Discovery, Total Synthesis, and Anti-inflammatory Evaluation of Naturally Occurring Naphthopyrone-Macrolide Hybrids as Potent NLRP3 Inflammasome Inhibitors.

Numerous clinical disorders have been linked to the etiology of dysregulated NLRP3 (NACHT, LRR, and PYD domain-containing protein 3) inflammasome activation. Despite its potential as a pharmacological target, modulation of NLRP3 activity remains challenging. Only a sparse number of compounds have been reported that can modulate NLRP3 and none of them have been developed into a commercially available drug. In this research, we identified three potent NLRP3 inflammasome inhibitors, gymnoasins A-C (1-3), with unprecedented pentacyclic scaffolds, from an Antarctic fungus Pseudogymnoascus sp. HDN17-895, which represent the first naturally occurring naphthopyrone-macrolide hybrids. Additionally, biomimetic synthesis of gymnoasin A (1) was also achieved validating the chemical structure and affording ample amounts of material for exhaustive bioactivity assessments. Biological assays indicated that 1 could significantly inhibited in vitro NLRP3 inflammasome activation and in vivo pro-inflammatory cytokine IL-1ß release, representing a valuable new lead compound for the development of novel therapeutics with the potential to inhibit the NLRP3 inflammasome.

Dissecting the association between gut microbiota and hypertrophic scarring: a bidirectional Mendelian randomization study.

Hypertrophic scars affect a significant number of individuals annually, giving rise to both cosmetic concerns and functional impairments. Prior research has established that an imbalance in the composition of gut microbes, termed microbial dysbiosis, can initiate the progression of various diseases through the intricate interplay between gut microbiota and the host. However, the precise nature of the causal link between gut microbiota and hypertrophic scarring remains uncertain. In this study, after compiling summary data from genome-wide association studies (GWAS) involving 418 instances of gut microbiota and hypertrophic scarring, we conducted a bidirectional Mendelian randomization (MR) to investigate the potential existence of a causal relationship between gut microbiota and the development of hypertrophic scar and to discern the directionality of causation. By utilizing MR analysis, we identified seven causal associations between gut microbiome and hypertrophic scarring, involving one positive and six negative causal directions. Among them, Intestinimonas, Ruminococcus2, Barnesiella, Dorea, Desulfovibrio piger, and Ruminococcus torques act as protective factors against hypertrophic scarring, while Eubacterium rectale suggests a potential role as a risk factor for hypertrophic scars. Additionally, sensitivity analyses of these results revealed no indications of heterogeneity or pleiotropy. The findings of our MR study suggest a potential causative link between gut microbiota and hypertrophic scarring, opening up new ways for future mechanistic research and the exploration of nanobiotechnology therapies for skin disorders.

Genome Mining Reveals a UbiA-Type Prenyltransferase Access to Farnesylation of Diketopiperazines.

UbiA-type prenyltransferases (PTases) are significant enzymes that lead to structurally diverse meroterpenoids. Herein, we report the identification and characterization of an undescribed UbiA-type PTase, FtaB, that is responsible for the farnesylation of indole-containing diketopiperazines (DKPs) through genome mining. Heterologous expression of the fta gene cluster and non-native pathways result in the production of a series of new C2-farnesylated DKPs. This study broadens the reaction scope of UbiA-type PTases and expands the chemical diversity of meroterpenoids.

Exploring the Diverse Landscape of Fungal Cytochrome P450-Catalyzed Regio- and Stereoselective Dimerization of Diketopiperazines.

Dimeric indole-containing diketopiperazines (di-DKPs) are a diverse group of natural products produced through cytochrome P450-catalyzed C-C or C-N coupling reactions. The regio- and stereoselectivity of these reactions plays a significant role in the structural diversity of di-DKPs. Despite their pivotal role, the mechanisms governing the selectivity in fungi are not fully understood. Employing bioinformatics analysis and heterologous expression experiments, five undescribed P450 enzymes (AmiP450, AcrP450, AtP450, AcP450, and AtuP450) responsible for the regio- and stereoselective dimerization of diketopiperazines (DKPs) in fungi are identified. The function of these P450s is consistent with phylogenetic analysis, highlighting their dominant role in controlling the dimerization modes. Combinatorial biosynthesis-based pathway reconstitution of non-native gene clusters expands the chemical space of fungal di-DKPs and reveals that the regioselectivity is influenced by the substrate. Furthermore, multiple sequence alignment and molecular docking of these enzymes demonstrate a C-terminal variable region near the substrate tunnel entrance in AtuP450 that is crucial for its regioselectivity. These findings not only reveal the secret of fungal di-DKPs diversity but also deepen understanding of the mechanisms and catalytic specificity involved in P450-catalyzed dimerization reactions.

