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Fetal growth restriction (FGR) affects approximately 8% of pregnancies in Western countries and is characterised by complex placental adaptations at both metabolic and transcriptional levels. In this study, we integrated RNA sequencing and metabolomic analyses to investigate alterations in steroidogenesis, NAD⁺ metabolism and ω-3/ω-6 polyunsaturated fatty acid (PUFA) pathways in placental biopsies and trophoblast organoids. Placentas from small-for-gestational-age (SGA10 and SGA3) infants, compared with appropriate-for-gestational-age (AGA) controls, showed increased cholesterol uptake and enhanced steroid biosynthesis. In SGA3 placentas, these changes were accompanied by activation of the NAD⁺ salvage pathway, supporting elevated steroidogenesis, redox balance and energy metabolism. Despite this compensatory response, concentrations of key steroid metabolites, including androstenedione sulfate and oestrogens, were reduced. Metabolomic profiling further revealed a marked depletion of lysophospholipids enriched in ω-3 and ω-6 PUFAs, along with decreased levels of free arachidonic acid (ARA), docosahexaenoic acid (DHA) and selected prostaglandins and thromboxanes. These alterations suggest mobilisation of lipid stores to counteract reduced PUFA-derived eicosanoid production, a process that may compromise placental vascular regulation and fetal neurodevelopment. Collectively, our results highlight the metabolic plasticity of the FGR placenta and identify coordinated alterations in lipid and NAD⁺ metabolism as key adaptive responses to placental insufficiency.

Germinal mono-allelic loss-of-function mutations of NEK1 drive amyotrophic lateral sclerosis (ALS) at variable penetrance, presumably through haploinsufficiency. Modeling the ALS-associated Arg812Ter mutation in mice revealed that the resulting truncated Nek1 (Nek1^t) is aggregation-prone, particularly in alpha-motoneurons (αMNs), and drives canonical ALS symptoms when bi-allelically expressed (Nek1^t/t). Promyelocytic leukemia (Pml) ablation allows for ALS symptoms to occur even in heterozygote Nek1^wt/t animals, mimicking the human situation. Pml precludes disease occurrence by promoting SUMO-facilitated degradation of Nek1^t proteins through PML nuclear bodies (NBs). Conversely, Pml induction, achieved by activating the interferon pathway via poly(I:C) treatment, clears Nek1^t puncta in αMNs, dramatically reducing ALS-associated symptoms and extending survival by 5 months. Our studies highlight the role of mutant NEK1 expression in ALS pathogenesis and identifies activation of interferon pathways as a candidate therapeutic strategy that promotes Pml-triggered SUMOylation/degradation of toxic misfolded proteins in vivo, yielding dramatic clinical improvement. These observations provide strong proof-of-concept support to validate PML as a relevant therapeutic target in neurodegenerative conditions associated with protein misfolding and putative aggregation.

The intestinal microbiota constitutes a crucial defense barrier against pathogenic invasion; however, the molecular mechanisms enabling pathogens to evade or modulate this defense remain poorly understood. Here, we established a coculture model combining the commensal Escherichia coli Y18J, isolated from the piglet gut, and the enterotoxigenic E. coli (ETEC) strain W25K to investigate microbe-pathogen interactions. Our findings reveal a bidirectional regulatory mechanism between Y18J and W25K mediated by bacteriocin and toxin signaling. Colicin B/M produced by Y18J upregulates the expression of heat-stable enterotoxin (ST) in W25K during the early phase of coculture, while ST suppresses colicin B/M synthesis in Y18J. At later stages, colicin B/M stimulates heat-labile enterotoxin (LT) expression, which in turn enhances colicin B/M production. Notably, LT markedly reduces intestinal colonization of W25K(ST^-LT⁺) in murine hosts. Leveraging metagenomic and bioinformatic analyses, we further identified a Ligilactobacillus strain within the murine gut microbiota capable of producing multiple bacteriocins that effectively inhibit W25K colonization. Transcriptomic profiling of Y18J revealed glutamine synthetase as a pivotal regulator of colicin B/M-mediated antagonism. Mechanistic investigations demonstrated that ST suppresses colicin B/M expression through the cGMP signaling pathway, whereas LT enhances it via the cAMP signaling pathway. Collectively, these findings uncover a dual regulatory mechanism through which bacterial enterotoxins modulate probiotic antimicrobial activity, providing new insights into the molecular dialog between commensal and pathogenic bacteria. This study establishes a conceptual framework for developing microbiota-based strategies to prevent and control enteric infections.

