Mining biosynthetic gene clusters in Paenibacillus genomes to discover novel antibiotics.

BACKGROUND: Bacterial antimicrobial resistance poses a severe threat to humanity, necessitating the urgent development of new antibiotics. Recent advances in genome sequencing offer new avenues for antibiotic discovery. Paenibacillus genomes encompass a considerable array of antibiotic biosynthetic gene clusters (BGCs), rendering these species as good candidates for genome-driven novel antibiotic exploration. Nevertheless, BGCs within Paenibacillus genomes have not been extensively studied. RESULTS: We conducted an analysis of 554 Paenibacillus genome sequences, sourced from the National Center for Biotechnology Information database, with a focused investigation involving 89 of these genomes via antiSMASH. Our analysis unearthed a total of 848 BGCs, of which 716 (84.4%) were classified as unknown. From the initial pool of 554 Paenibacillus strains, we selected 26 available in culture collections for an in-depth evaluation. Genomic scrutiny of these selected strains unveiled 255 BGCs, encoding non-ribosomal peptide synthetases, polyketide synthases, and bacteriocins, with 221 (86.7%) classified as unknown. Among these strains, 20 exhibited antimicrobial activity against the gram-positive bacterium Micrococcus luteus, yet only six strains displayed activity against the gram-negative bacterium Escherichia coli. We proceeded to focus on Paenibacillus brasilensis, which featured five new BGCs for further investigation. To facilitate detailed characterization, we constructed a mutant in which a single BGC encoding a novel antibiotic was activated while simultaneously inactivating multiple BGCs using a cytosine base editor (CBE). The novel antibiotic was found to be localized to the cell wall and demonstrated activity against both gram-positive bacteria and fungi. The chemical structure of the new antibiotic was elucidated on the basis of ESIMS, 1D and 2D NMR spectroscopic data. The novel compound, with a molecular weight of 926, was named bracidin. CONCLUSIONS: This study outcome highlights the potential of Paenibacillus species as valuable sources for novel antibiotics. In addition, CBE-mediated dereplication of antibiotics proved to be a rapid and efficient method for characterizing novel antibiotics from Paenibacillus species, suggesting that it will greatly accelerate the genome-based development of new antibiotics.

Contribution of monocyte and macrophage extracellular traps to neutrophilic airway inflammation in severe asthma.

BACKGROUND: Increased blood/sputum neutrophil counts are related to poor clinical outcomes of severe asthma (SA), where we hypothesized that classical monocytes (CMs)/CM-derived macrophages (Mφ) are involved. We aimed to elucidate the mechanisms of how CMs/Mφ induce the activation of neutrophils/innate lymphoid cells (ILCs) in SA. METHODS: Serum levels of monocyte chemoattractant protein-1 (MCP-1) and soluble suppression of tumorigenicity 2 (sST2) were measured from 39 patients with SA and 98 those with nonsevere asthma (NSA). CMs/Mφ were isolated from patients with SA (n = 19) and those with NSA (n = 18) and treated with LPS/interferon-gamma. Monocyte/M1Mφ extracellular traps (MoETs/M1ETs) were evaluated by western blotting, immunofluorescence, and PicoGreen assay. The effects of MoETs/M1ETs on neutrophils, airway epithelial cells (AECs), ILC1, and ILC3 were assessed in vitro and in vivo. RESULTS: The SA group had significantly higher CM counts with increased migration as well as higher levels of serum MCP-1/sST2 than the NSA group. Moreover, the SA group had significantly greater production of MoETs/M1ETs (from CMs/M1Mφ) than the NSA group. The levels of MoETs/M1ETs were positively correlated with blood neutrophils and serum levels of MCP-1/sST2, but negatively correlated with FEV1%. In vitro/in vivo studies demonstrated that MoETs/M1ETs could activate AECs, neutrophils, ILC1, and ILC3 by increased migration as well as proinflammatory cytokine production. CONCLUSIONS: CM/Mφ-derived MoETs/M1ETs could contribute to asthma severity by enhancing neutrophilic airway inflammation in SA, where modulating CMs/Mφ may be a potential therapeutic option.

