First Report of Lasiodiplodia theobromae Causing Stem Brown Rot of Loquat in China.

Loquat (Rhaphiolepis biabas, heterotypic synonym: Eriobotrya japonica) is an important edible and medicinal plant that is widely cultivated on 133 thousand hectares (recorded in 2022) in China. A stem brown rot was observed on young and old trees in Mengzi city (23°23' N; 103°23' E), Yunnan Province, southwest China, during October 2014 and September 2021. Incidence ranged from 20% of trees in surrounding plantations to 50% incidence of a 160 tree orchard that was the focal point of the disease survey. Circular brown lesions occurred initially on the stems and gradually covered all the epidermis of the stem, leading to irregular dents within the bark that developed a dark brown powdery appearance (Fig.1A). Larger lesions affected vascular tissues, causing diseased trees to wither and die. Diseased tissues were surface-disinfected in a 5% sodium hypochlorite solution for 3 min, rinsed three times with sterile distilled water, placed on potato dextrose agar (PDA), and incubated in the dark at 28°C. Twenty samples were collected for tissue isolation, and 11 isolates were single-spored on water agar. In culture, the colonies on PDA were white to dark-gray, velvet, with dense hyphae, diameter 7.64 cm after 5 days. After 18 days, spherical or subglobose pycnidia were developed and semi-buried in medium, their walls were thicker and dark-brown, which were black particles surrounded by gray-black hyphae. Conidiogenous cells were hyaline, cylindrical, holoblastic, slightly swollen at the base, with rounded apex. Conidia were initially hyaline and aseptate with elliptic or ovate shape, becoming dark brown with a single septate and developing longitudinal striations along thick walls at maturity. Conidia dimensions varied from 8.0 to 12.2 × 3.8 to 6.1µm (n=50) (Fig.1D). The morphological characteristics of eleven isolates were consistent with the description of Lasiodiplodia theobromae (Alves et al. 2008). Further confirmation was also determined by sequencing the internal transcribed spacer (ITS), ß-tubulin genes, partial translation elongation factor-1α (TEF-1α) (White et al. 1990, Carbone et al. 1999, Glass et al.1995). The isolate LSB-1 was selected for DNA sequence analysis. Based on BLASTn analysis, ITS sequences (OM617921) had 98.3% similarity with L. theobromae CBS164.96 (accession AY640255), CBS124.13(accession DQ458890), CAA006 (accession DQ458891) and CBS111530 (accession EF622074), ß-tubulin sequences (OM643838) showed 99.1% similarity with L. theobromae accessions EU673110. The TEF-1α (OM643839) had 99.0% identity with L. theobromae accession EF633054. The isolate LSB-1 clustered on the same clade with other L. theobromae. Pathogenicity testing of isolate LSB-1, LSB-2, LSB-3 was conducted by inoculating the stems of l-year-old seedlings growing in pots. The epidermis at the inoculation site, 15-20 cm below the crown, was wiped with 75% alcohol cotton ball, washed three times with sterile water, and then punctured (5mm diameter) with sterile inoculation needle. A 5mm block of each isolate cultured on PDA for seven days was attached to the inoculation site. Controls were inoculated with sterile PDA blocks. The inoculation area was covered with polyethylene cling film. All inoculated seedlings were kept in controlled greenhouse at 27°C with 80% relative humidity under natural daylight conditions, and watered weekly. Each treatment was repeated three times. Eight days after inoculation, all diseased plants showed dark brown discoloration at the point of inoculation (Fig. 1G) with the bark at the inoculation site gradually raising as the disease progressed. Thirty days after inoculation, all inoculated seedlings produced typical symptoms, whereas the control seedlings remained healthy. Fungal isolates were only recovered from symptomatic stems and were morphologically identical to L. theobromae, completing Koch's postulates. According to the relevant literature, Lasiodiplodia theobromae has a broad host range, causing numerous diseases, including canker and dieback of branch (Aguilera-Cogley et al., 2021), panicle blight (Mahadevakumar et al, 2022), root rot (Abd-El Ghani and Fatouh, 2005), fruit rot(Freire et al., 2011) in diverse geographical regions. To our knowledge, this is the first report of L. theobromae causing stem brown rot of loquat in China and provides a foundation for further study of the epidemiology and integrated management of this disease.

The preventive effect and mechanisms of adsorbent supplementation in low concentration aflatoxin B1 contaminated diet on subclinical symptom and histological lesions of broilers.

