Hydroxytyrosol (HT), a potent natural antioxidant in olive oil, has broad food and nutraceutical applications, but sustainable, scalable production remains challenging. Here, we achieved efficient de novo HT biosynthesis in Escherichia coli using glucose-glycerol dual carbon sources, combining metabolic engineering and rational enzyme design. We eliminated feedback inhibition, deleted competing pathways, and rationally mutated the rate-limiting enzyme HpaBC (S462A/M293Y), which enhanced catalytic efficiency by improving substrate entry, oxygen availability, and shortening reaction distance, as revealed by molecular dynamics simulations. Global cofactor optimization boosted HT titer to 4.90 g/L in shake flasks. Under optimized dissolved oxygen in a 5 L bioreactor, HT production reached 13.25 g/L, the highest reported titer in E. coli to date, providing a robust strategy for sustainable HT biomanufacturing.

Recent advances in the microbial production of L-arginine and its derivatives using engineering Corynebacterium glutamicum and Escherichia coli.

Bacteria regulate homeostatic growth by adjusting proteome composition. In Escherichia coli, this coordination is mediated by guanosine tetraphosphate and pentaphosphate, collectively termed (p)ppGpp, which couple amino acid supply with ribsosome production. We identified a distinct architecture in Bacillus subtilis, in which guanosine triphosphate (GTP), not (p)ppGpp, controls proteome allocation. Translational inhibition resulted in GTP depletion and suppressed amino acid biosynthesis through feedback inhibition without altering ribosome abundance, establishing a regulated decoupling between total amino acid flux and proteome composition, with flux deviating from proteome-based predictions. By artificially adjusting GTP concentrations, we recoupled flux and proteome, restoring growth to maximal amounts. The regulated suboptimality enables a trade-off to balance growth and stress resilience. Similar GTP-based strategies were present in other Firmicute species, indicating possible evolutionary conservation. Proteome composition and metabolic flux have distinct regulatory layers in some bacteria.

Thalamocortical (TC) cells relay sensory information to the cortex as well as driving their own feedback inhibition through collateral excitation of the thalamic reticular nucleus (TRN). Inhibitory TRN cells are extensively coupled through electrical synapses. While electrical synapses are most often noted for synchronizing rhythmic forms of neuronal activity, their modulation of transient neuronal signals is less understood. Here we sought to characterize how electrical synapses embedded within a network of TRN neurons regulate the processing of ongoing sensory inputs during relay from thalamus to cortex. We constructed a thalamocortical network consisting of reciprocally connected Hodgkin–Huxley-style TC and TRN cells and one cortical output cell summing the TC activity. TRN cells were each electrically coupled to two neighboring cells, forming a ring topology. TC cells received synaptic inputs in sequence, with inputs separated by 10–50 ms, allowing us to assess the functional radius of an electrical synapse by comparing the cumulative effects of each additional TRN electrical synapse on responses within the network. Electrical synapse strength altered both TRN and TC spike response rates and latencies with each additional electrical synapse. Coupling within TRN modulated cortical integration of TC inputs by unexpectedly increasing response rates, duration, and reducing spike correlation to the input sequence that was presented to the TC layer. Thus, embedded TRN electrical synapses exert powerful influence on thalamocortical relay, highlighting the multisynaptic influences of electrically coupled cells on more complex and realistic networks of the brain.

Autophagy constitutes a major adaptive response that preserves cellular and organismal homeostasis during stress. However, stress responses also engage systemic communication pathways that may either maintain resilience or propagate pathology. We previously identified acyl-CoA-binding protein, also known as diazepam-binding inhibitor (DBI/ACBP), as a phylogenetically conserved extracellular factor secreted by stressed cells through an unconventional autophagy-dependent pathway. Once released, extracellular DBI/ACBP acts as a feedback inhibitor of autophagy and promotes metabolic and inflammatory alterations. In our most recent work, we identify regulated cell death as an additional major mechanism responsible for extracellular DBI/ACBP accumulation. Plasma DBI/ACBP concentrations correlate with markers of inflammation, senescence and multiorgan dysfunction in hospitalized patients. Experimentally induced injury to liver, kidney, pancreas or skeletal muscle indistinguishably causes rapid increases in circulating DBI/ACBP. Mechanistically, apoptosis, ferroptosis and necroptosis all provoke loss of intracellular DBI/ACBP together with its extracellular release following plasma membrane permeabilization. Pharmacological inhibition of these death pathways suppresses DBI/ACBP liberation. Across large human cohorts, elevated plasma DBI/ACBP is associated with aging, systemic inflammation, multiorgan dysfunction and future morbidity. We propose that DBI/ACBP is not merely a biomarker of tissue damage but rather a systemic autophagy-inhibitory stress signal contributing to maladaptive interorgan communication during aging and disease.

