Repeated evolution of the same enzymatic activity represents a powerful model of determinism across diverse lineages, yet its underlying evolutionary trajectories remain poorly understood. Here we identified three 3-oxo-glutaric acid (OGA)-forming type III polyketide synthases (PKSs) from Brassicaceae plants through in vitro enzyme assays. Structure–function analysis combined with comparative genomics indicates a strong correlation between OGA- and triacetic acid lactone (TAL)-forming activities during the evolution of these PKSs. An evolutionary change in a key active-site residue enables a functional switch from TAL-forming activity to OGA-forming activity. Intriguingly, further chalcone synthase activity assays and expanded exploration of PKSs from additional plant lineages suggest that OGA-forming PKSs may have evolved repeatedly from the intrinsic TAL-forming activity of chalcone synthases in a lineage-specific manner. This evolutionary feature may help explain the recurrent emergence of OGA-forming PKSs in vascular plants and provide insight into the trajectories underlying the independent evolution of homologous isoenzymes in plants. This study suggests that 3-oxo-glutaric acid-forming polyketide synthases evolved repeatedly from the intrinsic triacetic acid lactone-forming activity of chalcone synthases through independent, lineage-specific evolutionary events.

ASH2L is a core component of KMT2 complexes, crucial for H3K4 trimethylation. However, its role in spermatogenesis remains elusive. Here, we demonstrate an essential role of Ash2l for meiotic prophase but dispensable for mitosis in differentiated spermatogonia. Using a germ cell-specific Ash2l knockout mouse model, we reveal that Ash2l deficiency leads to meiotic arrest and sterility in both sexes. Ash2l-deficient spermatocytes exhibit failures in chromosomal synapsis associated with persistent DMC1 foci and γH2AX, resulting in meiocyte loss due to apoptosis. Conversely, Ash2l-deficient differentiated spermatogonia show normal development. Mechanistically, Ash2l deficiency results in a global loss of H3K4me3 in promoter regions and significantly decreases expression of thousands of genes. Among these are genes involved in epigenetic silencing pathways like H3K9 di-methylation, DNA methylation, and piRNA pathways, which are crucial for transposon repression during meiotic prophase I progression. Supporting this, we observe that Ash2l mutant spermatocytes display ectopic expression of LINE1-ORF1P. Our findings thus reveal the previously unappreciated role of ASH2L-dependent H3K4me3 modification in spermatogenesis and provide clues to the molecular mechanisms in epigenetic disorders underlying male infertility.

In situ forming biodegradable hydrogel scaffolds are crucial in tissue engineering and drug delivery systems, with extensive applications in articular cartilage (AC) repair. Polysaccharide-based hydrogels are ideal materials for cartilage tissue engineering owning to their biochemical and structural resemblance to the native extracellular matrix (ECM). In this study, a double-network (DN) hydrogel was fabricated through Schiff base reaction and photocrosslinking, utilizing glycidyl methacrylate-modified chitosan (GCS) and aldehyde-modified hyaluronic acid methacrylate (AHM) as precursors. To prepare the drug-loaded hydrogel, we grafted the bioactive factor kartogenin (KGN) onto GCS, generating KGN-conjugated GCS (KGCS), which subsequently formed a crosslinked network with AHM. In contrast to conventional hydrogels encapsulating either drug alone or drug-loaded microspheres, the proposed hydrogel demonstrated a more stable and sustained release profile. Furthermore, the physicochemical properties of both hydrogels were systematically characterized, comprising morphology, mechanical properties and swelling rate. The biocompatibility of hydrogels was comprehensively investigated through in vitro cell proliferation assays, hemocompatibility analysis, and in vivo evaluation of subcutaneous implant. Subsequently, a cartilage defect model was utilized to assess their efficacy in cartilage regeneration. In summary, the two DN hydrogels exhibited suitable porous structure, mechanical properties, and swelling rates. Furthermore, they demonstrated excellent biocompatibility and significantly promoted cartilage defect regeneration.

