MIBiG 3.0: a community-driven effort to annotate experimentally validated biosynthetic gene clusters.

With an ever-increasing amount of (meta)genomic data being deposited in sequence databases, (meta)genome mining for natural product biosynthetic pathways occupies a critical role in the discovery of novel pharmaceutical drugs, crop protection agents and biomaterials. The genes that encode these pathways are often organised into biosynthetic gene clusters (BGCs). In 2015, we defined the Minimum Information about a Biosynthetic Gene cluster (MIBiG): a standardised data format that describes the minimally required information to uniquely characterise a BGC. We simultaneously constructed an accompanying online database of BGCs, which has since been widely used by the community as a reference dataset for BGCs and was expanded to 2021 entries in 2019 (MIBiG 2.0). Here, we describe MIBiG 3.0, a database update comprising large-scale validation and re-annotation of existing entries and 661 new entries. Particular attention was paid to the annotation of compound structures and biological activities, as well as protein domain selectivities. Together, these new features keep the database up-to-date, and will provide new opportunities for the scientific community to use its freely available data, e.g. for the training of new machine learning models to predict sequence-structure-function relationships for diverse natural products. MIBiG 3.0 is accessible online at https://mibig.secondarymetabolites.org/.

Metabolic Engineering of "Last-line Antibiotic" Colistin in Paenibacillus polymyxa.

Colistin, also known as polymyxin E, is a lipopeptide antibiotic used to treat infections caused by multidrug-resistant gram-negative bacteria. It is considered a "last-line antibiotic", but its clinical development is hindered by low titer and impurities resulting from the presence of diverse homologs in microbial fermentation. To ensure consistent pharmaceutical activity and kinetics, it is crucial to have high-purity colistin active pharmaceutical ingredient (API) in the pharmaceutical industry. This study focused on the metabolic engineering of a natural colistin producer strain to produce colistin with a high titer and purity. Guided by genome mining, we identified Paenibacillus polymyxa ATCC 842 as a natural colistin producer capable of generating a high proportion of colistin A. By systematically inactivating seven non-essential biosynthetic gene clusters (BGCs) of peptide metabolites that might compete precursors with colistin or inhibit colistin production, we created an engineered strain, P14, which exhibited an 82% increase in colistin titer and effectively eliminated metabolite impurities such as tridecaptin, paenibacillin, and paenilan. Additionally, we engineered the L-2,4-diaminobutyric acid (L-2,4-DABA) pathway to further enhance colistin production, resulting in the engineered strain P19, which boosted a remarkable colistin titer of 649.3 mg/L - a 269% improvement compared to the original strain. By concurrently feeding L-isoleucine and L-leucine, we successfully produced high-purity colistin A, constituting 88% of the total colistin products. This study highlights the potential of metabolic engineering in improving the titer and purity of lipopeptide antibiotics in the non-model strain, making them more suitable for clinical use. These findings indicate that efficiently producing colistin API in high purity directly from fermentation can now be achieved in a straightforward manner.

Enrichment of biofertilizer-type hydrogen-oxidizing bacteria on urea containing Cu(II).

With the utilization of pesticides and fertilizers (e.g. urea), the presence of nitrogen and heavy metals (e.g. copper) can enter and pollute the environment. Biofertilizers can be used to replace chemical fertilizers to increase crop yields and reduce environmental stress. The utilization of hydrogen-oxidizing bacteria (HOB) to be biofertilizers has recently attracted more attention. However, the enrichment of HOB on urea and the effect of copper are undetermined. HOB were successfully enriched using urea in this investigation. The average urea conversion rate (AUCR) was 180.08 mgN/L/d with a hydraulic retention time of 10 h. Microbial community (R1) was dominated by Hydrogenophaga (83.92%), a biofertilizer-type HOB. After addition of 5.47 mg/L Cu2+, the AUCR was decreased by 16%-151.18 mgN/L/d, and the growth of HOB was inhibited by 48%. Meanwhile, inhibition was also reflected by the increase of polysaccharide content (20.27 ± 0.57 to 33.45 ± 2.53 mg/gVSS) and protein content (106.19 ± 19.39 to 125.14 ± 24.73 mg/gVSS) of extracellular polymeric substances in the HOB. The resulting microbial community (R2) was changed to Azospiralium-dominated flora (91.33%). Both enriched microbial communities (R1 and R2) exhibited the abilities of ACC degradation and phosphate solubilization. This study demonstrates that employing urea can directly enrich biofertilizer-type HOB and copper-tolerant HOB can be obtained in a 5.47 mg/L Cu2+ environment. The results provide potential methods to obtain biofertilizer from copper-containing urea wastewater via HOB.