Prediction of the number of asthma patients using environmental factors based on deep learning algorithms.

BACKGROUND: Air pollution, weather, pollen, and influenza are typical aggravating factors for asthma. Previous studies have identified risk factors using regression-based and ensemble models. However, studies that consider complex relationships and interactions among these factors have yet to be conducted. Although deep learning algorithms can address this problem, further research on modeling and interpreting the results is warranted. METHODS: In this study, from 2015 to 2019, information about air pollutants, weather conditions, pollen, and influenza were utilized to predict the number of emergency room patients and outpatients with asthma using recurrent neural network, long short-term memory (LSTM), and gated recurrent unit models. The relative importance of the environmental factors in asthma exacerbation was quantified through a feature importance analysis. RESULTS: We found that LSTM was the best algorithm for modeling patients with asthma. Our results demonstrated that influenza, temperature, PM10, NO2, CO, and pollen had a significant impact on asthma exacerbation. In addition, the week of the year and the number of holidays per week were an important factor to model the seasonality of the number of asthma patients and the effect of holiday clinic closures, respectively. CONCLUSION: LSTM is an excellent algorithm for modeling complex epidemiological relationships, encompassing nonlinearity, lagged responses, and interactions. Our study findings can guide policymakers in their efforts to understand the environmental factors of asthma exacerbation.

Biomarkers for predicting type 2-high and uncontrolled asthma in real-world practice.

BACKGROUND: Blood eosinophil count (BEC), immunoglobulin (Ig) E, and fractional exhaled nitric oxide (FeNO) are key clinical indicators for identifying type 2 (T2) asthma. OBJECTIVE: To provide optimal cutoff points of T2 markers for assessing T2-high or uncontrolled asthma in real-world practice. METHODS: Various clinical and laboratory parameters were analyzed according to the result of T2 markers (BEC, serum-free IgE, and FeNO) in adults with asthma who had maintained antiasthmatic medications. The cutoff levels for representing uncontrolled asthma were determined using receiver operating characteristic analysis. Blood levels of periostin and eosinophil-derived neurotoxin were measured by enzyme-linked immunosorbent assay. Activation markers of circulating eosinophils (Siglec8+) and neutrophils (CD66+) were analyzed by flow cytometry. RESULTS: Of 133 patients with asthma, 23 (17.3%) had 3 T2 markers (BEC ≥ 300 cells/µL, serum-free IgE ≥ 120 ng/mL, and FeNO ≥ 25 parts per billion) and significantly higher levels of sputum eosinophils, blood eosinophil-derived neurotoxin, and Siglec8+ eosinophils but lower 1-second forced expiratory volume percentage, in addition to a higher rate of uncontrolled status (P < .05 for all). Furthermore, patients with uncontrolled asthma had significantly higher levels of FeNO and BEC with lower 1-second forced expiratory volume percentage (P < .05 for all). The optimal cutoff values for predicting uncontrolled asthma were found to be 22 parts per billion of FeNO levels, 161.4 cells/L of BECs, and 85.9 ng/mL of serum-free IgE levels. CONCLUSION: We suggest the optimal cutoff values of BEC, IgE, and FeNO for classifying T2-high or uncontrolled asthma, which could be applied as candidate biomarkers for targeting patients with asthma who require T2 biologics.

Streptooctatins A and B, fusicoccane-type diterpenoids with autophagic activity from Streptomyces sp. KCB17JA11.

Two new fusicoccane-type diterpenoids, streptooctatins A (1) and B (2), together with a known compound cyclooctatin (3) were isolated from Streptomyces sp. KCB17JA11. The structures of 1 and 2 were determined by analyzing spectroscopic and spectrometric data from 1D and 2D NMR and HRESIMS experiments. Compounds 1 and 2 induced EGFP-LC3 puncta indicating autophagic activities against HeLa cells without cytotoxicity.

Ulleungdolin, a Polyketide-Peptide Hybrid Bearing a 2,4-Di-O-methyl-ß-d-antiarose from Streptomyces sp. 13F051 Co-cultured with Leohumicola minima 15S071.