Epigenetic programming governs cell fate determination during development through intricately controlling sequential gene activation and repression. Although H3K4me3 is widely recognized as a hallmark of gene activation, its role in modulating transcription output and timing within a continuously developing system remains poorly understood. In this study, we provide a detailed characterization of the epigenomic landscapes in developing male germ cells. We identified thousands of spermatid-specific broad H3K4me3 domains regulated by the SETD1B-RFX2 axis, representing a previously underappreciated form of H3K4me3. These domains, overlapping with H3K27ac-marked enhancers and promoters, play critical roles in orchestrating robust transcription and accurate temporal control of gene expression. Mechanistically, these broad H3K4me3 compete effectively with regular H3K4me3 for transcriptional machinery, thereby ensuring robust levels and precise timing of master gene expression in mouse spermiogenesis. Disruption of this mechanism compromises the accuracy of transcription dosage and timing, ultimately impairing spermiogenesis. Additionally, we unveil remarkable changes in the distribution of heterochromatin marks, including H3K27me3 and H3K9me2, during the mitosis-to-meiosis transition and completion of meiotic recombination, which closely correlates with gene silencing. This work underscores the highly orchestrated epigenetic regulation in spermatogenesis, highlighting the previously unrecognized role of Setd1b in the formation of broad H3K4me3 domains and transcriptional control, and provides an invaluable resource for future studies toward the elucidation of spermatogenesis.

The eukaryotic translation inhibitor cycloheximide (CHX) and its analogue actiphenol (APN) feature a glutarimide moiety and a six-membered carbocyclic ring system. The biosynthesis of CHX and APN is not yet fully understood, particularly with respect to the mechanism of formation of the fully reduced cyclohexanone ring in CHX and the aromatic phenol ring in APN. In this work, a combination of gene inactivation, chemical synthesis, and in vitro biochemical experiments highlighted an ensemble of three tailoring redox enzymes as being responsible for the biosynthesis of the six-membered carbocyclic ring systems. Specifically, two redox enzymes (ChxJ and ChxI) alone can generate an active intermediate that undergoes a cascade of non-enzymatic transformations to create APN, while a reductive enzyme (ChxG) acts as a gatekeeper, directing the same intermediate down a different pathway toward CHX. Finally, the full nature of each biosynthetic pathway was established in detail, including the formation mechanisms of six-membered carbocyclic rings.

The conserved MRE11-RAD50-NBS1/Xrs2 complex is crucial for DNA break metabolism and genome maintenance. Although hypomorphic Rad50 mutation mice showed normal meiosis, both null and hypomorphic rad50 mutation yeast displayed impaired meiosis recombination. However, the in vivo function of Rad50 in mammalian germ cells, particularly its in vivo role in the resection of meiotic double strand break (DSB) ends at the molecular level remains elusive. Here, we have established germ cell-specific Rad50 knockout mouse models to determine the role of Rad50 in mitosis and meiosis of mammalian germ cells. We find that Rad50-deficient spermatocytes exhibit defective meiotic recombination and abnormal synapsis. Mechanistically, using END-seq, we demonstrate reduced DSB formation and abnormal DSB end resection occurs in mutant spermatocytes. We further identify that deletion of Rad50 in gonocytes leads to complete loss of spermatogonial stem cells due to genotoxic stress. Taken together, our results reveal the essential role of Rad50 in mammalian germ cell meiosis and mitosis, and provide in vivo views of RAD50 function in meiotic DSB formation and end resection at the molecular level.

Background: Long-chain non-coding RNA (LINC00261) in the treatment of papillary thyroid carcinoma (PTC) with 131I is still unknown despite its proven anti-tumour effect in thyroid cancer (TC) and other types of cancer. Methods: The database and RT-qPCR were used to analyze the expression level of LINC00261 in PTC and cell lines. PTC cells resistant to 131I (TPC-1/R) were created through ongoing exposure to a lethal dose of 131I, and a subcutaneous xenotransplantation model was developed using PTC mice. Bioinformatics analysis and dual-luciferase assays demonstrated the interaction between LINC00261, miR-23a-3p, and CELF2. RT-qPCR and Western blot were used to detect the expression of LINC00261, miR-23a-3p, and CELF2. Additionally, CCK-8, flow cytometry, immunofluorescence (IF), Western blot, and comet assay were employed to measure cell viability level and DNA damage. Results: PTC cell lines exhibited a decrease in the expression of LINC00261. The growth and progression through the S-phase of TPC-1/R cells were suppressed by LINC00261, leading to increased apoptosis and DNA damage. The objective of LINC00261 was to regulate the axis of miR-23a-3p/CELF2. Downregulating LINC00261 enhances the growth and advancement of 131I-resistant cells in the S-phase by activating the miR-23a-3p/CELF2 pathway while suppressing cell death and DNA harm. The miR-23a-3p/CELF2 axis activates DNA damage in 131I-resistant PTC cells by LINC00261. Conclusions: LINC00261 activates DNA damage in 131I-resistant PTC cells caused by miR-23a-3p/CELF2 axis, improving the progression of cancer cells of PTC.

