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Mechanisms driving acute food allergic reactions have not been fully characterized. We profile the dynamic transcriptome of acute peanut allergic reactions using serial peripheral blood samples obtained from 19 children before, during, and after randomized, double-blind, placebo-controlled oral challenges to peanut. We identify genes with changes in expression triggered by peanut, but not placebo, during acute peanut allergic reactions. Network analysis reveals that these genes comprise coexpression networks for acute-phase response and pro-inflammatory processes. Key driver analysis identifies six genes ( LTB4R , PADI4 , IL1R2 , PPP1R3D , KLHL2 , and ECHDC3 ) predicted to causally modulate the state of coregulated networks in response to peanut. Leukocyte deconvolution analysis identifies changes in neutrophil, naive CD4 + T cell, and macrophage populations during peanut challenge. Analyses in 21 additional peanut allergic subjects replicate major findings. These results highlight key genes, biological processes, and cell types that can be targeted for mechanistic study and therapeutic targeting of peanut allergy. Rising rates of peanut allergy pose a public health problem. Here, the authors profile blood transcriptomes during double-blind, placebo-controlled oral challenge in peanut-allergic children to identify gene and cell composition changes, and construct causal networks to detect key allergic reaction drivers.

Mobilization of seed storage compounds, such as starch and oil, is required to provide energy and metabolic building blocks during seedling development. Over 50% of fatty acids in Arabidopsis (Arabidopsis thaliana) seed oil have a cis-double bond on an even-numbered carbon. Degradation of these substrates requires peroxisomal fatty acid 𝛽-oxidation plus additional enzyme activities. Such auxiliary enzymes, including the enoyl-CoA hydratase ECH2, convert (R)-3-hydroxyacyl-CoA intermediates to the core 𝛽-oxidation substrate (S)-3-hydroxyacyl-CoA. ECH2 was suggested to function in the peroxisomal conversion of indole-3-butyric acid (IBA) to indole-3-acetic acid, because ech2 seedlings have altered IBA responses. The underlying mechanism connecting ECH2 activity and IBA metabolism is unclear. Here, we show that ech2 seedlings have reduced root length, smaller cotyledons, and arrested pavement cell expansion. At the cellular level, reduced oil body mobilization and enlarged peroxisomes suggest compromised 𝛽-oxidation. ech2 seedlings accumulate 3-hydroxyoctenoate (C8:1-OH) and 3-hydroxyoctanoate (C8:0-OH), putative hydrolysis products of catabolic intermediates for 𝛼-linolenic acid and linoleic acid, respectively. Wild-type seedlings treated with 3-hydroxyoctanoate have ech2-like growth defects and altered IBA responses. ech2 phenotypes are not rescued by Suc or auxin application. However, ech2 phenotypes are suppressed in combination with the core 𝛽-oxidation mutants mfp2 or ped1, and ech2 mfp2 seedlings accumulate less C8:1-OH and C8:0-OH than ech2 seedlings. These results indicate that ech2 phenotypes require efficient core 𝛽-oxidation. Our findings suggest that low ECH2 activity causes metabolic alterations through a toxic effect of the accumulating intermediates. These effects manifest in altered lipid metabolism and IBA responses leading to disrupted seedling development.

Despite the potential of biotechnological processes for one-carbon (C1) bioconversion, efficient biocatalysts required for their implementation are yet to be developed. To address intrinsic limitations of native C1 biocatalysts, here we report that 2-hydroxyacyl CoA lyase (HACL), an enzyme involved in mammalian α-oxidation, catalyzes the ligation of carbonyl-containing molecules of different chain lengths with formyl-coenzyme A (CoA) to produce C1-elongated 2-hydroxyacyl-CoAs. We discovered and characterized a prokaryotic variant of HACL and identified critical residues for this newfound activity, including those supporting the hypothesized thiamine pyrophosphate-dependent acyloin condensation mechanism. The use of formyl-CoA as a C1 donor provides kinetic advantages and enables C1 bioconversion to multi-carbon products, demonstrated here by engineering an Escherichia coli whole-cell biotransformation system for the synthesis of glycolate and 2-hydroxyisobutyrate from formaldehyde and formaldehyde plus acetone, respectively. Our work establishes a new approach for C1 bioconversion and the potential for HACL-based pathways to support synthetic methylotrophy. A bacterial 2-hydroxyacyl-CoA lyase catalyzes ligation of carbonyl-containing molecules of different chain lengths with formyl-CoA to produce elongated 2-hydroxyacyl-CoAs, enabling a one-carbon bioconversion pathway with formaldehyde as a substrate.