Phenylhydrazone Alkaloids from the Deep-Sea Cold Seep Derived Fungus Talaromyces amestolkiae HDN21-0307.

Alkaloids with a phenylhydrazone architecture are rarely found in nature. Four unusual phenylhydrazone alkaloids named talarohydrazones A-D (1-4) were isolated from the deep-sea cold seep derived fungus Talaromyces amestolkiae HDN21-0307 using the one strain-many compounds (OSMAC) approach and MS/MS-based molecular networking (MN) combined with network annotation propagation (NAP) and the unsupervised substructure annotation method MS2LDA. Their structures were elucidated by spectroscopic data analysis, single-crystal X-ray diffraction, and quantum chemical calculations. Talarohydrazone A (1) possessed an unusual skeleton combining 2,4-pyridinedione and phenylhydrazone. Talarohydrazone B (2) represents the first natural phenylhydrazone-bearing azadophilone. Bioactivity evaluation revealed that compound 1 exhibited cytotoxic activity against NCI-H446 cells with an IC50 value of 4.1 µM. In addition, compound 1 displayed weak antibacterial activity toward Staphylococcus aureus with an MIC value of 32 µg/mL.

Antiviral and cytotoxic indole diterpenoids from the antarctic sponge-derived fungus Aspergillus candidus HDN15-152.

One new indole diterpenoid, ascandinine T (1), and three known analogues (2-4) were isolated from an Antarctic sponge-derived fungus Aspergillus candidus HDN15-152. The structures, including absolute configurations, were established based on NMR, HRESIMS, and electronic circular dichroism (ECD) calculations. All isolated compounds were tested for antiviral and anticancer activity. Compound 4 displayed antiviral activity against influenza A virus (IAV) of A/PR/8/34(H1N1) strain with an IC50 value of 39.2 µM, while compound 2 showed cytotoxicity against NCI-H446, NCI-H446/EP and L-02 cells with IC50 values ranging from 9.77 to 13.91 µM.

Evolution-Based Discovery of Polyketide Acylated Valine from a Cytochalasin-Like Gene Cluster in Simplicillium lamelliciola HDN13430.

Utilizing a gene evolution-oriented approach for gene cluster mining, a cryptic cytochalasin-like gene cluster (sla) in Antarctic-derived Simplicillium lamelliciola HDN13430 was identified. Compared with the canonical cytochalasin biosynthetic gene clusters (BGCs), the sla gene cluster lacks the key α,ß-hydrolase gene. Heterologous expression of the sla gene cluster led to the discovery of a new compound, slamysin (1), characterized by an N-acylated amino acid structure and demonstrating weak anti-Bacillus cereus activity. These findings underscore the potential of genetic evolution in uncovering novel compounds and indicating specific adaptive evolution within specialized habitats.

Cyclo-diphenylalanine production in Aspergillus nidulans through stepwise metabolic engineering.

Cyclo-diphenylalanine (cFF) is a symmetrical aromatic diketopiperazine (DKP) found wide-spread in microbes, plants, and resulting food products. As different bioactivities continue being discovered and relevant food and pharmaceutical applications gradually emerge for cFF, there is a growing need for establishing convenient and efficient methods to access this type of compound. Here, we present a robust cFF production system which entailed stepwise engineering of the filamentous fungal strain Aspergillus nidulans A1145 as a heterologous expression host. We first established a preliminary cFF producing strain by introducing the heterologous nonribosomal peptide synthetase (NRPS) gene penP1 to A. nidulans A1145. Key metabolic pathways involving shikimate and aromatic amino acid biosynthetic support were then engineered through a combination of gene deletions of competitive pathway steps, over-expressing feedback-insensitive enzymes in phenylalanine biosynthesis, and introducing a phosphoketolase-based pathway, which diverted glycolytic flux toward the formation of erythrose 4-phosphate (E4P). Through the stepwise engineering of A. nidulans A1145 outlined above, involving both heterologous pathway addition and native pathway metabolic engineering, we were able to produce cFF with titers reaching 611 mg/L in shake flask culture and 2.5 g/L in bench-scale fed-batch bioreactor culture. Our study establishes a production platform for cFF biosynthesis and successfully demonstrates engineering of phenylalanine derived diketopiperazines in a filamentous fungal host.

