New sesquiterpenes and viridin derivatives from Penicillium sp. Ameliorates NAFLD by regulating the PINK1/Parkin mitophagy pathway.

Fungi from the plant rhizosphere microbiome are considered an important source of bioactive novel natural compounds. In this study, three new sesquiterpenes, penisterpenoids A-C (1-3), and three new viridin derivatives, peniviridiols A-C (4-6), along with twenty one known compounds (7-27), were isolated from the rhizosphere fungus Penicillium sp. SMU0102 of medicinal plant Bupleurum chinense DC. Their structures were elucidated by extensive spectroscopic analysis. The absolute configurations of compounds 1-6 were determined by experimental and calculated ECD spectra, DP4 + probability analysis, modified Mosher's method, and X-ray crystallography. All new compounds were screened for their cytotoxic and lipid-lowering activities in vitro. Among them, compound 1 (20 µM) remarkably alleviated lipid accumulation both in FFA-induced LO2 cells and TAA-induced zebrafish NAFLD models. Furthermore, compound 1 enhanced ATP production and mitochondrial membrane potential (MMP), suppressed reactive oxygen species (ROS) formation, restored mitochondrial structure, and induced autophagosome formation. Moreover, compound 1 significantly upregulated the expression of representative proteins for the mitochondrial homeostasis, including OPA1, DRP1, MFF, and Fis1, as well as mitophagy representative proteins PINK1, Parkin, and P62. Further mechanistic investigations indicated that compound 1 primarily alleviated lipid accumulation through selective activation of the PINK1/Parkin mitophagy signaling pathway.

Dectin-1 induces TRPV1 sensitization and contributes to visceral hypersensitivity of irritable bowel syndrome in male mice.

BACKGROUND: Visceral hypersensitivity is considered the core pathophysiological mechanism that causes abdominal pain in patients with irritable bowel syndrome (IBS). Fungal dysbiosis has been proved to contribute to visceral hypersensitivity in IBS patients. However, the underlying mechanisms for Dectin-1, a major fungal recognition receptor, in visceral hypersensitivity are poorly understood. This study aimed to explore the role of Dectin-1 in visceral hypersensitivity and elucidate the impact of Dectin-1 activity on the function of transient receptor potential vanilloid type 1 (TRPV1). METHODS: Visceral hypersensitivity model was established by the intracolonic administration of 0.1 mL TNBS (130 µg/mL in 30% ethanol) in the male mice. Fluconazole and nystatin were used as fungicides. Laminarin, a Dectin-1 antagonist and gene knockout (Clec7a-/-) mice were used to interrupt the function of Dectin-1. Colorectal distension-electromyogram recording was performed to assess visceral sensitivity. Immunostaining experiment was performed to determine the localization of Dectin-1 in dorsal root ganglion (DRG) neurons. Calcium imaging study was performed to assay TRPV1-mediated calcium influx in acutely dissociated DRG neurons. RESULTS: Pretreatment with fungicides, administration of laminarin or genetic deletion of Clec7a alleviated TNBS-induced visceral hypersensitivity in male mice. The expression of Dectin-1 was upregulated in the DRG and colon of TNBS-treated mice. Colocalization of Dectin-1 and TRPV1 was observed in DRG neurons. Importantly, pretreatment with curdlan, a Dectin-1 agonist, increased TRPV1-mediated calcium influx. CONCLUSIONS: Dectin-1 contributes to visceral hypersensitivity in IBS or in inflammatory bowel disease in remission and activation of Dectin-1 induces TRPV1 sensitization. SIGNIFICANCE STATEMENT: This work provides direct evidence for the functional regulation of TRPV1 channel by Dectin-1 activity, proposing a new mechanism underlying TRPV1 sensitization. Control of intestinal fungi might be beneficial for the treatment of refractory abdominal pain in patients with IBS or IBD in remission.

Molecular mechanism of Fe3+ binding inhibition to Vibrio metschnikovii ferric ion-binding protein, FbpA, by rosmarinic acid and its hydrolysate, danshensu.