Bacterial Cytochrome P450 Catalyzed Post-translational Macrocyclization of Ribosomal Peptides.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a fascinating group of natural products that exhibit diverse structural features and bioactivities. P450-catalyzed RiPPs stand out as a unique but underexplored family. Herein, we introduce a rule-based genome mining strategy that harnesses the intrinsic biosynthetic principles of RiPPs, including the co-occurrence and co-conservation of precursors and P450s and interactions between them, successfully facilitating the identification of diverse P450-catalyzed RiPPs. Intensive BGC characterization revealed four new P450s, KstB, ScnB, MciB, and SgrB, that can catalyze the formation of Trp-Trp-Tyr (one C-C and two C-N bonds), Tyr-Trp (C-C bond), Trp-Trp (C-N bond), and His-His (ether bond) crosslinks, respectively, within three or four residues. KstB, ScnB, and MciB could accept non-native precursors, suggesting they could be promising starting templates for bioengineering to construct macrocycles. Our study highlights the potential of P450s to expand the chemical diversity of strained macrocyclic peptides and the range of biocatalytic tools available for peptide macrocyclization.

Multicolor fluorescence assay of tetracycline: lanthanide complexed amino clay loaded with copper nanoclusters.

A multicolor fluorescent nanoprobe has been prepared by loading bovine serum albumin-stabilized copper nanoclusters (BSA-Cu NCs) onto amino clay (AC) and grafting Eu3+ with auxiliary ligand citric acid (Cit). Tetracycline (TC) can coordinate with Eu3+ by ß-diketone structure and transfer energy to Eu3+ through antenna effect. When the concentration of TC is in the range 0 to 13 µM, the blue emission intensity of BSA-CuNCs is basically unchanged, and the red emission of Eu3+ is remarkably enhanced due to the coordination with TC. The emission color gradually changes from blue to red under UV lamp (λ = 365 nm). However, when the TC concentration is in the range 13 to 350 µΜ, the internal amino acid residues of BSA sensitize TC, and the emission color gradually changed from red to green. The nanoprobe has rich color, is simple to prepare, portable, and provide a wide detection range. The limit of detection (LOD) is 3.04 nM, which could be used for real-time visual analysis of trace TC in actual samples (lake water, milk, honey, and bovine serum albumin). In addition, a visual test paper has been designed and combined with the color scanning APP of a smartphone to complete the qualitative and semi-quantitative test of TC. BSA-Cu NCs were loaded on amino clay and graft Eu3+ to establish ratiometric fluorescent nanoprobe for TC detection. With the increase of TC concentration, the emission color under 365 nm UV lamp gradually changed from blue to red and then to green, and the color changed obviously and can be observed by the naked eye. The visual test paper and smartphone application detection sensor were developed to realize rapid, convenient, real-time, and visual detection of TC in actual samples.

Expanded Sequence Space of Radical S-Adenosylmethionine-Dependent Enzymes Involved in Post-translational Macrocyclization.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent one of the largest but primarily underexplored natural product families in bacteria. The genetically encoded nature of RiPPs simplifies the prediction and prioritization of their biosynthetic gene clusters (BGCs). We report a small peptide and enzyme co-occurrence analysis workflow (SPECO), which allowed us to identify 32 220 prospective rSAM-catalyzed RiPP BGCs from 161 954 bacterial genomes and prioritize 25 families with new biosynthetic architectures or precursor patterns. We characterized three new enzymes that respectively catalyze cysteine-glycine (BlaB), histidine-aliphatic side chain (ScaB), and tyrosine/histidine-arginine (VguB) cross-links. The cyclophane-forming enzyme ScaB exhibits broad substrate selectivity, allowing it to catalyze diverse triceptide formation. These results demonstrate the strength of the SPECO workflow in discovering new enzymes for peptide macrocyclization.

Discovery of Antibiofilm Activity of Elasnin against Marine Biofilms and Its Application in the Marine Antifouling Coatings.