This study aimed to investigate the subclinical symptom and histological lesions of 21-day-old and 42-day-old broilers exposure to low concentration aflatoxin B1 (AFB1), and the preventive effect with adsorbent (Toxo-MX) supplementation. A total of 576 one-day-old Arbor Acres broilers were randomly allotted into 6 treatments 8 replicates and 12 birds per cage, fed with 0 ppb, 60 ppb and 120 ppb AFB1 contamination diet with or without Toxo-MX supplementation. Results showed both 60 ppb and 120 ppb AFB1 contamination significantly reduced growth performance in 21-day-old broilers (P < 0.05), but not in 42-day-old broilers (P > 0.05), however, AFB1 contamination in diet caused a higher feed to gain ratio (P < 0.05). Broilers of 21-day-old exposure to 60 ppb and 120 ppb AFB1 increased mRNA expression of hepatic inflammatory cytokines, and superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity (P < 0.05), 42-day-old broilers showed a same change in 120 ppb but not in 60 ppb of AFB1 contamination (P < 0.05). mRNA expressions of clauding-1, Zonula occludens-1 (ZO-1), and occludin decreased, but Bax, Bcl-2, and caspase-3 increased in 21-day-old broilers exposure to 60 ppb and 120 ppb AFB1 (P < 0.05), broilers of 42-day-old resisted on intestinal aflatoxicosis impairment against 60 ppb AFB1 contamination (P < 0.05), but not in 120 ppb (P < 0.05). Toxo-MX supplementation significantly reversed the detrimental effects on growth performance in both age broilers and reduced the accelerated feed to gain ratio caused by AFB1 (P < 0.05). Intestinal mRNA expression of tight junction and apoptotic genes in both age broilers were recovered by Toxo-MX supplementation (P < 0.05). However, Toxo-MX did not restore the accelerated expression of hepatic inflammation cytokines and SOD, GSH-Px in 120ppb AFB1 group (P < 0.05). The data demonstrated that diet supplementation with Toxo-MX reversed the detrimental effect on growth performance and intestine in broilers exposure to 60 ppb and 120 ppb AFB1. However, did not completely recovered hepatic inflammation induced by AFB1.

[Review on characteristics of acupuncture-activated network regulatory effect based on brain connectomics].

The network analysis method based on brain connectomics is an important entry point to explore the working mechanism of brain and is also the current trend of researches on acupuncture stimulation-induced changes of neuroimages. We, in the present review, summarized the common network analysis methods for exploring the underlying mechanisms of brain network-mediated regulatory effects of acupuncture interventions. Moreover, combining the current research development and our team's previous research findings, we extracted the characteristics of targeting, conditionity and dynamic of regulatory effects of acupuncture-activated brain network, and put forward our prospects about the future research from the aspects of new scanning modes (for instance, multimodal data acquisition of magnetic resonance, electroencephalogram, near infrared spectrum, etc.), network analysis (such as Granger causality analysis, complex network measures, whole-brain connectivity dynamics tracking, high-order resting-state functional connectivity analysis, etc.) and experimental research paradigms (for example, introduce of transcranial magnetic stimulation induced transient changes of brain functional activity, machine learning approach, etc.).

Comparisons of different vitamin D supplementation for prevention of osteoporotic fractures: a Bayesian network meta-analysis and meta-regression of randomised controlled trials.

Previous randomised controlled trials have shown the controversial effectiveness of oral vitamin D supplementation in preventing osteoporotic fractures. PubMed, EMBASE and Cochrane Library electronic databases were searched. Pairwise meta-analysis, Bayesian network meta-analysis and meta-regression were applied. A total of 33 studies containing 83,083 participants were included. Oral vitamin D supplementation showed no statistically significant on reducing the risk of total fractures (RR = 0.96, 95%CI = 0.87-1.05 p = 0.389). Vitamin D3 (700-800IU/d) plus calcium showed statistical significance in reducing the incidence of total, hip and non-vertebral fractures in the pairwise meta-analysis. Significant reductions were specifically identified in female in total and hip fractures. However, we did not observe any above significant results using Bayesian network meta-analyses. Strikingly, a meta-regression analysis identified an inverse association between the efficacy of fracture prevention and increased body mass index. Thus, we recommended that the vitamin D dose should be adjusted according to BMI based on further confirmation.