A new secondary metabolite, ulleungdolin (1), was isolated from the co-culture of an actinomycete, Streptomyces sp. 13F051, and a fungus, Leohumicola minima 15S071. Based on the NMR, UV, and MS data, it was deduced that the planar structure of 1 comprised an isoindolinone (IsoID) with an octanoic acid, a tripeptide, and a sugar. The tripeptide has the unprecedented amino acids norcoronamic acid, 3-hydroxy-glutamine, and 4-hydroxy-phenylglycine and is linked by a C-N bond with IsoID. The absolute configurations were determined by chemical derivatization, extensive spectroscopic methods, and electronic circular dichroism calculations and supported by bioinformatic analyses. Bioactivity evaluation studies indicated that 1 had an antimigration effect on MDA-MB-231 breast cancer cells.

Gwanakosides A and B, 6-Deoxy-α-l-talopyranose-Bearing Aromatic Metabolites from a Streptomyces sp. and Coculture with Pandoraea sp.

Single-strain cultivation of a mountain soil-derived Streptomyces sp. GA02 and its coculture with Pandoraea sp. GA02N produced two aromatic products, gwanakosides A and B (1 and 2, respectively). Their spectroscopic analysis revealed that 1 is a new dichlorinated naphthalene glycoside and 2 is a pentacyclic aromatic glycoside. The assignment of the two chlorine atoms in 1 was confirmed by the analysis of its band-selective CLIP-HSQMBC spectrum. The sugars in the gwanakosides were identified as 6-deoxy-α-l-talopyranose based on 1H-1H coupling constants, Rotating frame Overhauser enhancement spectroscopy (ROESY) NMR correlations, and chemical derivatization followed by spectroscopic and chromatographic analyses. The absolute configuration of 2, whose production was enhanced approximately 100-fold in coculture, was proposed based on a quantum mechanics-based chemical shift analysis method, DP4 calculations, and the chemically determined configuration of 6-deoxy-α-l-talopyranose. Gwanakoside A displayed inhibitory activity against pathogenic bacteria, including Staphylococcus aureus (MIC = 8 µg/mL) and Mycobacterium tuberculosis (MIC50 = 15 µg/mL), and antiproliferative activity against several human cancer cell lines (IC50 = 5.6-19.4 µM).

Angucyclines containing ß-á´ -glucuronic acid from Streptomyces sp. KCB15JA151.

Two angucyclines, pseudonocardones D (1) and E (2), were isolated from Streptomyces sp. KCB15JA151. The planar structure was elucidated by comprehensive spectroscopic analysis. The absolute configuration of the sugar unit was determined based on the basis of coupling constants, ROESY, chemical derivatization and HPLC analysis. The biological activities of compounds 1 and 2 were examined by performing a computational target prediction, which led to tests of the antiestrogenic activity. The result suggested that compound 1 might be an ERα antagonist.

Ulleunganilines A-C, Trichostatin Analogues Bearing a Modified Side Chain from Streptomyces sp. 13F051.

Three new trichostatin analogues, ulleunganilines A-C (1-3), and seven known trichostatins (4-10) were isolated from cultures of Streptomyces sp. 13F051. NMR, UV, and MS data indicated that the planar structures of 1-3 consisted of modified side chains in the trichostatic acid moiety. The absolute configuration of the 2,4-dimethyl-branched carbon chains in 1 and 2 was determined by the PGME method, while the amino acid group in 3 was identified by advanced Marfey's method. Based on the structure of the modified side chains, the origin of 1-3 is proposed. Further experiments indicated that 1 and 3 displayed moderate histone deacetylase inhibitory activity, suggesting that not only the hydroxamate group but also the N,N-dimethyl group were essential for the inhibitory activity.

Sargachromenol Purified from Sargassum horneri Inhibits Inflammatory Responses via Activation of Nrf2/HO-1 Signaling in LPS-Stimulated Macrophages.

