Multiomics-Based Biocargo Components Analysis in Enterococcus faecium Membrane Vesicles.

Enterococcus spp. have been shown to have gastrointestinal tract protective functions; our recent results suggest that membrane vesicles (MVs) play an important role in the gastric protection of Enterococcus faecium (E. faecium). The specific function is determined by molecular compositions of MVs. To resolve biocargo components in E. faecium MVs (EfmMVs), MVs were isolated from E. faecium culture. Transcriptomics, label-free quantitative proteomics, and untargeted metabolomics were performed to obtain information about the complexity of ribonucleic acids (RNAs), proteins, and metabolites biocargo they carry, respectively. RNA-sequencing identified a total of 2122 transcripts. The top 20 transcripts accounted for 27.63% of total counts, which, including enzymes, participate in glycolysis, ribosomal proteins, DNA-directed RNA polymerases, protein-synthesizing relative enzymes, molecules associated with protein post-translational processing and transport, and peptidoglycan lyases. Label-free quantitative proteomics analysis identified a total of 711 proteins. The top 20 proteins accounted for 48.02% of all identified proteins, which including ribosomal proteins, enzymes participate in glycolysis, DNA-directed RNA polymerases, protein-synthesizing relative enzymes, peptidoglycan lyases, and autolysin. Untargeted metabolomics analysis identified a total of 519 metabolites. The top 20 metabolites accounted for 79.55% of all identified metabolites, which included amino acids, substrates, or products in the metabolism of amino acids, natural organic acids, products in the metabolism of organic acids, ketone compounds, and two other compounds. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that the identified biocargo components enriched in metabolism, genetic, and environmental information processing. Overall, we hope that the current exploration of multiple "-omics" analyses of this EfmMVs will provide useful information and further groundwork for future studies on E. faecium application.

Effects of Salmonella Outer Membrane Vesicles on Intestinal Microbiota and Intestinal Barrier Function.

Salmonella enterica is one of the most important zoonotic pathogens causing foodborne gastroenteritis worldwide. Outer membrane vesicles (OMVs) are lipid-bilayer vesicles produced by Gram-negative bacteria, which contain biologically active components. We hypothesized that OMVs are an important weapon of S. enterica to initiate enteric diseases pathologies. In this study, the effects of S. enterica OMVs (SeOMVs) on intestinal microbiota and intestinal barrier function were investigated. In vitro fecal culture experiments showed that alpha diversity indexes and microbiota composition were altered by SeOMV supplementation. SeOMV supplementation showed an increase of pH, a decrease of OD630 and total short chain fatty acid (SCFA) concentrations. In vitro IPEC-J2 cells culture experiments showed that SeOMV supplementation did not affect the IPEC-J2 cell viability and the indicated genes expression. In vivo experiments in mice showed that SeOMVs had adverse effects on average daily gain (p < 0.05) and feed:gain ratio (p < 0.05), and had a tendency to decrease the final body weight (p = 0.073) in mice. SeOMV administration decreased serum interleukin-10 level (p < 0.05), decreased the relative abundance of bacteria belonging to the genera BacC-u-018 and Akkermansia (p < 0.05). Furthermore, SeOMV administration damaged the ileum mucosa (p < 0.05). These findings suggest that SeOMVs play an important role in the activation of intestinal inflammatory response induced by S. enterica, and downregulation of SCFA-producing bacteria is a possible mechanism.

YY1 cooperates with TFEB to regulate autophagy and lysosomal biogenesis in melanoma.

Autophagy is a self-proteolytic process that degrades intracellular material to maintain cellular homeostasis. Transcription factor EB (TFEB) is the master activator that regulates the transcription of genes involved in autophagy and lysosomal biogenesis. However, the cotranscriptional factors of TFEB are rarely identified. Here, we found that Yin Yang 1 (YY1) regulated autophagy and lysosome biogenesis in melanoma cells. YY1 cooperates with TFEB to regulate autophagy through controlling the transcription of autophagy and lysosome biogenesis related genes. Moreover, suppression of YY1 enhanced the antitumor efficiency of vemurafenib both in vitro and in vivo. Collectively, these studies identify YY1 as a novel cotranscription factor of TFEB in regulating autophagy and lysosomal functions and suggest YY1 could be a therapeutic target in cancer treatment.

miR-9 regulates ferroptosis by targeting glutamic-oxaloacetic transaminase GOT1 in melanoma.