Biofilms are surface-attached multicellular communities that play critical roles in inducing biofouling and biocorrosion in the marine environment. Given the serious economic losses and problems caused by biofouling and biocorrosion, effective biofilm control strategies are highly sought after. In a screening program of antibiofilm compounds against marine biofilms, we discovered the potent biofilm inhibitory activity of elasnin. Elasnin effectively inhibited the biofilm formation of seven strains of bacteria isolated from marine biofilms. With high productivity, elasnin-based coatings were prepared in an easy and cost-effective way, which exhibited great performance in inhibiting the formation of multi-species biofilms and the attachment of large biofouling organisms in the marine environment. The 16S amplicon analysis and anti-larvae assay revealed that elasnin could prevent biofouling by the indirect impact of changed microbial composition of biofilms and direct inhibitory effect on larval settlement with low toxic effects. These findings indicated the potential application of elasnin in biofilm and biofouling control in the marine environment.

Selection and environmental adaptation along a path to speciation in the Tibetan frog Nanorana parkeri.

Tibetan frogs, Nanorana parkeri, are differentiated genetically but not morphologically along geographical and elevational gradients in a challenging environment, presenting a unique opportunity to investigate processes leading to speciation. Analyses of whole genomes of 63 frogs reveal population structuring and historical demography, characterized by highly restricted gene flow in a narrow geographic zone lying between matrilines West (W) and East (E). A population found only along a single tributary of the Yalu Zangbu River has the mitogenome only of E, whereas nuclear genes of W comprise 89-95% of the nuclear genome. Selection accounts for 579 broadly scattered, highly divergent regions (HDRs) of the genome, which involve 365 genes. These genes fall into 51 gene ontology (GO) functional classes, 14 of which are likely to be important in driving reproductive isolation. GO enrichment analyses of E reveal many overrepresented functional categories associated with adaptation to high elevations, including blood circulation, response to hypoxia, and UV radiation. Four genes, including DNAJC8 in the brain, TNNC1 and ADORA1 in the heart, and LAMB3 in the lung, differ in levels of expression between low- and high-elevation populations. High-altitude adaptation plays an important role in maintaining and driving continuing divergence and reproductive isolation. Use of total genomes enabled recognition of selection and adaptation in and between populations, as well as documentation of evolution along a stepped cline toward speciation.

Publisher Correction: Resistance to nonribosomal peptide antibiotics mediated by D-stereospecific peptidases.

In the version of this article originally published, the links and files for the Supplementary Information, including Supplementary Tables 1-5, Supplementary Figures 1-25, Supplementary Note, Supplementary Datasets 1-4 and the Life Sciences Reporting Summary, were missing in the HTML. The error has been corrected in the HTML version of this article.

Resistance to nonribosomal peptide antibiotics mediated by D-stereospecific peptidases.

Nonribosomal peptide antibiotics, including polymyxin, vancomycin, and teixobactin, most of which contain D-amino acids, are highly effective against multidrug-resistant bacteria. However, overusing antibiotics while ignoring the risk of resistance arising has inexorably led to widespread emergence of resistant bacteria. Therefore, elucidation of the emerging mechanisms of resistance to nonribosomal peptide antibiotics is critical to their implementation. Here we describe a networking-associated genome-mining platform for linking biosynthetic building blocks to resistance components associated with biosynthetic gene clusters. By applying this approach to 5,585 complete bacterial genomes spanning the entire domain of bacteria, with subsequent chemical and enzymatic analyses, we demonstrate a mechanism of resistance toward nonribosomal peptide antibiotics that is based on hydrolytic cleavage by D-stereospecific peptidases. Our finding reveals both the widespread distribution and broad-spectrum resistance potential of D-stereospecific peptidases, providing a potential early indicator of antibiotic resistance to nonribosomal peptide antibiotics.

Activities of Nerol, a natural plant active ingredient, against Candida albicans in vitro and in vivo.

Candida albicans invasion is one of the most serious fungal infections in clinical history. In recent years, because of the widespread use of immunosuppressive drugs, chemotherapy drugs, glucocorticoids, and broad-spectrum antibiotics, serious drug resistance has been reported; therefore, a new type of antifungal drug needs to be developed. In this study, we found that Nerol (NEL) had strong antimicrobial activity and 0.77 µL/mL NEL was the minimum inhibitory concentration (MIC) effective against C. albicans. We determined the change of the growth curve of NEL for C. albicans, to identify the trend of NEL activity against C. albicans. Through the determination of the ergosterol content and glucose-induced extracellular fluid acidification of NEL on C. albicans, we found that NEL inhibits the growth of C. albicans by destroying cell membranes. This finding was also supported by the expression of SAP (secreted aspartyl proteinase) involved in cell membrane synthesis. Finally, demonstrations of phenotype investigation, colony-forming unit (CFU) counts, and PAS (periodic acid-Schiff) staining were conducted to prove that NEL had the ability to treated mouse oral C. albicans infection and vaginal C. albicans infection. This research may help us to investigate new antimicrobial agents for treating C. albicans infections. KEY POINTS: • NEL can inhibit the growth of C. albicans. • NEL destroys the cell membrane formation and permeability of C. albicans. • NEL can treat vulvovaginal candidiasis and oropharyngeal candidiasis in mice. • NEL could be used as a possible antifungal agent.