In this study, we isolated sargachromenol (SC) from Sargassum horneri and evaluated its anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. SC did not show cytotoxicity at all concentrations and effectively increased the cell viability by reducing the nitric oxide (NO) and intracellular reactive oxygen species (ROS) production in LPS-stimulated RAW 264.7 macrophages. In addition, SC decreased the mRNA expression levels of inflammatory cytokines (IL-1ß, IL-6, and TNF-α) and inflammatory mediators (iNOS and COX-2). Moreover, SC suppressed the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) and mitogen-activated protein kinase (MAPK) signaling, whereas activated the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling in LPS-stimulated RAW 264.7 macrophages. Interestingly, the anti-inflammatory effect of SC was abolished by the inhibition of HO-1 in LPS-stimulated RAW 264.7 macrophages. According to the results, this study suggests that the antioxidant capacity of SC leads to its anti-inflammatory effect and it potentially may be utilized in the nutraceutical and pharmaceutical sectors.

Regulation of Survival Motor Neuron Gene Expression by Calcium Signaling.

Spinal muscular atrophy (SMA) is caused by homozygous survival of motor neurons 1 (SMN1) gene deletion, leaving a duplicate gene, SMN2, as the sole source of SMN protein. However, a defect in SMN2 splicing, involving exon 7 skipping, results in a low level of functional SMN protein. Therefore, the upregulation of SMN protein expression from the SMN2 gene is generally considered to be one of the best therapeutic strategies to treat SMA. Most of the SMA drug discovery is based on synthetic compounds, and very few natural compounds have been explored thus far. Here, we performed an unbiased mechanism-independent and image-based screen of a library of microbial metabolites in SMA fibroblasts using an SMN-specific immunoassay. In doing so, we identified brefeldin A (BFA), a well-known inhibitor of ER-Golgi protein trafficking, as a strong inducer of SMN protein. The profound increase in SMN protein was attributed to, in part, the rescue of the SMN2 pre-mRNA splicing defect. Intriguingly, BFA increased the intracellular calcium concentration, and the BFA-induced exon 7 inclusion of SMN2 splicing, was abrogated by the depletion of intracellular calcium and by the pharmacological inhibition of calcium/calmodulin-dependent kinases (CaMKs). Moreover, BFA considerably reduced the expression of Tra2-ß and SRSF9 proteins in SMA fibroblasts and enhanced the binding of PSF and hnRNP M to an exonic splicing enhancer (ESE) of exon 7. Together, our results demonstrate a significant role for calcium and its signaling on the regulation of SMN splicing, probably through modulating the expression/activity of splicing factors.

Identification of Molecules from Coffee Silverskin That Suppresses Myostatin Activity and Improves Muscle Mass and Strength in Mice.

Coffee has been shown to attenuate sarcopenia, the age-associated muscle atrophy. Myostatin (MSTN), a member of the TGF-ß growth/differentiation factor superfamily, is a potent negative regulator of skeletal muscle mass, and MSTN-inhibition increases muscle mass or prevents muscle atrophy. This study, thus, investigated the presence of MSTN-inhibitory capacity in coffee extracts. The ethanol-extract of coffee silverskin (CSE) but not other extracts demonstrated anti-MSTN activity in a pGL3-(CAGA)12-luciferase reporter gene assay. CSE also blocked Smad3 phosphorylation induced by MSTN but not by GDF11 or Activin A in Western blot analysis, demonstrating its capacity to block the binding of MSTN to its receptor. Oral administration of CSE significantly increased forelimb muscle mass and grip strength in mice. Using solvent partitioning, solid-phase chromatography, and reverse-phase HPLC, two peaks having MSTN-inhibitory capacity were purified from CSE. The two peaks were identified as ßN-arachinoyl-5-hydroxytryptamide (C20-5HT) and ßN-behenoyl-5-hydroxytryptamide (C22-5HT) using mass spectrometry and NMR analysis. In summary, the results show that CSE has the MSTN-inhibitory capacity, and C20-5HT and C22-5HT are active components of CSE-suppressing MSTN activity, suggesting the potential of CSE, C20-5HT, and C22-5HT being developed as agents to combat muscle atrophy and metabolic syndrome.

Catenulisporidins A and B, 16-membered macrolides of the hygrolidin family produced by the chemically underexplored actinobacterium Catenulispora species.