Ferroptosis is a recently recognized form of regulated cell death driven by lipid-based reactive oxygen species (ROS) accumulation. However, the molecular mechanisms of ferroptosis regulation are still largely unknown. Here we identified a novel miRNA, miR-9, as an important regulator of ferroptosis by directly targeting GOT1 in melanoma cells. Overexpression of miR-9 suppressed GOT1 by directly binding to its 3'-UTR, which subsequently reduced erastin- and RSL3-induced ferroptosis. Conversely, suppression of miR-9 increased the sensitivity of melanoma cells to erastin and RSL3. Importantly, anti-miR-9 mediated lipid ROS accumulation and ferroptotic cell death could be abrogated by inhibiting glutaminolysis process. Taken together, our findings demonstrate that miR-9 regulates ferroptosis by targeting GOT1 in melanoma cells, illustrating the important role of miRNA in ferroptosis.

Protective effect of Enterococcus faecium against ethanol-induced gastric injury via extracellular vesicles.

Recently, Enterococcus has been shown to have gastric protective functions, and the mechanisms by which Enterococcus modulates gastric function are still being investigated. Herein, we investigated how Enterococcus faecium (Efm) and E. faecium-derived extracellular vesicles (EVs) (EfmEVs) exert protective effect against ethanol-induced gastric injury by investigating the effect of EfmEVs on gastric mucosal ulcer scoring, histological lesion, mucosal glycoprotein production, acidity, anti-oxidative function, and inflammatory responses in rat. Pretreatment with Efm showed significant reduction of ethanol-induced gastric injury, as evidenced by the lowering of ulcer index, histological lesion, gastric pH, and inflammatory responses and the enhancement of mucosal glycoprotein production and anti-oxidative function. Further functional studies on three bioactive components [inactivated Efm, EfmEVs (EVs), and EV-free supernatants] of the bacterial culture showed that EVs are mostly responsible for the gastroprotective effect. Moreover, EV secretion is beneficial for the gastroprotective effect of Efm. Hence, EVs mediated the protective effect of Efm against ethanol-induced gastric injury by lowering inflammatory responses and enhancing anti-oxidative function and may be a potent anti-inflammatory and anti-oxidative strategy to alleviate hyperinflammatory gastrointestinal tract conditions.IMPORTANCEThis study indicated that Enterococcus faecium provided a protective effect against rat gastric injury, which involved improvement of the mucosal glycoprotein production, anti-oxidative function, and inflammatory responses. Furthermore, we confirmed that three bioactive components (inactivated Efm, extracellular vesicles, and EV-free supernatants) of E. faecium culture also contributed to the gastroprotective effect. Importantly, E. faecium-derived EVs showed an effective impact for the gastroprotective effect.

Long noncoding RNA NEAT1 promotes ferroptosis by modulating the miR-362-3p/MIOX axis as a ceRNA.

Ferroptosis, a novel form of regulated cell death induced by iron-dependent lipid peroxidation, plays an essential role in the development and drug resistance of tumors. Long noncoding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) has been reported to be involved in the regulation of cell cycle, proliferation, apoptosis, and migration of tumor cells. However, the function and molecular mechanism of NEAT1 in regulating ferroptosis in tumors remain unclear. Here, we found that ferroptosis inducers erastin and RSL3 increased NEAT1 expression by promoting the binding of p53 to the NEAT1 promoter. Induced NEAT1 promoted the expression of MIOX by competitively binding to miR-362-3p. MIOX increased ROS production and decreased the intracellular levels of NADPH and GSH, resulting in enhanced erastin- and RSL3-induced ferroptosis. Importantly, overexpression of NEAT1 increased the anti-tumor activity of erastin and RSL3 by enhancing ferroptosis both in vitro and in vivo. Collectively, these data suggest that NEAT1 plays a novel and indispensable role in ferroptosis by regulating miR-362-3p and MIOX. Considering the clinical findings that HCC patients are insensitive to chemotherapy and immunotherapy, ferroptosis induction may be a promising therapeutic strategy for HCC patients with high NEAT1 expression.