Indoleamine 2,3-dioxygenase 2 (IDO2) is a heme enzyme in the kynurenine pathway that shares high structural similarity with IDO1 but exhibits markedly lower catalytic activity. To clarify the molecular basis of this difference, we performed spectroscopic, biochemical, and crystallographic analyses of human IDO2. We found that IDO2 binds L-tryptophan (L-Trp) in a flipped orientation stabilized by the IDO2-specific residue His143, which results in inefficient catalysis. Replacement of His143 with tyrosine, the corresponding residue in IDO1, restored an IDO1-like binding mode of L-Trp and enhanced activity by more than 1000-fold. Structural analyses further revealed that IDO2 accommodates various tryptophan derivatives, such as 5-methyl-l-Trp (5MT) and 5-methoxy-l-Trp (5MoT), in a productive conformation, while other ligands, including D-Trp and serotonin, adopt nonproductive poses. In addition, we observed that 5MT and 5MoT are metabolized by IDO2 at levels comparable to the metabolism of L-Trp by human tryptophan 2,3-dioxygenase. These results highlight the unique structural constraints that underlie IDO2's low activity and broadened substrate recognition, providing a molecular framework for understanding the functional divergence between IDO1 and IDO2.

Recently, enzymatic depolymerisation of synthetic polyesters has emerged as an attractive complement to current plastic recycling methods. However, the arsenal of robust enzymes available for these applications remains limited. Here, we identified and biochemically characterised three novel carboxylesterases (TA21, TA26 and TA29) with polyesterase activity from the genome of the thermophilic, obligately alkane-degrading bacterium Thermoleophilum album YS-3 (ATCC 35264). The purified proteins hydrolysed model para-nitrophenyl monoesters, favouring short-chain substrates (C2-C6), with TA21 showing the highest carboxylesterase activity. All three enzymes displayed maximal activity at 55-60 °C, with 20-25% activity remaining at 75 °C. Notably, they also retained substantial activity at moderate temperatures (15 °C), which is uncommon for thermophilic enzymes. In agarose-plate screens with emulsified polyesters, the enzymes hydrolysed amorphous poly(ethylene terephthalate) (aPET), bis(benzoyloxyethyl) terephthalate (3PET), polylactic acid (PLA) and polycaprolactone (PCL). HPLC analysis identified terephthalic acid (TA) and mono(2-hydroxyethyl) terephthalate (MHET) as the major degradation products of 3PET and aPET, with TA21 efficiently converting MHET to TA. TA21 was active on PLA and PCL, hydrolysing them to lactic acid and 6-hydroxyhexanoic acid, respectively, and preferred 3PET and PLA over aPET and PCL (0.25-1.5 mm products formed after overnight incubation). Structural analysis revealed the presence of a medium-sized lid domain in all three enzymes. In TA21, this domain contributes two aromatic residues and an arginine for coordinating MHET in the active site, which may account for its higher MHET-degrading activity. These findings introduce novel thermophilic polyesterases that may serve as promising candidates for further optimisation and protein engineering research on enzymatic depolymerisation of synthetic polyesters.

Neonatal regulatory T (Treg) cells in secondary lymphoid organs have greater proliferative capacity and more potent suppressive functions than adult Treg cells. However, the phenotypic and functional features of Tregs in neonatal nonlymphoid organs are not well understood. Our prior work demonstrated that thymus-derived Treg cells entering the neonatal mouse liver enhance immune tolerance and periportal liver maturation. Compared to splenic Treg cells, these hepatic Tregs have faster turnover and superior suppression of naïve T-cell proliferation. To further define this population, we conducted single-cell transcriptomic and immunophenotypic analyses of liver- and spleen-derived Tregs from neonatal and adult mice. Our analysis revealed a distinct T-box transcription factor Tbx21 (T-bet)⁺ effector Treg subset uniquely enriched in the neonatal liver. These cells possess liver-homing properties, clonal expansion of unique T-cell receptor (TCR) repertoires, and heightened suppressive activity. Interleukin-27 (IL-27) and interferon gamma (IFN-γ), cytokines highly expressed in the neonatal liver, play a critical role in promoting T-bet expression and the acquisition of an ectonucleoside triphosphate diphosphohydrolase 1 (CD39)⁺ C-X-C chemokine receptor type 3 (CXCR3)⁺ double-positive phenotype in Treg cells. Collectively, our findings characterize a liver-specific neonatal T-bet⁺ Treg population shaped by the hepatic microenvironment, highlighting its unique tissue-resident signature and immunoregulatory role.