Global warming has increased the growth of pathogenic Vibrio bacteria, which can cause foodborne illnesses and death. Vibrio bacteria require iron for growth and survival. They utilize a ferric ion-binding protein (FbpA) to bind and transport Fe3+ into the cell. FbpA from Vibrio metschnikovii (Vm) is a potential target for inhibiting its growth. Rosmarinic acid (RA) can block the binding of VmFbpA to Fe3+ ; however, the molecular mechanism of Fe3+ binding and RA inhibition to VmFbpA is unclear. In this study, we used x-ray crystallography to determine the Fe3+ -binding mode of VmFbpA and the mechanism of RA inhibition. The structures revealed that in the Fe3+ bound form, Fe3+ was coordinated to VmFbpA by two Tyr residues, two HCO3 - ions, and two water molecules in a six-coordinated geometry. In contrast, in the inhibitor bound form, RA was initially bound to VmFbpA following gel filtration purification, but it was hydrolyzed to danshensu (DSS), which occupied the binding site as shown in an electron density map and reverse phase chromatography (RPC) analysis. Both RA and DSS exhibited a stronger binding affinity to VmFbpA, higher Fe3+ reduction capacity, and more potent bacteriostatic effect on V. metschnikovii compared with caffeic acid (CA), another hydrolysis product of RA. These results provide insight into the mechanism of iron acquisition by V. metschnikovii and inhibition by RA and DSS. Our findings offer clues on the development of effective strategies to prevent Vibrio infections.

Antibacterial spirooxindole alkaloids from Penicillium brefeldianum inhibit dimorphism of pathogenic smut fungi.

Three new antibacterial spirooxindole alkaloids, spirobrefeldins A-C (1-3), together with four known analogs, spirotryprostatin M (4), spirotryprostatin G (5), 12ß-hydroxyverruculogen TR-2 (6), and 12α-hydroxyverruculogen TR-2 (7), were isolated from terrestrial fungus Penicillium brefeldianum. All the new compounds were elucidated extensively by the interpretation of their NMR (1D and 2D) spectra and high-resolution mass data, and their absolute configurations were determined by computational chemistry and CD spectra. The absolute configurations of spiro carbon C-2 in spirotryprostatin G (5) and spirotryprostatin C in literature were reported as S, which were revised to R based on experimental and calculated CD spectra. All the compounds were evaluated for their antimicrobial activities toward Pseudomonas aeruginosa PAO1, Dickeya zeae EC1, Staphylococcus epidermidis, Escherichia coli, and Sporisorium scitamineum. Compound 7 displayed moderate inhibitory activity toward dimorphic switch of pathogenic smut fungi Sporisorium scitamineum at 25 µM. Compounds 3 and 6 showed weak antibacterial activities against phytopathogenic bacterial Dickeya zeae EC1 at 100 µM.

The Comparison of Short- and Long-Term Outcomes for Laparoscopic Versus Open Gastrectomy for Patients With Advanced Gastric Cancer: A Meta-Analysis of Randomized Controlled Trials.

Objectives: The effect of laparoscopic gastrectomy (LG) for the treatment of advanced gastric cancer (AGC) is still controversial. The aim of this meta-analysis was to contrast the short- and long-term outcomes of laparoscopic versus conventional open gastrectomy (OG) for patients with AGC. Methods: Databases including PubMed, Embase, Scopus, and Cochrane Library were systematically searched until December 2021 for randomized controlled trial-enrolled patients undergoing LG or OG for the treatment of AGC. Short-term outcomes were overall postoperative complications, anastomotic leakage, number of retrieved lymph node, surgical time, blood loss, length of hospital stay, and short-term mortality. Long-term outcomes were survival rates at 1, 3, and 5 years. Results: A total of 12 trials involving 4,101 patients (2,059 in LG group, 2,042 in OG group) were included. No effect on overall postoperative complications (OR 0.84, 95% CI 0.67 to 1.05, p = 0.12, I2 = 34%) and anastomotic leakage (OR 1.26, 95% CI 0.82 to 1.95, p = 0.30, I2 = 0%) was found. Compared with the open approach, patients receiving LG had fewer blood loss (MD -54.38, 95% CI -78.09 to -30.67, p < 0.00001, I2 = 90%) and shorter length of hospital stay (MD -1.25, 95% CI -2.08 to -0.42, p = 0.003, I2 = 86%). However, the LG was associated with a lower number of retrieved lymph nodes (MD -1.02, 95% CI -1.77 to -0.27, p = 0.008, I2 = 0%) and longer surgical time (MD 40.87, 95% CI 20.37 to 54.44, p < 0.00001, I2 = 94%). Furthermore, there were no differences between LG and OG groups in short-term mortality and survival rate at 1, 3, and 5 years. Conclusions: LG offers improved short-term outcomes including shorter hospital stays and fewer blood loss, with comparable postoperative complications, short-term mortality, and survival rate at 1, 3, and 5 years when compared to the open approach. Our results support the implementation of LG in patients with AGC. Systematic Review Registration: PROSPERO (CRD 42021297141).