The Molecular Mechanism of Perillaldehyde Inducing Cell Death in Aspergillus flavus by Inhibiting Energy Metabolism Revealed by Transcriptome Sequencing.

Perillaldehyde (PAE), an essential oil in Perilla plants, serves as a safe flavor ingredient in foods, and shows an effectively antifungal activity. Reactive oxygen species (ROS) accumulation in Aspergillus flavus plays a critical role in initiating a metacaspase-dependent apoptosis. However, the reason for ROS accumulation in A. flavus is not yet clear. Using transcriptome sequencing of A. flavus treated with different concentrations of PAE, our data showed that the ROS accumulation might have been as a result of an inhibition of energy metabolism with less production of reducing power. By means of GO and KEGG enrichment analysis, we screened four key pathways, which were divided into two distinct groups: a downregulated group that was made up of the glycolysis and pentose phosphate pathway, and an upregulated group that consisted of MAPK signaling pathway and GSH metabolism pathway. The inhibition of dehydrogenase gene expression in two glycometabolism pathways might play a crucial role in antifungal mechanism of PAE. Also, in our present study, we systematically showed a gene interaction network of how genes of four subsets are effected by PAE stress on glycometabolism, oxidant damage repair, and cell cycle control. This research may contribute to explaining an intrinsic antifungal mechanism of PAE against A. flavus.

Positive N-Methyl-D-Aspartate Receptor Modulation by Rapastinel Promotes Rapid and Sustained Antidepressant-Like Effects.

BACKGROUND: Modulation of glutamatergic synaptic transmission by N-methyl-D-aspartate receptors can produce rapid and sustained antidepressant effects. Rapastinel (GLYX-13), initially described as a N-methyl-D-aspartate receptor partial glycine site agonist, exhibits rapid antidepressant effect in rodents without the accompanying dissociative effects of N-methyl-D-aspartate receptor antagonists. METHODS: The relationship between rapastinel's in vitro N-methyl-D-aspartate receptor pharmacology and antidepressant efficacy was determined by brain microdialysis and subsequent pharmacological characterization of therapeutic rapastinel concentrations in N-methyl-D-aspartate receptor-specific radioligand displacement, calcium mobilization, and medial prefrontal cortex electrophysiology assays. RESULTS: Brain rapastinel concentrations of 30 to 100 nM were associated with its antidepressant-like efficacy and enhancement of N-methyl-D-aspartate receptor-dependent neuronal intracellular calcium mobilization. Modulation of N-methyl-D-aspartate receptors by rapastinel was independent of D-serine concentrations, and glycine site antagonists did not block rapastinel's effect. In rat medial prefrontal cortex slices, 100 nM rapastinel increased N-methyl-D-aspartate receptor-mediated excitatory postsynaptic currents and enhanced the magnitude of long-term potentiation without any effect on miniature EPSCs or paired-pulse facilitation responses, indicating postsynaptic action of rapastinel. A critical amino acid within the NR2 subunit was identified as necessary for rapastinel's modulatory effect. CONCLUSION: Rapastinel brain concentrations associated with antidepressant-like activity directly enhance medial prefrontal cortex N-methyl-D-aspartate receptor activity and N-methyl-D-aspartate receptor-mediated synaptic plasticity in vitro. At therapeutic concentrations, rapastinel directly enhances N-methyl-D-aspartate receptor activity through a novel site independent of the glycine coagonist site. While both rapastinel and ketamine physically target N-methyl-D-aspartate receptors, the 2 molecules have opposing actions on N-methyl-D-aspartate receptors. Modest positive modulation of N-methyl-D-aspartate receptors by rapastinel represents a novel pharmacological approach to promote well-tolerated, rapid, and sustained improvements in mood disorders.

Cinnamaldehyde inhibits Candida albicans growth by causing apoptosis and its treatment on vulvovaginal candidiasis and oropharyngeal candidiasis.