Two new macrolide metabolites of the hygrolidin family, catenulisporidins A and B (1 and 2), together with a known compound hygrolidin (3), were isolated from the culture broth of the rare actinobacterium Catenulispora sp. KCB13F192. Their structures were elucidated on the basis of HRESIMS spectrometric and NMR spectroscopic analyses. Catenulisporidins A and B are the first example of natural hygrolidin and bafilomycin derivatives featuring a modified macrolide ring, and catenulisporidin A possesses a tetrahydrofuran ring through an ether linkage between C-7 and C-10. In cell-based fluorescent imaging and immunoblot assays, the three compounds were shown to inhibit autophagic flux in HeLa cells.

Mojabanchromanol Isolated from Sargassum horneri Attenuates Particulate Matter Induced Inflammatory Responses via Suppressing TLR2/4/7-MAPK Signaling in MLE-12 Cells.

Chromanols from marine algae are studied for drug development due to its prominent bioactive properties, and mojabanchromanol (MC), a chromanol isolated from a brown algae Sargassum horneri, is found to possess anti-oxidant potential. In this study, we hypothesized MC may attenuate particulate matter (PM)-induced and reactive oxygen species (ROS)-mediated inflammatory responses in airways and tried to identify its potential and underlying mechanism against PM (majority <2.5 µm in diameter)-induced inflammatory responses in a lung type II alveolar epithelial cell line, MLE-12. MC attenuated PM-induced malondialdehyde (MDA), a lipid peroxidation end product, and 8-hydroxydeoxyguanosine (8-OHdG), the most representative DNA oxidative damage product, further validating MC's potential in attenuating PM-induced oxidative stress. MC also suppressed PM-triggered TLR2/4/7 activation in MLE-12 cells. Moreover, MC reduced ROS-mediated phosphorylation of mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1/2 (Erk1/2) and c-Jun NH (2)-terminal kinase (JNK) that were also activated in PM exposed cells. MC further inhibited the secretion of pro-inflammatory cytokines (IL-6, IL-1ß and IL-33) in MLE-12 cells exposed to PM. These results provide a clear evidence for MC's potential in attenuating PM-triggered inflammatory responses in MLE-12 cells via repressing TLR2/4/7 and MAPK signaling. Therefore, MC can be developed as a therapeutic agent against PM induced airway inflammatory responses.

Cytotoxic Activities and Molecular Mechanisms of the Beauvericin and Beauvericin G1 Microbial Products against Melanoma Cells.

Melanoma is the most serious type of skin cancer and remains highly drug-resistant. Therefore, the discovery of novel effective agents against melanoma is in high demand. Herein, we investigated the cytotoxic activities in melanoma cells and underlying molecular mechanisms of beauvericin (BEA) and its analogue beauvericin G1 (BEA G1), which are cyclohexadepsipeptides isolated from fungi. BEA and BEA G1 significantly suppressed the growth, clonogenicity, migration, and invasion of A375SM human melanoma cells and promoted caspase-dependent apoptosis through upregulation of death receptors, as well as modulation of pro- and anti-apoptotic Bcl-2 family members. Furthermore, the effects of BEA and BEA G1 were associated with the suppression of multiple molecular targets that play crucial roles in melanoma oncogenesis, including ERK, JNK, p38, NF-κB, STAT3, and MITF. Notably, the cytotoxic efficacy of BEA G1 against A375SM cells was stronger than that of BEA. These findings suggest that BEA and BEA G1 can be further investigated as potent cytotoxic natural compounds for the suppression of melanoma progression.

Phosphorylation of human enhancer filamentation 1 (HEF1) stimulates interaction with Polo-like kinase 1 leading to HEF1 localization to focal adhesions.