The T-Type Calcium Channel Cav3.2 in Somatostatin Interneurons in Spinal Dorsal Horn Participates in Mechanosensation and Mechanical Allodynia in Mice.

Somatostatin-positive (SOM+) neurons have been proposed as one of the key populations of excitatory interneurons in the spinal dorsal horn involved in mechanical pain. However, the molecular mechanism for their role in pain modulation remains unknown. Here, we showed that the T-type calcium channel Cav3.2 was highly expressed in spinal SOM+ interneurons. Colocalization of Cacna1h (which codes for Cav3.2) and SOM tdTomato was observed in the in situ hybridization studies. Fluorescence-activated cell sorting of SOM tdTomato cells in spinal dorsal horn also proved a high expression of Cacna1h in SOM+ neurons. Behaviorally, virus-mediated knockdown of Cacna1h in spinal SOM+ neurons reduced the sensitivity to light touch and responsiveness to noxious mechanical stimuli in naïve mice. Furthermore, knockdown of Cacna1h in spinal SOM+ neurons attenuated thermal hyperalgesia and dynamic allodynia in the complete Freund's adjuvant-induced inflammatory pain model, and reduced both dynamic and static allodynia in a neuropathic pain model of spared nerve injury. Mechanistically, a decrease in the percentage of neurons with Aß-eEPSCs and Aß-eAPs in superficial dorsal horn was observed after Cacna1h knockdown in spinal SOM+ neurons. Altogether, our results proved a crucial role of Cav3.2 in spinal SOM+ neurons in mechanosensation under basal conditions and in mechanical allodynia under pathological pain conditions. This work reveals a molecular basis for SOM+ neurons in transmitting mechanical pain and shows a functional role of Cav3.2 in tactile and pain processing at the level of spinal cord in addition to its well-established peripheral role.

Citrinadin C, a new cytotoxic pentacyclic alkaloid from marine-derived fungus Penicillium citrinum.

A new cytotoxic pentacyclic alkaloid, citrinadin C (1), together with four known compounds (2-5), were isolated from deep-sea-derived fungus Penicillium citrinum. The structure of new compound 1 was elucidated by extensive 1D and 2D NMR and Mass spectroscopic data, and its absolute configuration was determined by CD spectrum. All the compounds were evaluated for their cytotoxic and antibacterial activities. Compound 1 showed cytotoxic activities against human liver cancer cell line MHCC97H, with IC50 value of 16.7 µM. Compound 4 displayed significant antibacterial activity against phytopathogen Xanthomonas campestris, with MIC value of 25 µM.

Novel Cyclohexyl Meroterpenes Produced by Combinatorial Biosynthesis.

Structurally diverse fungal meroterpenoids are promising drug seed compounds. To obtain unnatural, novel meroterpene scaffolds, we tested combinatorial biosynthesis by co-expressing functionally distinct terpene cyclase (TPC) genes, pyr4, ascF, andB, or cdmG, with the biosynthetic genes for the production of a TPC substrate, (10'R)-epoxyfarnesyl-dimethylorsellinic acid-3,5-methyl ester, in Aspergillus oryzae NSAR1 as a heterologous host. As a result, all of the tested TPCs afforded the same two novel mono-cyclization products. This study provides important information on the substrate scope of the TPCs, and will contribute to the production of unnatural, novel molecules for future drug discovery.