The invasion of Candida albicans is one of the most common fungal infections seen in clinical practice, and serious drug resistance has been reported in recent years. Therefore, new anti-C. albicans drugs must be introduced. In this research, it was demonstrated that cinnamaldehyde (CA) shows strong antimicrobial activity, with 0.26 mg/mL CA being the minimum inhibitory concentration to manage C. albicans. Extraordinarily, we detected that CA accumulated the intracellular reactive oxygen species (ROS) and enhanced the calcium concentration in the cytoplasm and mitochondria through flow cytometry. In addition, we observed that C. albicans cells released Cytochrome c from the mitochondria to the cytoplasm, depolarized the mitochondrial membrane potential, and activated the metacaspase when exposed to 0.065, 0.13, 0.26, and 0.52 mg/mL CA. Furthermore, to confirm that CA introduces the C. albicans apoptosis, we discovered that when the phosphatidylserine was exposed, DNA damage and chromatin condensation occurred, which were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and 4',6-diamidino-2-phenylindole (DAPI) staining. Finally, demonstrations of phenotype investigation, colony-forming unit (CFU) counts, and periodic acid-Schiff (PAS) staining were conducted to prove that CA possessed the ability to treat oropharyngeal candidiasis (OPC) and vulvovaginal candidiasis (VVC). From the above, our research indicates that CA is a promising antifungal candidate when applied to C. albicans infections.

Inhibitors of the Neutral Amino Acid Transporters ASCT1 and ASCT2 Are Effective in In Vivo Models of Schizophrenia and Visual Dysfunction.

The N-methyl-d-aspartate receptor coagonist d-serine is a substrate for the neutral amino acid transporters ASCT1 and ASCT2, which may regulate its extracellular levels in the central nervous system (CNS). We tested inhibitors of ASCT1 and ASCT2 for their effects in rodent models of schizophrenia and visual dysfunction, which had previously been shown to be responsive to d-serine. L-4-fluorophenylglycine (L-4FPG), L-4-hydroxyPG (L-4OHPG), and L-4-chloroPG (L-4ClPG) all showed high plasma bioavailability when administered systemically to rats and mice. L-4FPG showed good brain penetration with brain/plasma ratios of 0.7-1.4; however, values for L-4OHPG and L-4ClPG were lower. Systemically administered L-4FPG potently reduced amphetamine-induced hyperlocomotion in mice, whereas L-4OHPG was 100-fold less effective and L-4ClPG inactive at the doses tested. L-4FPG and L-4OHPG did not impair visual acuity in naive rats, and acute systemic administration of L-4FPG significantly improved the deficit in contrast sensitivity in blue light-treated rats caused by retinal degeneration. The ability of L-4FPG to penetrate the brain makes this compound a useful tool to further evaluate the function of ASCT1 and ASCT2 transporters in the CNS.

Divergent biosynthesis yields a cytotoxic aminomalonate-containing precolibactin.

Colibactin is an as-yet-uncharacterized genotoxic secondary metabolite produced by human gut bacteria. Here we report the biosynthetic discovery of two new precolibactin molecules from Escherichia coli, including precolibactin-886, which uniquely incorporates the highly sought genotoxicity-associated aminomalonate building block into its unprecedented macrocyclic structure. This work provides new insights into the biosynthetic logic and mode of action of this colorectal-cancer-linked microbial chemical.

[The Detection of Micro RNA346 Gene Polymorphism by Capillary Electrophoresis].

OBJECTIVE: To develop a method for the detection of micro RNA346 gene polymorphism by capillary electrophoresis (CE). METHODS: The genome DNA was extracted with the kit of blood/cell/tissue genome DNA extraction,then micro RNA346 gene was amplified by PCR,digested by BciT130Ⅰrestriction enzyme and detected by CE. The conditions for CE separation were optimized. Samples from rheumatoid arthritis patients and healthy persons were detected under the optimal conditions. RESULTS: Under the optimized experimental conditions of CE (sieving medium mass concentration was 10 g/L and the separation voltage was 12 kV),the detection of the digested products of microRNA346 gene could be completed within 25 min. The intra-day relative standard deviation (RSD) of the method was 0.43%-0.63% and inter-day RSD was 1.49%-1.56%.Samples from 96 rheumatoid arthritis patients and 43 healthy persons were analyzed by the proposed method. The results showed that only micro RNA346Ⅰtype was detected but micro RNA346 Ⅱ type wasn't. CONCLUSION: This method is easy to operate,and has the advantages of high efficiency,fast speed,less sample consumption and high automation level. This method is suitable for the determination of RNA gene polymorphism of mirco RNA.