Elevated expression of human enhancer filamentation 1 (HEF1; also known as NEDD9 or Cas-L) is an essential stimulus for the metastatic process of various solid tumors. This process requires HEF1 localization to focal adhesions (FAs). Although the association of HEF1 with FAs is considered to play a role in cancer cell migration, the mechanism targeting HEF1 to FAs remains unclear. Moreover, up-regulation of Polo-like kinase 1 (Plk1) positively correlates with human cancer metastasis, yet how Plk1 deregulation promotes metastasis remains elusive. Here, we report that casein kinase 1δ (CK1δ) phosphorylates HEF1 at Ser-780 and Thr-804 and that these phosphorylation events promote a physical interaction between Plk1 and HEF1. We found that this interaction is critical for HEF1 translocation to FAs and for inducing migration of HeLa cells. Plk1-docking phosphoepitopes were mapped/confirmed in HEF1 by various methods, including X-ray crystallography, and mutated for functional analysis in HeLa cells. In summary, our results reveal the role of a phosphorylation-dependent HEF1-Plk1 complex in HEF1 translocation to FAs to induce cell migration. Our findings provide critical mechanistic insights into the HEF1-Plk1 complex-dependent localization of HEF1 to FAs underlying the metastatic process and may therefore contribute to the development of new cancer therapies.

Inhibitory effects of flavonoids isolated from Sophora flavescens on indoleamine 2,3-dioxygenase 1 activity.

Indoleamine 2,3-dioxygenase 1 (IDO1), a tryptophan catabolising enzyme, is known as a tumour cell survival factor that causes immune escape in several types of cancer. Flavonoids of Sophora flavescens have a variety of biological benefits for humans; however, cancer immunotherapy effect has not been fully investigated. The flavonoids (1-6) isolated from S. flavescens showed IDO1 inhibitory activities (IC50 4.3-31.4 µM). The representative flavonoids (4-6) of S. flavescens were determined to be non-competitive inhibitors of IDO1 by kinetic analyses. Their binding affinity to IDO1 was confirmed using thermal stability and surface plasmon resonance (SPR) assays. The molecular docking analysis and mutagenesis assay revealed the structural details of the interactions between the flavonoids (1-6) and IDO1. These results suggest that the flavonoids (1-6) of S. flavescens, especially kushenol E (6), as IDO1 inhibitors might be useful in the development of immunotherapeutic agents against cancers.

Ulleungdin, a Lasso Peptide with Cancer Cell Migration Inhibitory Activity Discovered by the Genome Mining Approach.

The advances of genomic sequence analyses and genome mining tools have enabled the exploration of untapped microbial natural products. Through genome mining studies to discover cryptic natural products, we found biosynthetic genes encoding a new lasso peptide in the genome sequence of a soil bacterium, Streptomyces sp. KCB13F003 isolated from Ulleung Island (a small volcanic island), Korea. The production and purification of the encoded peptide, named ulleungdin, were achieved by optimizing the culture conditions followed by LC-MS-targeted isolation. Structure elucidation was performed by NMR spectroscopic and MS spectrometric analyses and chemical means (Marfey's and GITC derivatizations), proving ulleungdin to be a new 15-mer class II lasso peptide with a threaded structure. Biological evaluation with the cell invasion assay and time-lapse cell tracking analysis revealed that ulleungdin has significant inhibitory activities against cancer cell invasion and migration.

Aturanosides A and B, Glycosylated Anthraquinones with Antiangiogenic Activity from a Soil-Derived Streptomyces Species.

A chemical investigation of a culture extract from a soil-derived Streptomyces sp. RK88-1441 led to the isolation and characterization of two new glycosylated anthraquinones, aturanosides A (1) and B (2), and a new anthraquinone derivative, aturanocin (3). The structures of these compounds were elucidated by detailed NMR and MS spectroscopic analyses. The absolute configurations of the sugar units, based on the magnitudes of the coupling constants, ROESY correlations, and chemical derivatization, from 1 and 2 are 6- O-[ N-acetyl-α-d-glucosamino-(1→2)-α-l-rhamnoside] and 6- O-α-l-rhamnoside, respectively. Compounds 1 and 2 showed no cytotoxicity against human umbilical vein endothelial cells (HUVECs), but significantly suppressed vascular endothelial growth factor (VEGF)-induced tube formation and invasion of HUVECs. The down-regulation of both the phosphorylation of VEGF receptor 2 and the expression of vascular endothelial cadherin at the protein level were also observed.