Crocetin ameliorates chronic restraint stress-induced depression-like behaviors in mice by regulating MEK/ERK pathways and gut microbiota.

ETHNOPHARMACOLOGICAL RELEVANCE: This study aimed at determining the effects of saffron on depression as well as its neuroprotective and pharmacological effects on the intestinal function of crocetin in mice exposed to chronic restraint stress. MATERIALS AND METHODS: Chronic stress was induced in two-week-old ICR mice by immobilizing them for 6 h per day for 28 days. The mice were orally administered with crocetin (20, 40, 80 mg/kg), fluoxetine (20 mg/kg) or distilled water. The treatments were administered daily and open field and tail suspension tests were performed. Immunofluorescent and Western-bolt (WB) assays were conducted to determine the expression of mitogen-activated protein kinase phosphatase-1 (MKP-1), the precursor of brain-derived neurotrophic factor (proBDNF), extracellular signal-regulated kinase 1/2 (ERK1/2), phosphorylated cAMP response element-binding (CREB) protein in the hippocampus. Serum levels of dopamine (DA), proBDNF, MKP-1 and CREB were measured by Elisa kits. High-throughput sequencing was carried out to analyze the composition of intestinal microbiota. RESULTS: Crocetin ameliorated depressive-like behaviors caused by chronic restraint stress-induced depressive mice. It significantly attenuated the elevated levels of MKP-1, proBDNF, alanine transaminase, aspartate transaminase and increased the serum levels of DA as well as CREB. Histopathological analysis showed that crocetin suppressed hippocampus injury in restraint stress mice by protecting neuronal cells. Immunofluorescent and WB analysis showed elevated expression levels of ERK1/2, CREB and inhibited expression levels of MKP-1, proBDNF in the hippocampus. The intestinal ecosystem of the crocetin group partially recovered and was close to the control group. CONCLUSIONS: Crocetin has neuroprotective properties and ameliorates the effects of stress-associated brain damage by regulating the MKP-1-ERK1/2-CREB signaling and intestinal ecosystem.

Effects of Bread Yeast Cell Wall Beta-Glucans on Mice with Loperamide-Induced Constipation.

Constipation is a common gastrointestinal disorder characterized by changes in intestinal habits. Increasing evidence indicates that long-term use of irritant laxatives causes serious side effects. Meanwhile, more than 50% of patients are dissatisfied with sense of use of non-prescriptional laxatives. ß-glucans are natural polysaccharides widely found in yeast, fungus, and plants, which have been reported to exhibit various pharmacological effects. The aim of this study was to characterize the effect of ß-glucans extracted from the bread yeast cell wall on loperamide-induced constipation mice. Forty mice were fed with loperamide (10 mg/kg) to make the constipation model and a diet supplemented with 2.5, 5, and 10 mg/kg ß-glucan. We assessed the defecation frequency, intestinal transit function of mice, as well as used high-throughput sequencing to analyze the intestinal microbiota composition and functional biological profiles data. Meanwhile, we detected expression of neurotransmitters including acetylcholinesterase, substance P, and serotonin (5-HT) and expression of tight junction protein (TJP) including zonula occludens-1 and mucin-2 in distal colon to characterize the possible molecular mechanisms. ß-glucans significantly enhanced intestinal motility and provided a possibility to regulate the expression of neurotransmitters and TJP in mice. The intestinal microecological portion of the treatment group partially recovered and was closer to the normal group. This study showed that ß-glucans can influence the intestinal microbiota and restore microecological balance to regulate the express of neurotransmitters and TJP to recover intestinal epithelial mechanical barrier. We suggested that ß-glucans could be used as an active nutritional supplement to protect the damaged intestinal barrier and help patients who have constipation complications and dysbiosis.

Mycophenolic Acid as a Promising Fungal Dimorphism Inhibitor to Control Sugar Cane Disease Caused by Sporisorium scitamineum.