Lewis Acid-Catalyzed Selective [2 + 2]-Cycloaddition and Dearomatizing Cascade Reaction of Aryl Alkynes with Acrylates.

Combined Lewis acid, consisting of two or more Lewis acids, sometimes shows unique catalytic ability, and it may promote reactions which could not be catalyzed by any of the Lewis acids solely. On the other hand, the development of efficient methods for the facile synthesis of cyclobutenes and densely functionalized decalins is an attractive target for synthetic chemists due to their versatile synthetic utilities and widespread occurrence in natural products. Herein, we wish to report an efficient method for the assembly of cyclobutenes and densely functionalized decalin skeletons through In(tfacac)3-TMSBr catalyzed selective [2 + 2]-cycloaddition and dearomatizing cascade reaction of aryl alkynes with acrylates with high chemo- and stereoselectivity. The obtained cyclobutene could be easily converted into cyclobutane as well as synthetically useful 1,4- and 1,5-diketones with high chemo- and stereoselectivity. On the basis of mechanistic studies, plausible reaction mechanisms were proposed for both the [2 + 2]-cycloaddition and the dearomatizing cascade reaction. Finally, the computational studies of reaction mechanisms were conducted, and the results suggest that the combined Lewis acid could efficiently promote both reactions.

CTRP9 regulates hypoxia-mediated human pulmonary artery smooth muscle cell proliferation, apoptosis and migration via TGF-ß1/ERK1/2 signaling pathway.

Hypoxia is an important risk factor for pulmonary arterial remodeling in pulmonary arterial hypertension (PAH). Vascular remodeling in hypoxia-induced PAH is driven by excessive proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs). The purpose of the present study was to explore the expression of CTRP9 in rats model of hypoxia-induced PAH and investigate the effects of CTRP9 on HPASMCs function in vitro and determine the underlying mechanisms. We established a rat model of hypoxic PAH, which showed a downregulation of CTRP9 expression. In HPASMCs cultured under the condition of hypoxia, treatment with CTRP9 notably restrained cell proliferation responses to hypoxia accompanied with decreased two biomarkers of cell proliferation Ki-67 and PCNA. Meanwhile, CTRP9 strikingly promoted hypoxia-mediated cell apoptosis as reflected by upregulation of Bax and downregulation of Bcl-2, as well as enhanced Caspase 3 activity. Additionally, CTRP9 treatment dramatically prevented the migratory potential by declined the expression of MMP-2 and MMP-9. Moreover treatment with CTRP9 augmented hypoxia-mediated differentiation by elevating the expression level of differentiation markers α-SMA and SM22. Mechanistically, anti-proliferative effects conferred by CTRP9 are mediated through suppression of TGF-ß1/ERK1/2 pathway. Collectively, we identified CTRP9 as a novel mediator of PASMC growth in hypoxia-mediated PAH, indicating that CTRP9 in the pulmonary vasculature may be an underlying mechanism in the development of hypoxia-induced PAH. Our study, for the first time, established that CTRP9 plays a protective role of CTRP9 in pulmonary vascular remodeling, pointing to its potential clinical value for patients with PAH.

Silencing of NRAGE induces autophagy via AMPK/Ulk1/Atg13 signaling pathway in NSCLC cells.

NRAGE has been reported to be overexpressed in cancer cells, especially in lung cancer cells. To determine the role of NRAGE in non-small-cell lung cancer cells, we investigated the effects of NRAGE on autophagy in non-small-cell lung cancer cells. Human A549 and H1299 cells were transfected with NRAGE-specific small-interfering RNA. The Cell Counting Kit-8 and plate clone assay showed that downregulation of NRAGE could induce the proliferation in A549 and H1299 cells. In addition, our data suggested that downregulation of NRAGE enhances autophagosome formation by immunofluorescence. We found that knockdown of NRAGE induced autophagy, together with downregulation of P62 and upregulation of LC3-II protein. Furthermore, to elucidate the mechanism of NRAGE in suppressing autophagy, the protein expressions of AMPK, Ulk1, and Atg13 were assessed. Collectively, these results demonstrate the effective anti-autophagic of NRAGE in non-small-cell lung cancer cells through AMPK/Ulk1/Atg13 autophagy signaling pathways. Therefore, NRAGE could be used as a potential therapeutic target for lung cancer.