Highly Bioactive MXene-M2-Exosome Nanocomposites Promote Angiogenic Diabetic Wound Repair through Reconstructing High Glucose-Derived Immune Inhibition.

The repair of diabetic wounds remains challenging, primarily due to the high-glucose-derived immune inhibition which often leads to the excessive inflammatory response, impaired angiogenesis, and heightened susceptibility to infection. However, the means to reduce the immunosuppression and regulate the conversion of M2 phenotype macrophages under a high-glucose microenvironment using advanced biomaterials for diabetic wounds are not yet fully understood. Herein, we report two-dimensional carbide (MXene)-M2 macrophage exosome (Exo) nanohybrids (FM-Exo) for promoting diabetic wound repair by overcoming the high-glucose-derived immune inhibition. FM-Exo showed the sustained release of M2 macrophage-derived exosomes (M2-Exo) up to 7 days and exhibited broad-spectrum antibacterial activity. In the high-glucose microenvironment, relative to the single Exo, FM-Exo could significantly induce the optimized M2a/M2c polarization ratio of macrophages by activating the PI3K/Akt signaling pathway, promoting the proliferation, migration of fibroblasts, and angiogenic ability of endothelial cells. In the diabetic full-thickness wound model, FM-Exo effectively regulated the polarization status of macrophages and promoted their transition to the M2 phenotype, thereby inhibiting inflammation, promoting angiogenesis through VEGF secretion, and improving proper collagen deposition. As a result, the healing process was accelerated, leading to a better healing outcome with reduced scarring. Therefore, this study introduced a promising approach to address diabetic wounds by developing bioactive nanomaterials to regulate immune inhibition in a high-glucose environment.

Extending the Structural Diversity of Labdane Diterpenoids from Marine-Derived Fungus Talaromyces sp. HDN151403 Using Heterologous Expression.

Heterologous biosynthesis has become an effective means to activate fungal silent biosynthetic gene clusters (BGCs) and efficiently utilize fungal genetic resources. Herein, thirteen labdane diterpene derivatives, including five undescribed ones named talarobicins A-E (3-7), were discovered via heterologous expression of a silent BGC (labd) in Aspergillus nidulans. Their structures with absolute configurations were elucidated using extensive MS and NMR spectroscopic methods, as well as electronic circular dichroism (ECD) calculations. These labdanes belong to four skeleton types, and talarobicin B (4) is the first 3,18-dinor-2,3:4,18-diseco-labdane diterpene with the cleavage of the C2-C3 bond in ring A and the decarboxylation at C-3 and C-18. Talarobicin B (4) represents the key intermediate in the biosynthesis of penioxalicin and compound 13. The combinatorial heterologous expression and feeding experiments revealed that the cytochrome P450 enzymes LabdC, LabdE, and LabdF were responsible for catalyzing various chemical reactions, such as oxidation, decarboxylation, and methylation. All of the compounds are noncytotoxic, and compounds 2 and 8 displayed inhibitory effects against methicillin-resistant coagulase-negative staphylococci (MRCNS) and Bacillus cereus.

A thermostable type I-B CRISPR-Cas system for orthogonal and multiplexed genetic engineering.

Thermophilic cell factories have remarkably broad potential for industrial applications, but are limited by a lack of genetic manipulation tools and recalcitrance to transformation. Here, we identify a thermophilic type I-B CRISPR-Cas system from Parageobacillus thermoglucosidasius and find it displays highly efficient transcriptional repression or DNA cleavage activity that can be switched by adjusting crRNA length to less than or greater than 26 bp, respectively, without ablating Cas3 nuclease. We then develop an orthogonal tool for genome editing and transcriptional repression using this type I-B system in both thermophile and mesophile hosts. Empowered by this tool, we design a strategy to screen the genome-scale targets involved in transformation efficiency and established dynamically controlled supercompetent P. thermoglucosidasius cells with high efficiency ( ~ 108 CFU/µg DNA) by temporal multiplexed repression. We also demonstrate the construction of thermophilic riboflavin cell factory with hitherto highest titers in high temperature fermentation by genome-scale identification and combinatorial manipulation of multiple targets. This work enables diverse high-efficiency genetic manipulation in P. thermoglucosidasius and facilitates the engineering of thermophilic cell factories.

