In 2024, a mortality event affected farmed fourfinger threadfin (Eleutheronema tetradactylum) in Pingtung County, Taiwan, resulting in significant economic losses. Affected fish displayed anorexia and lethargy, with internal lesions characterized by multifocal white nodules. Histopathological examination confirmed systemic granulomatous disease. Two bacterial isolates, designated F5 and F69, were recovered from internal organs and identified as Francisella spp. by PCR. Experimental challenge demonstrated that strain F69 was less virulent than F5, exhibiting a higher LD50 (5.6 × 106 CFU/fish vs. 2.5 × 106 CFU/fish). Whole-genome average nucleotide identity analysis confirmed that both strains share >95% ANI with the Francisella salimarina group, classifying them within this species. Comparative genomic analysis identified two distinct polysaccharide-associated regions in strains F5 and F69. One region comprises a sugar biosynthetic gene cluster that is intact in F5 but contains several disrupted genes in strain F69. A second region encodes surface architecture characteristic of a KpsMT-dependent polysaccharide export system (kpsD, kpsE, kpsM, and kpsS), along with multiple glycosyltransferases and enzymes involved in the biosynthesis of UDP-activated amino sugars. Both strains contained a single copy of the Francisella pathogenicity island; however, key components such as pdpC were absent, and the pdpD gene was disrupted in F69. Genomic analysis identified a class A β-lactamase gene (blaOXY-2) in both strains, while F5 additionally harbored an FPH-family carbapenem-hydrolyzing class A β-lactamase. Consistent with these findings, both strains were phenotypically resistant to penicillins and cephalosporins, whereas only F5 exhibited resistance to imipenem. This study represents the first documented case of F. salimarina infection in Asia, affecting fourfinger threadfin in Taiwan, and provides important insights into its pathogenicity, antimicrobial resistance, and genomic features.

The efficacy of immune checkpoint blockade is limited in hepatocellular carcinoma (HCC) due to tumor-intrinsic mechanisms. Here we identify the deubiquitinase MINDY1 as a central regulator of metabolic-immune crosstalk that drives immunotherapy resistance in HCC. MINDY1 is significantly upregulated in immunotherapy non-responders and correlates with poor prognosis and increased M2-like macrophage infiltration. Mechanistically, MINDY1 stabilizes O-GlcNAc transferase (OGT) by removing K48-linked ubiquitin chains, thereby enhancing O-GlcNAcylation of uridine diphosphate glucose pyrophosphorylase 2 (UGP2) at T152. This modification promotes UGP2 enzymatic activity and increases uridine diphosphate glucose (UDPG) synthesis and extracellular accumulation. Tumor-derived UDPG acts as an extracellular immunometabolite that directly binds to PPARγ in macrophages, promoting its nuclear translocation and transcriptional activation. UDPG-driven PPARγ signaling enhances fatty acid oxidation and induces M2-like polarization, suppressing CD8+ T-cell cytotoxicity and establishing an immunosuppressive tumor microenvironment. Targeting MINDY1 remodels macrophage metabolism, restores CD8+ T-cell effector function, and significantly enhances anti-PD-1 efficacy in vivo. Our findings uncover a previously unrecognized mechanism that links tumor proteostasis to immune suppression and identify MINDY1 as a promising therapeutic target for overcoming immunotherapy resistance in HCC.

Aberrant N-glycosylation contributes to hepatocellular carcinoma (HCC) progression, yet the upstream regulators that coordinate nucleotide-sugar transport and site-specific N-glycosylation remain poorly characterized. Here, we identified SLC35A3, a Golgi-resident UDP-GlcNAc transporter, as a glycosylation-related tumor suppressor in HCC. SLC35A3 was downregulated in HCC tissues, and its restoration suppressed cell proliferation and tumorigenicity. Mechanistically, SLC35A3 overexpression increased Golgi-resident UDP-GlcNAc levels, indicating enhanced substrate availability for glycosyltransferases. Intact N-glycopeptide profiling demonstrated that SLC35A3 overexpression reprogrammed site-specific N-glycan processing, characterized by a predominant reduction in high-mannose (HM) glycoforms and selective remodeling of fucosylated-sialylated (FS) glycoforms. Molecular docking identified poliumoside as a putative SLC35A3 binder with anti-tumor effect in HCC models. Together, our findings establish SLC35A3 as a critical node in Golgi-dependent N-glycan remodeling and highlight its potential as a target for pharmacological activation.