Ceruloplasmin suppresses ferroptosis by regulating iron homeostasis in hepatocellular carcinoma cells.

Ferroptosis is a regulated form of cell death characterized by the iron-dependent accumulation of lipid hydroperoxides. Ceruloplasmin (CP) is a glycoprotein that plays an essential role in iron homeostasis. However, whether CP regulates ferroptosis has not been reported. Here, we show that CP suppresses ferroptosis by regulating iron homeostasis in hepatocellular carcinoma (HCC) cells. Depletion of CP promoted erastin- and RSL3-induced ferroptotic cell death and resulted in the accumulation of intracellular ferrous iron (Fe2+) and lipid reactive oxygen species (ROS). Moreover, overexpression of CP suppressed erastin- and RSL3-induced ferroptosis in HCC cells. In addition, a novel frameshift mutation (c.1192-1196del, p.leu398serfs) of CP gene newly identified in patients with iron accumulation and neurodegenerative diseases lost its ability to regulate iron homeostasis and thus failed to participate in the regulation of ferroptosis. Collectively, these data suggest that CP plays an indispensable role in ferroptosis by regulating iron metabolism and indicate a potential therapeutic approach for hepatocellular carcinoma.

Nedd4 ubiquitylates VDAC2/3 to suppress erastin-induced ferroptosis in melanoma.

Ferroptosis is a newly defined form of regulated cell death characterized by the iron-dependent accumulation of lipid hydroperoxides. Erastin, the ferroptosis activator, binds to voltage-dependent anion channels VDAC2 and VDCA3, but treatment with erastin can result in the degradation of the channels. Here, the authors show that Nedd4 is induced following erastin treatment, which leads to the ubiquitination and subsequent degradation of the channels. Depletion of Nedd4 limits the protein degradation of VDAC2/3, which increases the sensitivity of cancer cells to erastin. By understanding the molecular mechanism of erastin-induced cellular resistance, we can discover how cells adapt to new molecules to maintain homeostasis. Furthermore, erastin-induced resistance mediated by FOXM1-Nedd4-VDAC2/3 negative feedback loop provides an initial framework for creating avenues to overcome the drug resistance of ferroptosis activators.

A wheat caffeic acid 3-O-methyltransferase TaCOMT-3D positively contributes to both resistance to sharp eyespot disease and stem mechanical strength.

Plant caffeic acid 3-O-methyltransferase (COMT) has been implicated in the lignin biosynthetic pathway through catalyzing the multi-step methylation reactions of hydroxylated monomeric lignin precursors. However, genetic evidence for its function in plant disease resistance is poor. Sharp eyespot, caused primarily by the necrotrophic fungus Rhizoctonia cerealis, is a destructive disease in hexaploid wheat (Triticum aestivum L.). In this study, a wheat COMT gene TaCOMT-3D, is identified to be in response to R. cerealis infection through microarray-based comparative transcriptomics. The TaCOMT-3D gene is localized in the long arm of the chromosome 3D. The transcriptional level of TaCOMT-3D is higher in sharp eyespot-resistant wheat lines than in susceptible wheat lines, and is significantly elevated after R. cerealis inoculation. After R. cerealis inoculation and disease scoring, TaCOMT-3D-silenced wheat plants exhibit greater susceptibility to sharp eyespot compared to unsilenced wheat plants, whereas overexpression of TaCOMT-3D enhances resistance of the transgenic wheat lines to sharp eyespot. Moreover, overexpression of TaCOMT-3D enhances the stem mechanical strength, and lignin (particular syringyl monolignol) accumulation in the transgenic wheat lines. These results suggest that TaCOMT-3D positively contributes to both wheat resistance against sharp eyespot and stem mechanical strength possibly through promoting lignin (especially syringyl monolignol) accumulation.

miR-216b enhances the efficacy of vemurafenib by targeting Beclin-1, UVRAG and ATG5 in melanoma.