The morphological changes from single-cell yeast to filamentous hypha form are critical in plant pathogenic smut fungi. This dimorphic switch is tightly regulated by complex gene pathways in pathogenic development. The phytopathogenic basidiomycetes Sporisorium scitamineum displays a morphological transition from budding growth of haploid cells to filamentous growth of the dikaryon, which enables fungi to forage for nutrients and evade the host plant immune system. In the search for compounds that affect dimorphic switch instead of killing the cell directly, a natural product, mycophenolic acid (MPA), was purified and exhibited significant dimorphism inhibitory activities with minimum effective concentrations of 0.3 µg/mL. RNA sequencing and real-time quantitative transcription-PCR analysis showed that treatment of 100 µg/mL MPA dramatically repressed the expression of the ammonium transporter gene Ssa2 . A further subcellular localization experiment, ammonium response assay, and Western blot assay confirmed that Ssa2 could be one of the most important molecular targets of MPA in regulating dimorphism of S. scitamineum. These observations suggest that Ssa2 serves as a molecular target of MPA and could be used in the treatment of sugar cane smut diseases caused by S. scitamineum.

Upregulated EBI3 Correlates with Poor Outcome and Tumor Progression in Breast Cancer.

BACKGROUND AND AIM: So far, the understanding of the role of Epstein-Barr virus-induced gene 3 (EBI3) in breast cancer has been limited. This study uncovers the functional role and clinical significance of EBI3 in breast cancer patients. PATIENTS AND METHODS: The expression levels of EBI3, IL-27p28, and IL-12p35 were measured by quantitative real-time reverse transcription (RT)-polymerase chain reaction (PCR). Correlations of EBI3 expression with IL-27p28 and IL-12p35 expression were analyzed using Pearson's correlation assay. The prognostic performance of EBI3 was assessed via Kaplan-Meier survival assay and Cox regression analysis. RESULTS: EBI3 expression was increased in cancerous tissues compared with the controls (P < 0.05). This overexpression of EBI3 was correlated with lymph node metastasis and clinical stage (both P < 0.05). Besides, elevated expression of EBI3 was usually found in patients with positive lymph node metastasis (P < 0.05), and similar results were obtained in advanced clinical-stage breast cancer cases (P < 0.05). Increases in both IL-27p28 and IL-12p35 expression were identified in breast cancer tissues (all P < 0.05), and IL-12p35 expression was found to be associated with EBI3 expression (R = 0.888, P < 0.001). Survival curves revealed that high EBI3 expression was correlated with poor overall survival (log-rank P < 0.05). The Cox analysis indicated that EBI3 was an independent prognostic factor in breast cancer. CONCLUSION: Taken together, overexpression of EBI3 was associated with poor prognosis and might be involved in the progression of breast cancer.

Diaphanous-related formin-3 overexpression inhibits the migration and invasion of triple-negative breast cancer by inhibiting RhoA-GTP expression.

Triple-negative breast cancer (TNBC) is associated with a high risk of metastasis, recurrence, and poor prognosis. TNBC is insensitive to existing endocrine and targeted breast cancer therapies because it lacks the expression of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Therefore, there is an urgent need for identifying novel targets to improve the efficacy of TNBC treatment. Diaphanous-related formin-3 (DIAPH3) regulates cytoskeleton formation to regulate cell adhesion, migration, and differentiation. A previous study showed that DIAPH3 promoted the metastasis of prostate cancer. However, DIAPH3 expression in TNBC and its effect on TNBC development have not been reported to date. In present study, we investigated the expression and functions of DIAPH3 in TNBC. Results of immunohistochemical staining showed that DIAPH3 expression significantly decreased in breast cancer tissues, especially TNBC tissues, compared with that in paired non-tumor tissues. In addition, DIAPH3 expression was associated with TNM stage and lymph node metastasis, but not with tumor size in patients with TNBC. Further, DIAPH3 overexpression clearly suppressed the migration and invasion of MDA-MB-231 cells and decreased the expression of Ras Homolog Family Member A (RhoA), RhoA-GTP, Matrix Metallopeptidase 2 (MMP-2), and MMP-9. Thus, we predict that DIAPH3 overexpression inhibits the migration and invasion of TNBC by inhibiting RhoA-GTP expression and the results of the present study provide new insights for developing DIAPH3-targeting therapies for TNBC.

Does hospital-based transitional care reduce the postoperative complication in patients with enterostomy? A meta-analysis.