Cell-free multi-enzyme cascades enable precise biosynthesis, but using them for cost-efficient bioproduction remains challenging. This issue can be addressed through model-based process optimization using kinetic models. However, the predictive power of kinetic models is often limited by structural and parametric uncertainties. In this work, we present MoRSel, a novel method that tackles structural uncertainties through automated refinement and selection of kinetic models. MoRSel starts with a kinetic core model, a library of potential model changes, and available experimental data. The algorithm iteratively expands the model to achieve an optimal balance between fit quality and model complexity according to information-theoretic criteria. We applied MoRSel to a kinetic model of a cell-free enzyme cascade for producing the nucleotide sugar UDP-GalNAc. An initial core model showed significant inconsistencies with measured experimental data. By providing candidates for potential stoichiometric and regulatory changes in the model, MoRSel identified a model that more accurately captured the observed behavior. We then used this model to inform a recently introduced robust (ensemble-based) process optimization strategy that accounts for parametric uncertainty. We addressed two economically relevant problems. First, we determined the optimal initial substrate and enzyme concentrations to maximize the product titer, resulting in a 45 % increase in product titer and yield in a validation experiment. In a second optimization, we minimized the enzyme load and tested the computed optimal enzyme concentrations in another validation experiment. With enzyme costs reduced by 53 %, the relative titer and yield could be improved by 80 % and 75 %, respectively, with small reductions in absolute titer and yield. Overall, MoRSel advances the development of predictive models for complex biochemical systems, with particular relevance for the robust optimization of cell-free bioproduction processes.

Plants redirect their secondary metabolism when herbivores feed on them, and the galls that the aphid Schlechtendalia chinensis induces on Rhus chinensis carry this response to an extreme, with hydrolyzable tannins (HTs) reaching 74.5% of gall wall dry weight, about 32 times the level in ungalled leaves. To find out how the plant builds this much tannin and how the aphid survives it, we followed HT accumulation and candidate gene expression through gall development, expressed the genes in Arabidopsis thaliana and characterised the aphid enzymes acting on tannin. Expression of phosphoglucomutase, UDP-glucosyltransferase BX9 and gallate 1-beta-d-glucosyltransferase (GDG) rose and fell with HT content, and GDG, whose product β-glucogallin is the first committed intermediate, tracked it most closely. In Arabidopsis, the galloyl compound pool rose up to about four-fold, so the three genes enlarge the galloyl precursor supply in this host. In the aphid, tannase activity stayed close to the detection limit of the assay throughout gall development, whereas laccase activity ran several orders of magnitude higher and was concentrated in the abdomen. Crude aphid extract removed 41.6%-54.2% of the HT from gall extracts, and heating it or adding the copper-oxidase inhibitor NaN3 prevented this. Recombinant Sc-Lac5, one of the three aphid laccases, oxidised 1,2,3,4,6-penta-O-galloyl-β-d-glucose, tannic acid and gall HT, consuming O2 equivalent to 3.77 electrons per PGG molecule and about two per galloyl group of gall tannin (Km = 187 µM, kcat = 2.83 s[-1]). Silencing Sc-Lac5 made 2% dietary tannic acid lethal to aphids that otherwise tolerate it and allowed HT to accumulate in their bodies, and feeding the purified enzyme reversed both. Laccase activity, and Sc-Lac5 in particular, underpins the ability of S. chinensis to live in the tannin its feeding creates.

Uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes play a critical role in hepatic and other metabolism of endogenous and exogenous small molecules. The addition of glucuronic acid via UGT increases the polarity and subsequent solubility of small molecules, helping direct them for biliary excretion and renal excretion by organic anion transporter OAT3 (SLC22A8). Mutations in UGTs are associated with disorders in biliary metabolism (e.g., Gilbert's syndrome, Crigler-Najjar syndrome). Here, we used comprehensive serum metabolomic profiling of pan Ugt1-knockout (Ugt1[-/-]) mice to define the metabolic consequences of UGT deficiency. All 7 knockout mice exhibited markedly elevated bilirubin (mean = 5.96 mg/dL). Partial Least Squares Discriminant Analysis (PLS-DA) showed clear separation between Ugt1[-/-] and wild type (WT), and volcano plots revealed significant endogenous metabolite alterations. These included upregulation of numerous bilirubin degradation products as well as major disturbances in lipid and amino acid metabolism. In particular, pathway enrichment analyses identified disruptions in fatty acid biosynthesis, glycine-serine metabolism, heme breakdown, porphyrin metabolism, and β-oxidation of fatty acids. Thus, the UGT1 proteins regulate different aspects of endogenous metabolism and signaling. Consistent with this notion, there were increases in liver expression of several nuclear receptors (e.g., HNF4a, PPARa, PXR) involved in metabolic signaling. These results support the remote sensing and signaling theory (RSST), which proposes that UGT1 proteins play a key role in organ crosstalk via routing small molecules between the liver and kidney, with glucuronidated molecules being mainly directed to the multispecific SLC kidney transporter OAT3. We propose that this glucuronidation-regulated RSST loop is essential to signaling along the gut-liver-kidney axis.