Animals respond to tactile stimulations of the body with location-appropriate behavior, such as aimed grooming. These responses are mediated by mechanosensory neurons distributed across the body, whose axons project into somatotopically organized brain regions corresponding to body location. How mechanosensory neurons interface with brain circuits to transform mechanical stimulations into location-appropriate behavior is unclear. We previously described the somatotopic organization of bristle mechanosensory neurons (BMNs) around the Drosophila head that elicit a sequence of location-aimed grooming movements (Eichler et al., 2024). Here, we use a serial section electron microscopy reconstruction of a full adult fly brain to identify nearly all of BMN pre- and postsynaptic partners uncovering circuit pathways that control head grooming. Postsynaptic partners dominate the connectome and are both excitatory and inhibitory. We identified an excitatory cholinergic hemilineage (hemilineage 23b), a developmentally related group of neurons that elicits aimed head grooming and exhibits differential connectivity with BMNs from distinct head locations, revealing a lineage-based somatotopically organized parallel circuit architecture. Presynaptic partners provide extensive BMN presynaptic inhibition, consistent with models of sensory gain control as a mechanism of suppressing grooming movements and controlling the sequence. This work provides the first comprehensive map of a somatotopically organized connectome, and reveals how this organization could shape grooming. It also reveals the mechanosensory interface with the brain, illuminating fundamental features of mechanosensory processing, including feedforward excitation and inhibition, feedback inhibition, somatotopic circuit organization, and developmental origins.

NO-donating Bergenin derivatives with enhanced anti-inflammatory activity.

Cysteine biosynthesis is increasingly recognized as a critical determinant of bacterial virulence, highlighting this pathway as a promising reservoir of novel antimicrobial targets. In Pseudomonas aeruginosa, however, the molecular basis of cysteine production has only recently begun to emerge. Here, we identify PA3816 as the major P. aeruginosa serine acetyltransferase (PaCysE), the enzyme responsible for generating the activated serine intermediate that feeds O-acetylserine sulfhydrylase-mediated cysteine synthesis. Through a combination of biochemical and genetic approaches, we demonstrate that PaCysE efficiently catalyzes L-serine acetylation in vitro, and in turn, deletion mutants exhibit cysteine auxotrophy, underscoring its essential contribution to O-acetylserine production. Notably, PaCysE is less sensitive to feedback inhibition by cysteine and does not appear to form the canonical cysteine synthase complex, suggesting a regulatory architecture that diverges from well-characterized orthologs. Loss of PaCysE function has broad physiological consequences, including enhanced biofilm formation, reduced pyocyanin production, and attenuated infectivity in an animal model, linking cysteine biosynthesis directly to pathogen fitness. Finally, we identify a thiazole derivative that inhibits PaCysE activity (IC50 ≈ 30 µM) and suppresses bacterial growth in a cysteine-dependent manner, providing a proof-of-concept for therapeutically targeting this pathway.

In situ immunomodulation prior to surgery represents a promising strategy to elicit potent antitumor immunity and prevent postoperative recurrence, yet it faces the fundamental challenge of balancing sufficient immunostimulation against local hyperinflammation. To address this, we developed a multifunctional nanocomposite hydrogel that synchronizes immunogenic cell death (ICD) induction with multi-target active species regulation. Upon peritumoral co-injection of gallic acid-grafted chitosan (CS-GA), N-aminoethyl-N'-benzoylthiourea-functionalized oxidized hyaluronic acid (OHA-BTU), and allomelanin nanoparticles (pDHN NPs), near-infrared irradiation triggers photothermal ablation and generates reactive oxygen species (ROS), leading to tumor ICD and in situ antigen release. Simultaneously, OHA-BTU sequesters extracellular Cu²⁺ released from ROS-disrupted copper-associated proteins, while pDHN NPs capture cfDNA liberated from damaged nuclei and mitochondria (capture capacity = 258.4 μg mg⁻¹), thereby suppressing M1 macrophage hyperactivation and pro-inflammatory cytokine secretion. The catechol groups in CS-GA further scavenge extracellular ROS via redox cycling. This coordinated modulation of active species alleviates local hyperinflammation while preserving the viability and function of tumor-infiltrating T cells. After 14 days of treatment, significant tumor downstaging was achieved, with a volume reduction rate > 60.2% and a mitotic index of 4.2 per high-power field. In a contralateral rechallenge model, elevated infiltration of CD8⁺ T cells and CD8⁺ effector memory T cells correlated with 72.5% recurrence inhibition and 86.8% metastasis suppression. By integrating in situ vaccination with precise regulation of the immunoinflammatory microenvironment, this hydrogel-based platform functions as an effective preoperative in situ therapeutic vaccine, eliciting robust immune memory that prevents postsurgical recurrence and metastasis in breast cancer. STATEMENT OF SIGNIFICANCE: In situ immunomodulation prior to surgery faces the fundamental challenge of balancing sufficient immunostimulation against local hyperinflammation. Insufficient release diminishes immune stimulation, excessive release triggers local hyperinflammation and negative feedback inhibition of adaptive immunity. To address this, allomelanin nanoparticles were employed to induce photothermal immunogenic cell death under NIR irradiation and capture extracellular cell-free DNA to block TLR-9-mediated pro-inflammatory signaling in macrophages. N-aminoethyl-N'-benzoylthiourea-functionalized oxidized hyaluronic acid captures extracellular Cu2⁺ to inhibit CD44-accelerated metal uptake and subsequent pro-inflammatory metabolic reprogramming. Gallic acid-grafted chitosan scavenges extracellular reactive oxygen species via catechol redox reactions. These components synergistically suppress excessive local inflammation, prevent T-cell exhaustion, and elicit robust immune memory that prevents postsurgical recurrence and metastasis in breast cancer.