The ECHS1 (short-chain enoyl-CoA hydratase 1) gene is critical for mitochondrial fatty acid β-oxidation and branched-chain amino acid metabolism. Mutations in ECHS1 lead to severe mitochondrial dysfunction and are implicated in rare metabolic and neurodegenerative disorders. This review summarizes current understanding of how ECHS1 participates in key molecular processes, including energy metabolism, oxidative stress regulation, and apoptosis, and discusses its influence on mitochondrial function. It also highlights advances in experimental models, including mouse, Drosophila, and induced pluripotent stem cell (iPSC) -based systems, which have illuminated the gene’s physiological roles while revealing model-specific limitations. Therapeutic approaches, such as dietary interventions, gene therapy, enzyme replacement therapy, and stem cell therapy, are critically evaluated, emphasizing their potential and current challenges. Despite significant progress, gaps remain in understanding ECHS1 ’s tissue-specific and developmental-stage-specific functions. This review underscores the need for advanced human-relevant models and integrative technologies to address these gaps and foster the development of personalized treatments for ECHS1 -related disorders.

Production of flavors from natural substrates by microbial transformation has become a growing and expanding field of study over the past decades. Vanillin, a major component of vanilla flavor, is a principal flavoring compound used worldwide. Streptomyces sp. strain V-1 is known to be one of the most promising microbial producers of natural vanillin from ferulic acid. Although identification of the microbial genes involved in the biotransformation of ferulic acid to vanillin has been previously reported, purification and detailed characterization of the corresponding enzymes with important functions have rarely been studied. In this study, we isolated and identified 2 critical genes, fcs and ech, encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase, respectively, which are involved in the vanillin production from ferulic acid. Both genes were heterologously expressed in Escherichia coli, and the resting cell reactions for converting ferulic acid to vanillin were performed. The corresponding crucial enzymes, Fcs and Ech, were purified for the first time and the enzymatic activity of each purified protein was studied. Furthermore, Fcs was comprehensively characterized, at an optimal pH of 7.0 and temperature of 30°C. Kinetic constants for Fcs revealed the apparent Km, kcat, and Vmax values to be 0.35 mM, 67.7 s(-1), and 78.2 U mg(-1), respectively. The catalytic efficiency (kcat/Km) value of Fcs was 193.4 mM(-1) s(-1) for ferulic acid. The characterization of Fcs and Ech may be helpful for further research in the field of enzymatic engineering and metabolic regulation.

The transition between motility-associated acute infection and biofilm-associated chronic infection is crucial for bacterial proliferation and survival within hosts. This process of transition can be influenced by various host and environmental cues, however, whether bacteria could actively modulate the switch remains poorly understood. Here we identify a novel enoyl-CoA hydratase/isomerase, PA0744 (designated as DspII), which is co-upregulated with the previously characterized enoyl-CoA hydratase/isomerase DspI in a cell density-dependent manner to induce chronic-to-acute virulence switch in Pseudomonas aeruginosa . The two proteins form a heterocomplex that synthesizes the DSF-family quorum sensing signal cis −2-decenoic acid (CDA), which positively regulates the expression of the phosphodiesterase RbdA, and subsequently, activates biofilm dispersion and swarming motility through the c-di-GMP/FleQ pathway. Intriguingly, the DspII-DspI complex also synergistically antagonizes the GacA-upregulated sRNAs, RsmY and RsmZ, leading to overproduction of type III secretion system (T3SS) and augment of acute pathogenesis. Notably, the affinity of DspII-DspI interaction and consequently T3SS gene expression are fine-tuned by CDA levels. This study unveils the novel enzymatic role of the DspII-DspI complex in CDA biosynthesis as well as its non-enzymatic role in T3SS gene regulation, which presents a simple yet sophisticated bacterial signal-driven auto-regulatory mechanism that governs the chronic-to-acute virulence switch in P. aeruginosa . Here, the authors identify DspII, a functionally equivalent and indispensable partner of DspI in mediating the chronic-to-acute virulence switch in Pseudomonas aeruginosa , which is a key adaptive strategy that facilitates response to the host.

Focal segmental glomerulosclerosis (FSGS) is a common cause of nephrotic syndrome and often leads to end-stage renal disease. However, the underlying pathophysiological mechanisms that contribute to disease progression require further investigation to establish appropriate therapeutic targets and biomarkers. This study aimed to clarify the molecular mechanisms underlying FSGS by focusing on differentially expressed genes (DEGs) and lipid metabolism-related genes (LMRGs). We utilized the GSE69814, GSE129973, and GSE121233 datasets, which comprise glomerular transcriptomes from patients with FSGS, minimal change disease (MCD), and unaffected kidney tissues. We identified 2,459 DEGs from the GSE69814 dataset and 982 DEGs from the GSE129973 dataset. These DEGs intersected 1,450 LMRGs, resulting in 56 differentially expressed LMRGs (DELMRGs). Enrichment analysis revealed that these DELMRGs were primarily involved in fatty acid metabolic processes; localized in microbodies, peroxisomes, and mitochondrial matrices; and exhibited oxidoreductase activity. Protein-protein interaction networks were constructed using Cytoscape, and five hub DELMRGs (enoyl-CoA hydratase, short chain 1 [ ECHS1 ] , EHHADH, IDH1, SUCLG1 , and ALDH3A2 ) were identified using multiple algorithms. We assessed the diagnostic performance using receiver operating characteristic curves and expression levels from the GSE121233 dataset, and found that ECHS1 and ALDH3A2 showed strong diagnostic potential. Immunohistochemical verification of clinical specimens from children confirmed significant expression of ECHS1 in FSGS compared with that in normal and MCD tissues. This study highlights ECHS1 as a potential biomarker for pediatric FSGS, suggesting a potential role in early diagnosis or personalized treatment, offering insights into its pathogenesis and paving the way for targeted therapeutic strategies.

