seq analysis results in the GEO database, the normal colon mucosal epithelial cell line NCM460 possessed no active CYR61 enhancers, whereas colon cancer cells exhibited different patterns of active CYR61 enhancers. HCT116 cells had an active Enhancer3, whereas RKO cells had both Enhancer1 and Enhancer3 active. Pioneer factor FOXA1 promoted CYR61 expression by recruiting CBP histone acetyltransferase binding and increasing promoter-enhancer looping frequencies and enhancer activity. CBP knockdown attenuated H3K27ac enrichment, promoter-enhancer looping frequencies, and enhancer activity. Small molecule compound 12-O-tetradecanoyl phorbol-13-acetate (TPA) treatment, which stimulated CYR61 expression, and verteporfin (VP) treatment, which inhibited CYR61 expression, confirmed that the enhancers regulated CYR61 expression. Knockdown and ectopic expression of CYR61 rescued cell migration changes induced by over-expressing and knockdown of FOXA1, respectively. CONCLUSIONS: CYR61 enhancer activation, mediated by FOXA1 and CBP, occurs during CRC progression to up-regulate CYR61 expression and promote cell migration in CRC, suggesting inhibition of recruitment of FOXA1 and/or CBP to CYR61 enhancers may have therapeutic implications.