Angiotensin-converting enzyme inhibitors are widely used for treatment of hypertension and related diseases. Here, six karnamicins E 1 -E 6 ( 1 – 6 ), which bear fully substituted hydroxypyridine and thiazole moieties are characterized from the rare actinobacterium Lechevalieria rhizosphaerae NEAU-A2. Through a combination of isotopic labeling, genome mining, and enzymatic characterization studies, the programmed assembly of the fully substituted hydroxypyridine moiety in karnamicin is proposed to be due to sequential operation of a hybrid polyketide synthase-nonribosomal peptide synthetase, two regioselective pyridine ring flavoprotein hydroxylases, and a methyltransferase. Based on AlphaFold protein structures predictions, molecular docking, and site-directed mutagenesis, we find that two pyridine hydroxylases deploy active site residues distinct from other flavoprotein monooxygenases to direct the chemo- and regioselective hydroxylation of the pyridine nucleus. Pleasingly, karnamicins show significant angiotensin-converting enzyme inhibitory activity with IC 50 values ranging from 0.24 to 5.81 μM, suggesting their potential use for the treatment of hypertension and related diseases.

Six new azoxy-aromatic compounds (o-alkylazoxymycins A-F, 1-6) and two new nitrogen-bearing phenylvaleric/phenylheptanoic acid derivatives (o-alkylphemycins A and B, 7 and 8) were isolated from Streptomyces sp. Py50. Their structures were elucidated based on HRESIMS, NMR, UV spectroscopic analyses, and X-ray crystallographic data. O-Alkylazoxymycins A-F (1-6) are the first natural examples of azoxy compounds with the azoxy bond attached to the ortho-position of the phenylheptanoic acid or phenylvaleric acid moiety. Compounds 1, 5, and 6 were active against Epidermophyton floccosum with MIC50 values ranging from 10.1 to 51.2 μM. A plausible biosynthetic pathway of 2 and 3 was proposed.

Quinolizidomycins A (1) and B (2), two unprecedented quinolizidine alkaloids featuring a tricyclic 6/6/5 ring system, were isolated from Streptomyces sp. KIB-1714. Their structures were assigned by detailed spectroscopic data analyses and X-ray diffraction. Stable isotope labeling experiments suggested that compounds 1 and 2 are derived from lysine, ribose 5-phosphate, and acetate units, which indicates an unprecedented manner of assembly of the quinolizidine (1-azabicyclo[4.4.0]decane) scaffold in quinolizidomycin biosynthesis. Quinolizidomycin A (1) was active in an acetylcholinesterase inhibitory assay.

Trichotherosin A (1), a new trichothecene precursor derivative, along with six known trichothecene mycotoxins (2-7), was isolated from Trichothecium roseum KIB-093, a plant pathogenic fungus associated with a rotten moldy apple core. Their structures were elucidated based on their HRMS (high-resolution mass spectrometry) and NMR (nuclear magnetic resonance) data as well as theoretical calculations. Trichothecene mycotoxins are widely recognized for their potent toxicity. Compounds 1-7 were all active in a cytotoxicity screening, with no selectivity toward either normal human cell lines or human cancer cell lines. Among them, 2 was the most cytotoxic, with the smallest IC50 value being 33 nM. T. roseum is recognized as one of the main pathogenic fungi causing apple core rot. It is of great possibility for infected apples to be utilized in the apple-based beverage industry. According to the cytotoxicity results, qualitative determination of compounds 2-7, the isolated members characterized by a 12,13-epoxytrichothec-9-ene (EPT) core skeleton with certain cytotoxic effects, was performed using HPLC-HR-ESI-Q-TOF-MS (high-performance liquid chromatography-high resolution-electrospray ionization-quadrupole-time-of-flight-mass spectrometry) in 10 different commercial apple juices. All compounds 2-7 were detectable, with the detection rates ranging from 13.33 to 80%, indicating the potential contamination of trichothecene mycotoxins in certain apple juices, which could pose health risks to consumers.