Ameliorative effects and molecular mechanisms of vine tea on western diet-induced NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is a disease that is prevalent worldwide, and its prevention by dietary administration has recently been considered as an important strategy. In this study, we administered mice with vine tea polyphenol (VTP) extracted from Ampelopsis grossedentata, a Chinese herb, to investigate the preventive effect on western diet (WD)-induced NAFLD. Male C57BL/6N mice were fed either a normal diet (ND) or WD with or without VTP for 12 weeks. The results revealed that VTP supplementation decreased the serum levels of cholesterol and triglycerides, and reduced the accumulation of hepatic lipid droplets caused by WD. Molecular data revealed that VTP enhanced fatty acid oxidation by reactivating the WD-suppressed phosphorylation of AMP-activated protein kinaseα (AMPKα) and the expressions of peroxisome proliferator-activated receptor alpha (PPARα), carnitine palmitoyl transferase IA (CPT1A) and cytochrome P450, family 4, subfamily a1 (CYP4A1). VTP inhibited hepatic lipogenesis by reducing the WD-enhanced level of mature sterol regulatory element-binding protein 1 (SREBP1) and fatty acid synthase (FAS). Moreover, VTP activated nuclear factor (erythroid-derived 2)-like 2 (Nrf2)-mediated expressions of hemeoxygenase-1 (HO-1) and quinone oxidoreductase (NQO1), and reduced hepatic TBARS levels to prevent hepatic oxidative stress. On the other hand, VTP also increased intestinal zonula occludens-1 (ZO-1) expression and the relative abundance of gut Akkermansia, and reduced the ratio of Firmicutes/Bacteroidetes. Thus, VTP might prevent WD-induced NAFLD by balancing fatty acid oxidation and lipogenesis, hepatic oxidative stress, and gut microbiome, at least. These results suggest that vine tea, containing a high content of the bioactive compound dihydromyricetin, is a potential food resource for preventing NAFLD.

Modulation of Allicin-Free Garlic on Gut Microbiome.

The allicin diallyldisulfid-S-oxide, a major garlic organosulfur compound (OSC) in crushed garlic (Allium sativum L.), possesses antibacterial effects, and influences gut bacteria. In this study, we made allicin-free garlic (AFG) extract and investigated its effects on gut microbiome. C57BL/6N male mice were randomly divided into 6 groups and fed normal diet (ND) and high-fat diet (HFD) supplemented with or without AFG in concentrations of 1% and 5% for 11 weeks. The genomic DNAs of feces were used to identify the gut microbiome by sequencing 16S rRNA genes. The results revealed that the ratio of p-Firmicutes to p-Bacteroidetes increased by aging and HFD was reduced by AFG. In particular, the f-Lachnospiraceae, g-Akkermansia, and g-Lactobacillus decreased by aging and HFD was enhanced by AFG. The g-Dorea increased by aging and HFD decreased by AFG. In addition, the ratio of glutamic-pyruvic transaminase to glutamic-oxaloacetic transaminase (GPT/GOT) in serum was significantly increased in the HFD group and decreased by AFG. In summary, our data demonstrated that dietary intervention with AFG is a potential way to balance the gut microbiome disturbed by a high-fat diet.

Aqueous extract of Fritillariae cirrhosae induces cellular apoptosis through activation of STATs-mediated immunomodulation.

ETHNOPHARMACOLOGICAL RELEVANCE: Fritillariae cirrhosae (FC), referred to'Chuan beimu'in China. As an important edible and medicinal plant, the bulbs of F.cirrhosae is used traditionally in the treatment of pulmonary diseases associated with lung heat, inflammation and tumors. In the study, we investigated the effect of aqueous extract of FC (FC-AE) and elucidated its mechanism in non-small cell lung cancer A549 cells and a xenograft model of nude mice. MATERIALS AND METHODS: CCK-8 and plate colony formation assay were used to evaluate the effect of FC-AE in A549 cells in vitro, and the gene expression profile of FC-AE on A549 cells was assessed by RNA sequencing system. Then, the effects of FC-AE on cell cycle and apoptosis of A549 cells were analyzed by flow cytometry. In combination with RNA-seq data, RT-PCR and western blot were used to evaluate the expression of proteins related to apoptosis and immune regulation. A xenograft model of nude mice was used to assess the effect of FC-AE in vivo. RESULTS: CCK-8 and plate cloning assays showed that FC-AE inhibited the proliferation and colony formation of A549 cells. A549 cells treated with FC-AE can triggered apoptosis. GO and KEGG pathway enrichment analysis of RNA-seq data showed that most of the differentially expressed genes (DEGs) were related to immune response, apoptosis and cell cycle process. Several immune and apoptotic DEGs were identified by qRT-PCR which were consistented with RNA-seq data. In nude mice, FC-AE reduced the tumor size and promoted the secretion of cytokines IL12 and IFNγ. FC-AE up-regulated the two members (STAT1 and STAT4) of STATs and their target genes (IFNγ and IL-12, respectively) protein expressions, and actively regulates Bcl-2/Bax family proteins which resulted in cellular apoptosis in A549 cells. CONCLUSION: Our finding suggests that FC-AE mediates apoptosis through a STAT1 and STAT4-mediated co-regulatory network, which may be the key novel mechanism for its antitumor activity. The F. cirrhosa may be a promising antitumor drug for modulating immune responses to improve cancer therapy.