Microglia, the resident macrophages of the central nervous system, continuously survey the parenchyma with their highly motile processes. While calcium (Ca[2+]) signaling is a potential regulator of this process, the intrinsic mechanisms controlling process dynamics remain poorly defined. Here, we characterized microglial Ca[2+] signals evoked by purinergic receptor activation both in vitro and in situ. In cultured microglia, UDP/UTP-induced P2Y6 receptor activation evoked robust Ca[2+] signals with a sustained component mediated by store-operated Ca[2+] entry (SOCE). In contrast, Ca[2+] signals induced by P2Y6 and P2Y12/P2Y13 receptor activation in acutely isolated microglia or in situ microglia were dominated by intracellular store release, with minimal SOCE contribution. Functionally, Ca[2+] signaling induced by P2Y6 activation or store depletion suppressed dorsal ruffling-an F-actin-dependent process-in cultured microglia. In brain slices, these Ca[2+] signals triggered the retraction of fine processes and reduced process surveillance, while chemogenetic activation of Ca[2+] signaling in vivo similarly decreased process complexity. Crucially, this microglial structural remodeling was associated with exacerbated status epilepticus. Thus, our study demonstrates that microglial Ca[2+] signals act as a potent suppressor of process dynamics, thereby regulating neuronal excitability.

This study presents an asymmetric cooperative perception-and-control architecture in which a sensor-rich mobile master vehicle performs external perception, state estimation, motion planning, and path-tracking control for sensor-light slave vehicles. The proposed vehicle-model-aided LiDAR tracking (VMALT) method combines ego-motion-compensated LiDAR measurements with transmitted speed and steering commands through a kinematic bicycle model. In physical-vehicle experiments, VMALT reduced the slave-position RMSE from 0.36 m with a LiDAR-only constant-velocity Kalman filter to 0.32 m, corresponding to an 11.1% improvement. The estimated state was subsequently used for closed-loop speed and path-tracking control of the physical slave vehicle. Scalability was evaluated using a one-master-two-slave configuration over three separate runs comprising circular and linear paths and geometrically identified line-of-sight-overlap candidates. Concurrent two-target LiDAR availability ranged from 96.465% to 99.934%, with a maximum interior observation gap of 0.10 s. The VMALT trajectories expressed in the GPS coordinate frame followed the position and direction of both slave vehicles, with heading RMSEs ranging from 1.835∘ to 4.609∘. In an LTE-tethering communication test, all 2400 UDP packets were returned, with median and 99th-percentile round-trip times of 4.654 and 8.220 ms, respectively. These results demonstrate the feasibility of centralized perception and control for multiple sensor-light vehicles under the evaluated low-speed operating conditions.

Ansamitocin P-3 (AP-3) is an ansamycin antibiotic produced by Actinosynnema pretiosum with high clinical and commercial value. Its biosynthesis is tightly controlled, yet the underlying transcriptional network is not fully understood. Here, we identified the two-component system CrsAB and characterized the response regulator CrsB. Deletion of crsB resulted in a 57% reduction in AP-3 yield without impairing morphological development or biomass accumulation. RNA‑Seq showed that CrsB acts as a global regulator, influencing energy metabolism, cell integrity, and biosynthetic capacity. At the fermention at 48 h, genes asm23 and asm43-47 within the biosynthetic gene cluster, which are primarily responsible for the synthesis of AHBH from UDP-glucose, were significantly downregulated upon crsB deletion. Conversely, genes of the pentose phosphate pathway, zwf, opcA and pgl, were markedly upregulated, whereas their expression levels showed no significant differences between the two strains at 72 h. These temporal transcriptional changes were confirmed by qRT‑PCR. Collectively, our data suggest that CrsB positively coordinates primary and secondary metabolism primarily during the early stage to promote AP-3 production, offering new targets for metabolic engineering.