Plastoquinone synthesis inhibition by tetrabromo biphenyldiol as a widespread algicidal mechanism of marine bacteria.

Algae and bacteria have complex and intimate interactions in the ocean. Besides mutualism, bacteria have evolved a variety of molecular-based anti-algal strategies. However, limited by the unknown mechanism of synthesis and action of these molecules, these strategies and their global prevalence remain unknown. Here we identify a novel strategy through which a marine representative of the Gammaproteobacteria produced 3,3',5,5'-tetrabromo-2,2'-biphenyldiol (4-BP), that kills or inhibits diverse phytoplankton by inhibiting plastoquinone synthesis and its effect cascades to many other key metabolic processes of the algae. Through comparative genomic analysis between the 4-BP-producing bacterium and its algicidally inactive mutant, combined with gene function verification, we identified the gene cluster responsible for 4-BP synthesis, which contains genes encoding chorismate lyase, flavin-dependent halogenase and cytochrome P450. We demonstrated that in near in situ simulated algal blooming seawater, even low concentrations of 4-BP can cause changes in overall phytoplankton community structure with a decline in dinoflagellates and diatoms. Further analyses of the gene sequences from the Tara Oceans expeditions and 2750 whole genome sequences confirmed the ubiquitous presence of 4-BP synthetic genes in diverse bacterial members in the global ocean, suggesting that it is a bacterial tool potentially widely used in global oceans to mediate bacteria-algae antagonistic relationships.

Activation of a Silent Polyketide Synthase SlPKS4 Encoding the C7-Methylated Isocoumarin in a Marine-Derived Fungus Simplicillium lamellicola HDN13-430.

Coumarins, isocoumarins and their derivatives are polyketides abundant in fungal metabolites. Although they were first discovered over 50 years ago, the biosynthetic process is still not entirely understood. Herein, we report the activation of a silent nonreducing polyketide synthase that encodes a C7-methylated isocoumarin, similanpyrone B (1), in a marine-derived fungus Simplicillium lamellicola HDN13-430 by heterologous expression. Feeding studies revealed the host enzymes can change 1 into its hydroxylated derivatives pestapyrone A (2). Compounds 1 and 2 showed moderate radical scavenging activities with ED50 values of 67.4 µM and 104.2 µM. Our discovery fills the gap in the enzymatic elucidation of naturally occurring C7-methylated isocoumarin derivatives.

[Development of highly efficient electrocompetent cells for electroporation of Geobacillus thermoglucosidasius NCIMB 11955].

Geobacillus thermoglucosidasius is a kind of Gram-positive facultative anaerobic bacteria. The fast growth rate under high temperature and less susceptibility to microbial contamination enable G. thermoglucosidasius to be a desirable producer of biofuels and high-value-added chemicals for the next-generation industrial biotechnology. However, compared with the classical model strain Escherichia coli, the applications of G. thermoglucosidasius are hampered by its low transformation efficiency. This study aimed at obtaining competent cells with high transformation efficiency through inactivating restriction enzymes, adding cell membrane inhibitors and cell wall weakening agents. The results showed that the electro-transformation efficiency achieved 1.2×104 CFU/(µg DNA) by knocking out four genes encoding restriction enzymes. Adding a certain amount of tween 80, dl-threonine and glycine further increased the competent efficiency about 22.5, 44, and 334 times, respectively. The electro-transformation efficiency was enhanced to 4.6×106 CFU/(µg DNA) under the optimized conditions, laying a foundation for genetic manipulation and metabolic engineering of G. thermoglucosidasius.

New cyclic dipeptide discovered from deep-sea derived Aspergillus sp. HDN20-1401.

A new alkaloid named aspergilalkaloid A (1) with pyridoindole hydroxymethyl piperazine dione structure along with six known compounds 2-7 were isolated from deep-sea derived fungus Aspergillus sp. HDN20-1401. The structure including absolute configuration was elucidated by extensive NMR analyses, HRESIMS, ECD calculation, and theoretical NMR calculation with DP4+ analysis. All isolated compounds were tested for antimicrobial and anticancer activity. Aspergilalkaloid A (1) showed inhibitive activity against Bacillus cereus with MIC value of 12.5 µM and weak activity against MRCNS.

Nuciferine improves random skin flap survival via TFEB-mediated activation of autophagy-lysosomal pathway.