Guvermectin is a novel plant growth regulator that has been registered as a new agrochemical in China. It is an adenosine analogue with an unusual psicofuranose instead of ribose. Herein, the gene cluster responsible for guvermectin biosynthesis in Streptomyces caniferus NEAU6 is identified using gene interruption and heterologous expression experiments. A key intermediate psicofuranine 6'-phosphate (PMP) is chemically synthesized, and the functions of GvmB, C, D, and E are verified by individual stepwise enzyme reactions in vitro. The results also show that the biosynthesis of guvermectin is coupled with adenosine production by a single cluster. The higher catalytic efficiency of GvmB on PMP than AMP ensures the effective biosynthesis of guvermectin. Moreover, a phosphoribohydrolase GvmA is employed in the pathway that can hydrolyze AMP but not PMP and shows higher catalytic efficiency for the AMP hydrolysis than that of the AMP dephosphorylation by GvmB, leading to shunting of adenosine biosynthesis toward the production of guvermectin. Finally, the crystal structure of GvmE in complex with the product PMP has been solved. Glu160 at the C-terminal is identified as the acid/base for protonation/deprotonation of N7 of the adenine ring, demonstrating that GvmE is a noncanonical adenine phosphoribosyltransferase.

During a screening for antifungal secondary metabolites, six new mono-/bis-alkenoic acid derivatives ( 2 – 7 ) and one known alkenoic acid derivative ( 1 ) were isolated from an endophytic fungi Scopulariopsis candelabrum . Their chemical structures were identified by 1 H-NMR, 13 C-NMR, 2D NMR, and high-resolution mass spectrometry, as well as comparisons with previously reported literatures. Among them, fusariumesters C‒F ( 2 – 5 ) are bis-alkenoic acid derivatives dimerized by an ester bond, while acetylfusaridioic acid A ( 6 ) and fusaridioic acid D ( 7 ) are alkenoic acid monomers. All the isolates were submitted to an antifungal assay against Candida albicans and the corn pathogen Exserohilum turcicum using the filter paper agar diffusion method. As a result, only compound 1 decorating with β -lactone ring turned out to be active against these two tested fungi. The broth microdilution assay against Candida albicans showed the minimum inhibitory concentration (MIC) value of 1 to be 20 μ g/ml, while the minimum inhibitory concentration value of the positive control (naystatin) was 10 μ g/ml. And the half maximal inhibitory concentration (IC 50 ) value (21.23 μ g/ml) of 1 against Exserohilum turcicum was determined by analyzing its inhibition effect on the mycelial growth, using cycloheximide (IC 50 = 46.70 μ g/ml) as the positive control.

Tropane alkaloids such as hyoscyamine and cocaine are of importance in medicinal uses. Only recently has the hyoscyamine biosynthetic machinery become complete. However, the cocaine biosynthesis pathway remains only partially elucidated. Here we characterize polyketide synthases required for generating 3-oxo-glutaric acid from malonyl-CoA in cocaine biosynthetic route. Structural analysis shows that these two polyketide synthases adopt distinctly different active site architecture to catalyze the same reaction as pyrrolidine ketide synthase in hyoscyamine biosynthesis, revealing an unusual parallel/convergent evolution of biochemical function in homologous enzymes. Further phylogenetic analysis suggests lineage-specific acquisition of polyketide synthases required for tropane alkaloid biosynthesis in Erythroxylaceae and Solanaceae species, respectively. Overall, our work elucidates not only a key unknown step in cocaine biosynthesis pathway but also, more importantly, structural and biochemical basis for independent recruitment of polyketide synthases in tropane alkaloid biosynthesis, thus broadening the understanding of conservation and innovation of biosynthetic catalysts.

Flavin-dependent enzymes enable a broad range of redox transformations and generally act as monofunctional and stereoselective catalysts. Herein, we report the investigation of a multifunctional and non-stereoselective FMN-dependent oxidoreductase RubE7 from the rubrolone biosynthetic pathway. Our study outlines a single RubE7-catalysed sequential reduction of three spatially distinct bonds in a tropolone ring and a reversible double-bond reduction and dehydrogenation. The crystal structure of IstO (a RubE7 homologue) with 2.0 Å resolution reveals the location of the active site at the interface of two monomers, and the size of active site is large enough to permit both flipping and free rotation of the substrate, resulting in multiple nonselective reduction reactions. Molecular docking and site mutation studies demonstrate that His106 is oriented towards the substrate and is important for the reverse dehydrogenation reaction.