Autophagy maintains cells survival in many stressful conditions including starvation, growth factor deprivation and misfolded protein accumulation. Additionally, autophagic survival mechanisms are used by transformed tumor cells to inhibit cell death, limit drug effectiveness and possibly generate drug resistance. However, the mechanism of how cells utilize autophagy during drug resistance is not fully understood. Here, we demonstrate that miR-216b plays an important role in alleviating drug resistance by regulating autophagy in melanoma. We show that miR-216b attenuates autophagy by directly targeting three key autophagy genes Beclin-1, UVRAG and ATG5. Overexpression of these genes from miRNA immune cDNA constructs rescue autophagic activity in the presence of miR-216b. Antagomir-mediated inactivation of endogenous miR-216b led to an increase of Beclin-1, UVRAG, ATG5, and subsequent autophagic activity. More importantly, we have discovered that BRAF(V600E) inhibitor vemurafenib suppresses miR-216b activity, which in turn activates autophagy to generate drug resistance in both BRAFi-sensitive and -resistant cells. Strikingly, ectopic expression of miR-216b increases the efficacy of vemurafenib both in vitro and in vivo. Taken together, these data indicate that miR-216b regulates autophagy by suppressing three key autophagy genes, and enhances the antitumor activity of vemurafenib in BRAF(V600E) melanoma cells.

miR-137 regulates ferroptosis by targeting glutamine transporter SLC1A5 in melanoma.

Ferroptosis is a regulated form of cell death driven by small molecules or conditions that induce lipid-based reactive oxygen species (ROS) accumulation. This form of iron-dependent cell death is morphologically and genetically distinct from apoptosis, necroptosis, and autophagy. miRNAs are known to play crucial roles in diverse fundamental biological processes. However, to date no study has reported miRNA-mediated regulation of ferroptosis. Here we show that miR-137 negatively regulates ferroptosis by directly targeting glutamine transporter SLC1A5 in melanoma cells. Ectopic expression of miR-137 suppressed SLC1A5, resulting in decreased glutamine uptake and malondialdehyde (MDA) accumulation. Meanwhile, antagomir-mediated inactivation of endogenous miR-137 increased the sensitivity of melanoma cells to erastin- and RSL3-induced ferroptosis. Importantly, knockdown of miR-137 increased the antitumor activity of erastin by enhancing ferroptosis both in vitro and in vivo. Collectively, these data indicate that miR-137 plays a novel and indispensable role in ferroptosis by inhibiting glutaminolysis and suggest a potential therapeutic approach for melanoma.

A Wheat Cinnamyl Alcohol Dehydrogenase TaCAD12 Contributes to Host Resistance to the Sharp Eyespot Disease.

Sharp eyespot, caused mainly by the necrotrophic fungus Rhizoctonia cerealis, is a destructive disease in hexaploid wheat (Triticum aestivum L.). In Arabidopsis, certain cinnamyl alcohol dehydrogenases (CADs) have been implicated in monolignol biosynthesis and in defense response to bacterial pathogen infection. However, little is known about CADs in wheat defense responses to necrotrophic or soil-borne pathogens. In this study, we isolate a wheat CAD gene TaCAD12 in response to R. cerealis infection through microarray-based comparative transcriptomics, and study the enzyme activity and defense role of TaCAD12 in wheat. The transcriptional levels of TaCAD12 in sharp eyespot-resistant wheat lines were significantly higher compared with those in susceptible wheat lines. The sequence and phylogenetic analyses revealed that TaCAD12 belongs to IV group in CAD family. The biochemical assay proved that TaCAD12 protein is an authentic CAD enzyme and possesses catalytic efficiencies toward both coniferyl aldehyde and sinapyl aldehyde. Knock-down of TaCAD12 transcript significantly repressed resistance of the gene-silenced wheat plants to sharp eyespot caused by R. cerealis, whereas TaCAD12 overexpression markedly enhanced resistance of the transgenic wheat lines to sharp eyespot. Furthermore, certain defense genes (Defensin, PR10, PR17c, and Chitinase1) and monolignol biosynthesis-related genes (TaCAD1, TaCCR, and TaCOMT1) were up-regulated in the TaCAD12-overexpressing wheat plants but down-regulated in TaCAD12-silencing plants. These results suggest that TaCAD12 positively contributes to resistance against sharp eyespot through regulation of the expression of certain defense genes and monolignol biosynthesis-related genes in wheat.