Due to their simplicity and reliability, random-pattern skin flaps are commonly utilized in surgical reconstruction to repair cutaneous wounds. However, the post-operative necrosis frequently happens because of the ischemia and high-level of oxidative stress of random skin flaps, which can severely affect the healing outcomes. Earlier evidence has shown promising effect of Nuciferine (NF) on preventing hydrogen peroxide (H2O2)-induced fibroblast senescence and ischemic injury, however, whether it can function on promoting ischemic flap survival remains unknown. In this work, using network pharmacology analysis, it was possible to anticipate the prospective targets of NF in the context of ischemia. The results revealed that NF treatment minimized H2O2-induced cellular dysfunction of human umbilical vein endothelial cells (HUVECs), and also improved flap survival through strengthening angiogenesis and alleviating oxidative stress, inflammation and apoptosis in vivo. These outcomes should be attributed to TFEB-mediated enhancement of autophagy-lysosomal degradation via the AMPK-mTOR signaling pathway, whilst the restriction of autophagy stimulation with 3MA effectively diminished the above advantages of NF treatment. The increased nuclear translocation of TFEB not only restored lysosome function, but also promoted autophagosome-lysosome fusion, eventually restoring the inhibited autophagic flux and filling the high energy levels. The outcomes of our research can provide potent proof for the application of NF in the therapy of vascular insufficiency associated disorders, including random flaps.

Overexpression of Global Regulator SCrp Leads to the Discovery of New Angucyclines in Streptomyces sp. XS-16.

Six angucyclines including three unreported compounds (1-3) were isolated from Streptomyces sp. XS-16 by overexpressing the native global regulator of SCrp (cyclic AMP receptor). The structures were characterized based on nuclear magnetic resonance (NMR) and spectrometry analysis and assisted by electronic circular dichroism (ECD) calculations. All compounds were tested for their antitumor and antimicrobial activities, and compound 1 showed different inhibitory activities against various tumor cell lines with IC50 values ranging from 0.32 to 5.33 µM.

Microbial Dimerization and Chlorination of Isoflavones by a Takla Makan Desert-Derived Streptomyces sp. HDN154127.

Sixteen new biisoflavones, bisoflavolins A-N (1-16), were discovered from cultures of the Takla Makan desert-derived strain Streptomyces sp. HDN154127. The chemical structures, including axial chirality, were elucidated by NMR, MS, and ECD analyses. Antibacterial activity of dimerized compounds was tested against seven different bacteria. The dimerized compounds showed better activity (MIC from 0.8 to 50.0 µM) than the corresponding monomers (daidzein and genistein, MIC > 50.0 µM). The rare dimeric and chlorinated structures in 1-16 were proved to be biotransformation products obtained from soy isoflavones and sodium chloride, which constituted the culture medium. This is the first report of an actinomycete that promotes both dimerization and chlorination utilizing natural isoflavones as skeletons sources.

Study on Regional Strata Movement during Deep Mining of Erdos Coal Field and Its Control.

Disasters such as rock bursts and mine earthquakes became increasingly serious with the increase in mining depth in Erdos Coal Field and became serious problems that restrict high-strength continuous mining of coal mines. In this study, strata movement and energy polling distribution of ultrathick weak-bonding sandstone layers were controlled by the local filling−caving multi-faces coordinated mining technique, which was based on the analysis of subsidence and overlying structural characteristics in the Yingpanhao mining area. Moreover, the influencing factors and the control effect laws were investigated. Surface subsidence and energy polling distribution control effects of different mining modes were compared, which confirmed the superiority of local filling based on the main key stratum. According to the results, the maximum surface subsidence velocity of the first mining face was 1.24 mm/d, which indicates the presence of a logistic functional relationship between the mining degree and subsidence factors. When the mining degree was close to full mining, the practical surface subsidence was smaller than the corresponding logistic functional value. The largest influencing factor for the strata movement control effect of partial filling mining based on the main key stratum was the width of the caving face, followed by the filling ratio, section pillar width, and width of the filling face, successively. With respect to the influencing degree on the energy polling distribution of partial filling mining based on the main key stratum, the order followed as section pillar width > filling ratio > caving working face > width of backfilling working face. Additionally, the comparative analysis from the perspectives of control effect, resource utilization, and cost-effectiveness demonstrated that partial filling mining based on the main key stratum was one of the techniques with high cost-effectiveness in controlling strata movement and relieving rock bursts, mining earthquakes, and subsidence disasters.