Examining the fundamental structure and processes of living cells at the nanoscale poses a unique analytical challenge, as cells are dynamic, chemically diverse, and fragile. A case in point is the cell membrane, which is too small to be seen directly with optical microscopy and provides little observational contrast for other methods. As a consequence, nanoscale characterization of the membrane has been performed ex vivo or in the presence of exogenous labels used to enhance contrast and impart specificity. Here, we introduce an isotopic labeling strategy in the gram-positive bacterium Bacillus subtilis to investigate the nanoscale structure and organization of its plasma membrane in vivo. Through genetic and chemical manipulation of the organism, we labeled the cell and its membrane independently with specific amounts of hydrogen (H) and deuterium (D). These isotopes have different neutron scattering properties without altering the chemical composition of the cells. From neutron scattering spectra, we confirmed that the B. subtilis cell membrane is lamellar and determined that its average hydrophobic thickness is 24.3 ± 0.9 Ångstroms (Å). Furthermore, by creating neutron contrast within the plane of the membrane using a mixture of H- and D-fatty acids, we detected lateral features smaller than 40 nm that are consistent with the notion of lipid rafts. These experiments-performed under biologically relevant conditions-answer long-standing questions in membrane biology and illustrate a fundamentally new approach for systematic in vivo investigations of cell membrane structure.

Microbial transformation has enabled phytosterols as readily available and bio-renewable starting materials for the industrial synthesis of steroidal active pharmaceutical ingredients (APIs). Editing the phytosterol side chain would create various steroidal compounds with a specific C17-side chain, which will greatly facilitate the synthesis of steroidal APIs. Precise cleavage of the phytosterol side chain requires identification of the key enzymes and the reaction pathways of phytosterol side chain metabolism. In this study, a hydratase EchA19 was identified in Mycolicibacterium neoaurum NRRL B-3805, a strain which was engineered by traditional mutation and screening or genetic manipulation, generating recombinant strains for the industrial-scale production of androstenedione (AD), androstadienedione (ADD), and 9α-hydroxy-androstenedione (9α-OH-AD) from phytosterols. It was found that EchA19 is the key hydratase affecting the first β-oxidation pathway of phytosterol side chain metabolism. The previously proposed carboxylation at the C28 position might occur after the cleavage of the C24 branched alkyl side chain, rather than after the dehydrogenation reaction. This study has provided us with new insights and a deeper understanding of the metabolic pathways of phytosterol side chain, and laid a foundation for synthesizing valuable steroid drug intermediates from phytosterols through metabolic regulation by precisely editing the side chain. Graphical Abstract

Vanillin is one of the most commonly used natural-occurring flavors in the world. This study successfully constructed an efficient whole-cell catalytic system for vanillin biosynthesis from ferulic acid by regulating feruloyl-CoA synthetase (FCS) and enoyl-CoA hydratase (ECH). First, we constructed an efficient cell-free catalytic system with FCS-Str ( fcs from Streptomyces sp. V-1) and ECH-Str ( ech from Streptomyces sp. V-1) combination at 1:1. The efficient cell-free catalytic system provided necessary strategies for optimizing the whole-cell catalytic system. Then, we constructed the recombinant Escherichia coli by heterologously expressing the fcs -Str and ech -Str combination. Moreover, E. coli JM109 was a better recombinant Escherichia coli than E. coli BL21 with higher vanillin production. Finally, we first adjusted the ratio of FCS and ECH in E. coli JM109 to 1:1 using two copies of fcs -Str. For higher vanillin production, we further optimized the induction conditions of E. coli JM109 to increase the amount of FCS and ECH. The optimized E. coli JM109-FE-F constructed in this study has the highest vanillin synthesis rate of converting 20 mM ferulic acid to 15 mM vanillin in 6 h among all of the E. coli catalytic systems. Our study made a significant contribution to the construction of the vanillin biosynthesis system and provided a valuable strategy for increasing vanillin production. Key points • The efficient cell-free vanillin biosynthesis system was constructed by FCS-Str and ECH-Str combination at 1:1. • Escherichia coli JM109 was determined as a better recombinant Escherichia coli than E. coli BL21 with higher vanillin production. • Escherichia coli JM109-FE-F with two copies of fcs-Str and one copy of ech-Str has the highest catalytic efficiency for vanillin production.