Chemical potentials (molar Gibbs energies) are usually extrapolated to the remote physical-chemical reference state and then stored. Subsequent use under in vivo conditions requires a similarly substantial, reverse extrapolation, again with significant potential errors. In order to shrink both extrapolations drastically and thereby enhance both biological meaning and accuracy, we propose a transformation to a more biological reference state: pH = 7, pMg = 3, 99.5% water, with 1 mm each of the additional 'precursors' inorganic phosphate, sulfate, ammonium, and bicarbonate, and with twin temperatures 37 and 25 °C, ionic strength 0.15 m and mm as concentration unit. These precursors substitute for reference compounds alien to biology such as H₂ at 1 bar, and solid graphite, sulfur, and phosphorus. The standard chemical potentials are herewith increased by the magnitudes of the chemical potentials of protons, Mg²⁺, water, and the four precursors, each multiplied by the number of corresponding atoms in the molecule. This defines standard 'metabolic potentials'. We make these potentials findable and accessible as 1360 collated standard chemical potentials for 320 compounds of biochemical interest at the twin metabolic reference states. We do this for 3 reference pH's: We present the metabolic reference state as a convenient anchor, not a universal intracellular milieu. All datasets must continue to report the actual experimental state (T, pH, pMg, I, osmolarity, concentrations), yet aim at (also) reporting parameter values for this anchor state; we supply algorithms to transform between states. This preserves interoperability across diverse organelles, media and between enzymology and chemical engineering, while facilitating reuse.

Four formate dehydrogenases (FDHs) from Pseudomonas sp. 101, Myceliophthora thermophila, Chaetomium thermophilum, and Ogataea parapolymorpha were recombinantly produced, purified, and characterized to investigate their catalytic properties and reaction mechanisms. The enzymes were studied for their ability to oxidize formate to carbon dioxide (CO₂) coupled with NAD⁺ reduction. In contrast, their CO₂ reduction activity was undetectable under the tested conditions. Oxidative reactions revealed significant differences in catalytic efficiency and substrate specificity, prompting further investigation through molecular dynamics (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) ONIOM calculations. Structural models were derived from high-resolution structural data available for enzymes from Pseudomonas sp. 101 (pseFDH) and Chaetomium thermophilum (ctFDH) and extended to all four variants. Comparative analyses of the transition states revealed distinct interaction patterns within the active sites, allowing us to discriminate between high- and low-performing catalysts, in full agreement with the experimental k_cat values. These findings provide a mechanistic rationale for the observed disparities in catalytic performance and offer structural insights into the determinants of FDH activity. Notably, ctFDH emerged as a potential candidate for the development of CO₂-reducing reactions, with QM/MM data guiding the rational design of transition-state stabilizing mutations.

Proteostasis is the finely tuned balance of protein synthesis, folding and degradation essential for cellular health. When this equilibrium is disrupted, misfolded proteins accumulate, triggering adaptive stress responses such as the unfolded protein response and the integrated stress response (ISR). Central to the ISR is the kinase GCN2, a sensor of amino acid deprivation and ribosomal stress. Upon activation, GCN2 phosphorylates eIF2α, dampening global translation while selectively enhancing the synthesis of the stress-responsive transcription factors ATF4 and CHOP. ATF4 orchestrates a broad transcriptional programme that supports amino acid metabolism, redox homeostasis, autophagy and proteasomal degradation, which are key processes for restoring proteostasis. Beyond its canonical role, GCN2 interfaces with other regulatory networks modulating mTORC1 to promote autophagic clearance of damaged proteins and organelles, facilitating stress granule formation, and integrating signals from oxidative and endoplasmic reticulum stress to rebalance the proteome. Dysregulated GCN2 activity has been implicated in diverse pathologies including neurodegeneration, cancer and pulmonary vascular disease, positioning it as a promising therapeutic target. In this review, we explore how GCN2 links nutrient sensing to translational control and metabolic adaptation, and how its central role in proteostasis may inform new strategies for treating diseases driven by protein misfolding and stress pathway imbalance.

Female breast development occurs at puberty and undergoes many cyclic changes under normal physiological conditions, like pregnancy, lactation and involution. The breast epithelium is surrounded by a heterogenous stroma that encompasses various cell types, including fibroblasts, immune cells, adipocytes, endothelial cells and an extracellular matrix (ECM). The ECM is a complex molecular meshwork composed of a variety of matricellular proteins and ECM remodelling enzymes, including proteases. Dynamic remodelling of the ECM is fundamental to the organisation and function of the mammary gland and is associated with stemness. It is often aberrantly regulated in breast cancer, which still has the highest incidence and mortality rates in women worldwide. Improved models could contribute to a better understanding of cell-matrix interactions, including in the stem cell niche, and ECM remodelling in health and tumorigenesis. This could lay the groundwork for therapeutic strategies that also target the breast cancer ECM for improved precision medicine tools.