This study used light-mediated comparative transcriptomics to identify the biosynthetic gene cluster of beticolin 1 in Cercospora. It contains an anthraquinone moiety and an unusual halogenated xanthone moiety connected by a bicyclo[3.2.2]nonane. During elucidation of the biosynthetic pathway of beticolin 1, a novel non-heme iron oxygenase BTG13 responsible for anthraquinone ring cleavage was discovered. More importantly, the discovery of non-heme iron oxygenase BTG13 is well supported by experimental evidence: (i) crystal structure and the inductively coupled plasma mass spectrometry revealed that its reactive site is built by an atypical iron ion coordination, where the iron ion is uncommonly coordinated by four histidine residues, an unusual carboxylated-lysine (Kcx377) and water; (ii) Kcx377 is mediated by His58 and Thr299 to modulate the catalytic activity of BTG13. Therefore, we believed this study updates our knowledge of metalloenzymes.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.

Two novel diarylcyclopentenones daturamycin A and B (1 and 2), and one new p-terphenyl daturamycin C (3), along with three known congeners (4-6), were isolated from a rhizosphere soil-derived Streptomyces sp. KIB-H1544. The structures of new com-pounds were elucidated via a joint use of spectroscopic analyses and single-crystal X-ray diffractions. Compounds 1 and 2 belong to a rare class of tricyclic 6/5/6 diarylcyclopentenones, and compounds 3-6 possess a C-18 tricyclic aromatic skeleton. The biosyn-thetic gene cluster of daturamycins was identified through gene knockout and biochemical characterization experiments and the biosynthetic pathway of daturamycins was proposed.

Dichroa febrifuga has been used in traditional Chinese medicine for centuries to treat productive cough and fevers caused by malaria infection, however the antimicrobial activity is still unknown. In this study, the crude alka-loids extract of D. febrifuga leaves showed potent anti-oomycete activity against phytopathogen Phytophthora capsici. Subsequent bioassay-guided fractionation led to identify febrifugine as the main active component. Febrifugine showed specific in vitro anti-oomycete activity against P. capsici, Phytophthora sojae and Phytophthora infestans with EC50 of 1.741, 1.674 and 0.413 mu g/mL, respectively, but showed no or weak antifungal activity against twelve test phytopathogenic fungi. Scanning electron microscope and optical microscope results indi-cated that febrifugine could alter the morphology of P. infestans hyphae, but did not destroy the cell wall. The electrical conductivity and intracellular component leakage experiments suggested the integrity of the cell membrane after the treatment of febrifugine. Moreover, febrifugine possessed inhibitory effects on zoospore release and germination of P. infestans. In vivo bioassay using detached leaves demonstrated that febrifugine had better protective and curing activities than commercialized drug metalaxyl. The application of febrifugine at a dose of 60 mu g/mL provided protective efficacies of 93.1% and 91.9%, and curing efficacies of 86.2% and 86.6% for pepper phytophthora blight and potato late blight, respectively. Taken together, this study showed that the crude alkaloids extract of D. febrifuga and its active component febrifugine represent alternatives to control plant diseases caused by oomycetes, and febrifugine is expected to be a new leading structure for the development of oomyceticides.

Objective: For exploring the influence of PD-L1 inhibitors on Thyroid carcinoma(TC) and the related mechanism of action. Methods: Nude mice models bearing subcutaneous TC were established with 20 BALB/c-Nu nude mice, and randomized into two groups. One group was injected with PD-L1 inhibitors (treatment group), and the other group was injected with the same amount of normal saline (model group). During the intervention period, the subcutaneous tumour volume and inflammatory factors of the two groups were measured. After the fourth injection, all rats were killed, and their tumours were weighed. In addition, TC cells were purchased, followed by quantification of their PD-1 and PD-L1 proteins. The growing ability and apoptosis of the TC cells intervened with PD-L1 inhibitor were evaluated. Results: After injection of PD-L1 inhibitors, TC-bearing nude mice showed notable decreases in tumour volume and weight and an increase in tumour inhibition rate (all P < 0.05). The treatment group presented lower levels of inflammatory factors and higher levels of survival rate and survival time than the model group (all P < 0.05). In in vitro experiment, TC cells showed higher PD-1 and PD-L1 proteins than normal thyroid epithelial cells (P < 0.05), and intervention with PD-L1 inhibitors inhibited cell growing ability and accelerated cell apoptosis (both P < 0.05). Conclusion: PD-L1 inhibitors can improve the development of subcutaneous tumour in tumour-bearing nude mice through accelerating the apoptosis of TC cells and suppressing their growth, which lays a foundation for the future clinical application of PDL1 inhibitors in the therapy of TC.