Preventive Effects and Mechanisms of Garlic on Dyslipidemia and Gut Microbiome Dysbiosis.

Garlic (Allium sativum L.) contains prebiotic components, fructans, antibacterial compounds, and organosulfur compounds. The complex ingredients of garlic seem to impart a paradoxical result on the gut microbiome. In this study, we used a mouse model to clarify the effects of whole garlic on the gut microbiome. C57BL/6N male mice were fed with or without whole garlic in normal diet (ND) or in high-fat diet (HFD) for 12 weeks. Supplementation with whole garlic attenuated HFD-enhanced ratio of serum GPT/GOT (glutamic-pyruvic transaminase/glutamic-oxaloacetic transaminase), levels of T-Cho (total cholesterol) and LDLs (low-density lipoproteins), and index of homeostatic model assessment for insulin resistance (HOMA-IR), but had no significant effect in the levels of serum HDL-c (high density lipoprotein cholesterol), TG (total triacylglycerol), and glucose. Moreover, garlic supplementation meliorated the HFD-reduced ratio of villus height/crypt depth, cecum weight, and the concentration of cecal organic acids. Finally, gut microbiota characterization by high throughput 16S rRNA gene sequencing revealed that whole garlic supplementation increased the α-diversity of the gut microbiome, especially increasing the relative abundance of f_Lachnospiraceae and reducing the relative abundance of g_Prevotella. Taken together, our data demonstrated that whole garlic supplementation could meliorate the HFD-induced dyslipidemia and disturbance of gut microbiome.

Magnolol additive as a replacer of antibiotic enhances the growth performance of Linwu ducks.

Magnolol rich in Magnolia officinalis is a bioactive polyphenolic compound. The aim of this study was to examine the effects of magnolol additive (MA) on growth performance, expression levels of antioxidant-related genes, and intestinal mucosal morphology of Linwu ducks aged from 49 to 70 days, comparing with that of an antibiotic additive (colistin sulfate [CS]). A total of 275, 49-day-old ducks were assigned to 5 groups with 5 cages of 11 ducks each and fed diets supplemented with 0, 100, 200 and 300 mg of MA/kg and 300 mg of CS/kg for 3 weeks, respectively. The results showed that the average daily body weight gain (ADG) was increased significantly in MA-fed groups (200 and 300 mg/kg), compared with the basal diet (BD) group (P < 0.05). The mRNA levels of superoxide dismutase-1 (SOD1), manganese superoxide dismutase-2 (MnSOD2) and catalase (CAT) were also increased significantly in MA groups (P < 0.05). In addition, hematoxylin and eosin staining revealed that Linwu ducks fed the diets with MA had more intact intestinal mucosa than those fed the BD and CS diets. In addition, ileal villus height, ileal villus height/crypt depth ratio (V/C) and duodenal V/C were also improved significantly (P < 0.05). Taken together, these data demonstrated that MA is an effective feed additive to enhance the growth performance of the Linwu ducks by improving the antioxidant and intestinal mucosal status, suggesting that MA will be a potential additive to replace antibiotic (CS).

Yin Yang-1 suppresses invasion and metastasis of pancreatic ductal adenocarcinoma by downregulating MMP10 in a MUC4/ErbB2/p38/MEF2C-dependent mechanism.

BACKGROUND: Increasing evidence indicates an important role of transcription factor Yin Yang-1 (YY1) in human tumorigenesis. However, its function in cancer remains controversial and the relevance of YY1 to pancreatic ductal adenocarcinoma (PDAC) remains to be clarified. METHODS: In this study, we detected YY1 expression in clinical PDAC tissue samples and cell lines using quantitative RT-PCR, immunohistochemistry and western blotting. We also detected MUC4 and MMP10 mRNA levels in 108 PDAC samples using qRT-PCR and analyzed the correlations between YY1 and MUC4 or MMP10 expression. The role of YY1 in the proliferation, invasion and metastatic abilities of PDAC cells in vitro was studied by CCK-8 assay, cell migration and invasion assays. In vivo pancreatic tumor growth and metastasis was studied by a xenogenous subcutaneously implant model and a tail vein metastasis model. The potential mechanisms underlying YY1 mediated tumor progression in PDAC were explored by digital gene expression (DGE) sequencing, signal transduction pathways blockage experiments and luciferase assays. Statistical analysis was performed using the SPSS 15.0 software. RESULTS: We found that the expression of YY1 in PDACs was higher compared with their adjacent non-tumorous tissues and normal pancreas tissues. However, PDAC patients with high level overexpression of YY1 had better outcome than those with low level overexpression. YY1 expression levels were statistically negatively correlated with MMP10 expression levels, but not correlated with MUC4 expression levels. YY1 overexpression suppressed, whereas YY1 knockdown enhanced, the proliferation, invasion and metastatic properties of BXPC-3 cells, both in vitro and in vivo. YY1 suppresses invasion and metastasis of pancreatic cancer cells by downregulating MMP10 in a MUC4/ErbB2/p38/MEF2C-dependent mechanism. CONCLUSIONS: The present study suggested that YY1 plays a negative role, i.e. is a tumor suppressor, in PDAC, and may become a valuable diagnostic and prognostic marker of PDAC.