OBJECTIVE: The objective of the study is to investigate whether hospital-based transitional care can reduce the postoperative complication in patients who received enterostomy or not by pooling the published prospective clinical studies. MATERIALS AND METHODS: Prospective clinical studies related to hospital-based transitional care for reducing the postoperative complication in patients with enterostomy were searched in the electronic databases of PubMed, Medline, EMBASE, CNKI, and Wanfang. The postoperative complications in the experiment and control groups were extracted from the original studies and pooled by fixed effects model. The publication bias was evaluated by Begg's funnel plot and Egger's line regression test. RESULTS: After searching through the electronic databases of PubMed, Medline, EMBASE, CNKI, and Wanfang, we finally included in eight studies with 600 cases related to hospital-based transitional care and postoperative complication in patients with enterostomy. The pooled result showed that hospital-based transitional care could significantly reduce the postoperative complication in patients with enterostomy (risk ratio = 0.42, 95% confidence interval: 1.0.32, ~0.55, P = 0.005) by fixed effects model. The Begg's funnel plot demonstrated a litter left-right asymmetry, which indicated potential publication bias. Moreover, Egger's line regression test showed that there were significant publications (t = -3.04, P = 0.023). CONCLUSION: Hospital-based transitional care can significantly reduce the postoperative complication in patients with enterostomy.

Inhibition of WWP2 suppresses proliferation, and induces G1 cell cycle arrest and apoptosis in liver cancer cells.

Primary liver cancer is one of the most common and aggressive human malignancies worldwide. As numerous studies have revealed that WW domain containing E3 Ub­protein ligase 2 (WWP2) exerts cancer­specific functions, the present study assessed the role of WWP2 in liver cancer. WWP2 was revealed to be significantly overexpressed in liver cancer tissues compared with paired normal tissues at the mRNA as well as at the protein level. Furthermore, small interfering RNA-mediated WWP2 knockdown in liver cancer cell lines was demonstrated to inhibit cell proliferation, cause cell cycle arrested in G1 phase and to induce apoptosis as revealed by a Cell Counting Kit-8 assay and flow cytometric analysis. In addition, western blot analysis revealed that WWP2 knockdown significantly increased the expression of apoptosis-associated markers caspase­7, caspase­8 and B-cell lymphoma 2 (Bcl-2)-associated X in liver cancer cell lines, while Bcl­2 was significantly decreased. In conclusion, the present study suggested that WWP2 may exert important functions in the over­proliferation and evasion of apoptosis of liver cancer, likely through regulating the expression of apoptosis-associated markers. Furthermore, WWP2 may represent a novel diagnostic marker and molecular therapeutic target for liver cancer.

Silencing of WWP2 inhibits adhesion, invasion, and migration in liver cancer cells.

The role and clinical implication of the WWP2 E3 ubiquitin ligase in liver cancer are poorly understood. In the current study, we investigated the expression level of WWP2 and its functions in cell adhesion, invasion, and migration in liver cancer. We used real-time PCR to detect the expression of WWP2 in liver cancer and adjacent samples from the People's Hospital of Lishui and also analyzed The Cancer Genome Atlas (TCGA) RNA-seq data by bioinformatics. Migration and invasion were detected by transwell analysis. We detected a strong WWP2 expression in tumor tissues of the People's Hospital of Lishui, and the survival rate was significantly higher in patients with lower WWP2-expressing tumors. WWP2 small hairpin RNA (shRNA) lentivirus stably infected cells (shWWP2), Huh7, showed slower growth speed compared with scramble control-infected cells in a xenograft mouse model. Knockdown of WWP2 Huh7 and BEL-7404 cells demonstrated a reduction in adhesion, invasion, and migration. Gene set enrichment analysis (GSEA) showed that WWP2 is positively correlated to cancer-related pathways including the chemokine signaling pathway. WWP2 also regulated MMP-9, caspase-9, CXCR3, and CCR5 expression in liver cancer cells. In addition, knockdown of CXCR3 and CCR5 significantly inhibited cell proliferation, adhesion, invasion, and migration in Huh7 and BEL-7404 cells. Our data suggest that targeting of WWP2 may be a therapeutic strategy for liver cancer treatment.