Tobacco root rot is a destructive soil-borne disease that constrains sustainable tobacco production in Yunnan. Fusarium oxysporum and Fusarium solani species complex are currently considered the principal pathogens associated with this disease. Therefore, effective biocontrol agents are needed, and understanding their pathogen-side modes of action is essential for improving biological management strategies. This study screened 100 bacterial strains isolated from the tobacco rhizosphere using plate confrontation assays and identified A5 as a broad-spectrum antagonistic strain. A5 consistently inhibited four tobacco root rot-associated Fusarium species, with hyphal growth inhibition rates of 63.31%, 59.42%, 69.64%, and 68.82%, respectively. Phylogenetic analyses of 16S rRNA and gyrB sequences placed A5 close to Bacillus aerophilus, supporting its classification as a B. aerophilus-related biocontrol candidate. To investigate the fungal response to A5, F. solani was exposed to 10% sterile A5 fermentation supernatant and analyzed using transcriptomic sequencing. GO and KEGG enrichment analyses showed differentially expressed genes (DEGs) involved in UDP-N-acetylglucosamine biosynthesis and associated cell wall precursor metabolic processes. Because F. oxysporum has a more established genetic manipulation system in tobacco root rot research, this species was used for downstream functional validation. RT-qPCR confirmed the significant expression responses of FOXG_19023, FOXG_10443, and FOXG_12345 after the treatment with A5 supernatant. Subsequent construction of knockout mutants demonstrated that deletion of FOXG_19023 significantly restricted colony growth, suggesting that FOXG_19023 may contribute to vegetative growth of F. oxysporum, although its specific role in UDP-N-acetylglucosamine-related cell wall metabolism remains to be explored. Overall, A5 was identified as a tobacco root rot biocontrol bacterium with activity against Fusarium pathogens. These results further suggest that A5 may affect expression of genes involved in pathogen UDP-N-acetylglucosamine-related metabolism, and this response may be associated with impaired Fusarium hyphal growth. This study provides a potential biocontrol resource for sustainable tobacco root rot management and offers candidate pathogen-side targets for future studies on the molecular mechanisms underlying Bacillus-mediated suppression of Fusarium.

Background: Polyploidization generates extensive gene redundancy, but how duplicated metabolic genes are retained and subsequently diversified remains poorly understood. UDP-glycosyltransferases (UGTs) provide a suitable system for examining this process because they participate in specialized metabolism, plant development, and environmental responses. This study aimed to characterize the retention, expansion, and regulatory divergence of the UGT family in allotetraploid Panax ginseng at subgenome resolution. Methods: We integrated telomere-to-telomere (T2T) genome annotation, phylogenetic and chromosomal analyses, duplication classification, collinearity and Ka/Ks analyses, promoter cis-acting element prediction, developmental co-expression networks, and transcriptomic responses to biotic and abiotic treatments. Results: A total of 212 PgUGT genes were identified, including 104 and 108 members in the A and B subgenomes, respectively. The family exhibited an overall near-mirrored retention pattern between the two subgenomes, accompanied by local copy-number asymmetry. Whole-genome and segmental duplication accounted for 64.2% of the family, and 99.5% of the gene pairs with valid Ka/Ks estimates had values below 1, suggesting pervasive purifying selection. PgUGT-containing co-expression modules were associated with bud, stem, leaf, and fruit developmental conditions, while promoter cis-acting element compositions exhibited member-specific variation. Transcriptional responses to fungal pathogens and abiotic, hormone, and chemical treatments were concentrated in particular members and local gene arrays rather than being coordinated across entire clades or subgenomes. Conclusions: The PgUGT family is characterized by extensive ancestral copy retention accompanied by local copy-number changes and copy-specific regulatory divergence. These findings provide a subgenome-resolved framework for understanding UGT family evolution in allotetraploid ginseng and prioritize candidate PgUGT genes for subsequent functional validation.

Lymphatic filariasis remains a major neglected tropical disease and there is an urgent need for parasite-specific therapeutic targets. The enzyme UDP-galactopyranose mutase (UGM) is essential for galactofuranose biosynthesis in Brugia malayi and is absent in humans, which makes it a promising drug target. In this study, we investigated phytochemicals from the bark of Tecomella undulata to find potential drug candidates against B. malayi UGM using an in silico approach. The structure of the target protein was predicted using the Alphafold model, and the active site was predicted using the combination of PrankWeb, DoGSiteScorer, and FTMap servers and further supported by superposition with a homologous experimentally determined UGM structure. Forty phytochemicals of T. undulata bark were screened by molecular docking using AutoDock Tools. Docking analysis showed Dehydrotectol (- 11.73 kcal/mol), Daucosterol (- 11.35 kcal/mol), urs-12-ene (- 10.87 kcal/mol), β-sitosterol (- 10.72 kcal/mol), and betulinic acid (- 10.47 kcal/mol) are the top-scoring ligands. Based on predicted binding free energy and interaction with key catalytic residues of UGM, Daucosterol was selected for molecular dynamics simulation using the GROMACS package. MD analysis and MM/PBSA calculations supported the thermodynamic stability of the docked complex. ADMET predictions indicated acceptable drug-likeness and low toxicity of several phytoconstituents of T. undulata bark. This study provides computational evidence supporting T. undulata bark phytochemicals can be a new drug formulation against B. malayi.