Transcriptional regulation of human MUC4 gene: identification of a novel inhibitory element and its nuclear binding protein.

The human mucin 4 (MUC4) is aberrantly expressed in pancreatic adenocarcinoma and tumor cell lines, while remaining undetectable in normal pancreas, indicating its important role in pancreatic cancer development. Although its transcriptional regulation has been investigated in considerable detail, some important elements remain unknown. The aim of the present study was to demonstrate the existence of a novel inhibitory element in the MUC4 promoter and characterize some of its binding proteins. By luciferase reporter assay, we located the inhibitory element between nucleotides -2530 and -2521 in the MUC4 promoter using a series of deletion and mutant reporter constructs. Electrophoretic mobility shift assay (EMSA) with Bxpc-3 cell nuclear extracts revealed that one protein or protein complex bind to this element. The proteins binding to this element were purified and identified as Yin Yang 1 (YY1) by mass spectrometry. Supershift assay and chromatin immunoprecipitation (ChIP) assay confirmed that YY1 binds to this element in vitro and in vivo. Moreover, transient YY1 overexpression significantly inhibited MUC4 promoter activity and endogenous MUC4 protein expression. In conclusion, we reported here a novel inhibitory element in the human MUC4 promoter. This provides additional data on MUC4 gene regulation and indicates that YY1 may be a potential target for abnormal MUC4 expression.

[Antibacterial activity and mechanism of baicalein].

Baicalein (BAI) is an effective bactericide. The antibacterial activity and mechanism experiments were carried out by determining conductivity and content of macromolecules of membrane penetrability, the oxidative respiratory metabolism and protein synthesis changes and the inhibition of DNA topoisomerase activities. Electrical conductivity and the number of large molecules of BAI increased 2.48% and 1.8%, respectively, than that of the control. However, the membrane integrity did not destroyed by BAI directly. With BAI treatment, inhibition rates of activities for SDH and MDH were 56.2% and 57.4%, respectively, demonstrating that BAI could inhibit cell respiratory. After treated with BAI for 20 h, the total soluble content of proteins decreased by 42.83%. Moreover, the activities of DNA topoisomerase I and II were inhibited completely by 0.2 mmol x L(-1) BAI. These results indicated that BAI had obvious antibacterial activity on Staphylococcus aureus. The mechanism is that it could affect bacterial membrane penetrability, inhibit protein synthesis and influence SDH, MDH and DNA topoisomerase I and II activities to exert its antibacterial functions.

[Point-through-point acupuncture for treatment of peripheral facial paralysis].

OBJECTIVE: To search for a more effective penetration needling method for peripheral facial paralysis. METHODS: The patients were divided into a treatment group and a control group according to visiting sequence. The treatment group of 50 cases were treated with Yangbai (GB 14)-through-Touwei (ST 8), Yangbai (a little above the original point)-through-Shangxing (GV 23), Sizhukong (TE 23)-through-Yuyao (EX-HN 4), Qianzheng-through-Yingxiang (LI 20), Dicang (ST 4)-through-Jiache (ST 6) and Jiache-through-Yingxiang, at an angle about 45 degrees between the penetration needling direction and local paralysis muscle bundle, and the control group (n=45) with Yangbai-through-Yuyao, Zanzhu (BL 2)-through-Yuyao, Dicang-through-Jiache, Jiache-through-Dicang, Qianzheng-through-Dicang, Sibai (ST 2)-through-Yingxiang. RESULTS: The cured rate and the cured and markedly effective rate were 76.0% and 92.0% in the treatment group, and 55.6% and 75.6% in the control group respectively, with significant differences between the two groups in the cured rate, the cured and markedly effective rate and the therapeutic times for cure. CONCLUSION: The penetration needling method at an angle about 45 degrees between the penetration needling and paralysis muscle bundle is more beneficial to recovery of facial paralysis.