Purpose This paper aims to design a memristor-based associative memory neural network circuit with emotional overshadowing and congruent effects, considering overshadowing and recovery processes under multiple emotions. Design/methodology/approach The proposed system simulates both overshadowing and recovery under multiple emotional conditions and it mainly includes the following three parts The memory block can realize the learning and forgetting process in Pavlovian associative memory The emotion block can recognize different emotional stimuli which in turn produce different emotional outputs and return to normal when the stimuli disappear The feedback inhibition block can realize the overshadowing between positive music and negative music. Findings The memory block, the emotion block and the feedback inhibition block can work together to realize the functions of overshadowing and recovering from overshadowing under different emotions as well as the obstructing phenomenon caused by long-term overshadowing. Originality/value In this paper, the authors design a memristor-based associative memory neural network circuit with overshadowing and congruent effects to realize the overshadowing and recovery of different emotions. The designed circuit realizes the functions of memory generation and forgetting, as well as judgment of musical stimuli in different emotions, overshadowing and obstructing caused by long-term overshadowing, and recovery after overshadowing, individually.

Suppressor of cytokine signaling 2 (SOCS2) is a classical feedback inhibitor of JAK-STAT signaling, but its role in porcine adipose-derived mesenchymal stem cells (PADSCs) and fat deposition remains unclear. Here, we investigated the function and mechanism of SOCS2 in PADSC proliferation and adipogenic differentiation. SOCS2 overexpression reduced colony number, slowed CCK-8 growth curves, and induced G0/G1 arrest, whereas SOCS2 knockdown enhanced proliferation and promoted the G1/S transition. In parallel, SOCS2 overexpression decreased lipid droplet accumulation and downregulated adipogenic markers, while SOCS2 silencing produced the opposite effects, indicating that SOCS2 negatively regulates both PADSC expansion and adipocyte formation. Transcriptomic analysis showed that SOCS2 mainly influenced pathways related to extracellular matrix organization, cell cycle, and cytokine/inflammatory signaling. At the protein level, SOCS2 silencing increased total JAK2 and STAT3, whereas SOCS2 overexpression reduced JAK2 abundance and STAT3 activation, accompanied by concordant changes in Cyclin D1, CDK2, and PCNA. Collectively, these results suggest that SOCS2 exerts dual negative effects on PADSC proliferation and adipogenic differentiation, accompanied by changes in JAK2-STAT3 signaling. These findings provide functional evidence supporting SOCS2 as a candidate gene related to porcine fat deposition and provide a basis for further genetic validation in pig breeding populations.

Iron dysregulation in the brain is a pathological hallmark of Parkinson's disease (PD) and contributes to oxidative stress and neurodegeneration. How the blood-brain barrier (BBB), the principal regulator of brain iron homeostasis, is implicated in this process particularly in the context of dopaminergic therapies remains unclear. In the brain interstitial space, apo-transferrin (apo-Tf), signals iron deficiency and promotes endothelial iron release, whereas holo-transferrin (holo-Tf) signals iron sufficiency and suppresses export. Here, we investigated how dopaminergic drugs modulate this transferrin-dependent regulatory system using human iPSC-derived brain endothelial cells. We quantified 55Fe release, intracellular iron retention, and expression of key iron-regulatory proteins. Our findings showed that L-DOPA acts as a potent driver of iron release, significantly increasing 55Fe efflux within just 1h and sustaining it through 24h. Importantly, apo- and holo-Tf preserved their opposing regulatory roles, however, the overall magnitude of iron export was increased under both conditions. These results demonstrated that L-DOPA acts by altering the normal parenchymal fine-tuning of endothelial iron release. Additionally, L-DOPA treatment reduced expression of the iron export protein ferroportin (FPN1) but intracellular 55Fe levels decreased, suggesting that FPN1 downregulation reflects a compensatory response to iron depletion rather than a limitation on export. Holo-Tf selectively downregulated transferrin receptor 1 (TfR1) and ferritin light chain (FTL), consistent with feedback inhibition of iron import and storage under iron-sufficient signaling. Selegiline, by contrast, produced delayed and modest effects and preserved transporter and ferritin expression across both apo- and holo-Tf conditions. These findings identify a previously unrecognized direct interaction between dopaminergic therapy and parenchymal transferrin signaling, positioning L-DOPA as a significant modulator of BBB iron export and revealing a mechanism through which PD treatments may negatively influence regional brain iron balance.

Maintenance of S-adenosylmethionine (SAM) homeostasis is essential for methylation of biomolecules, nucleotide and polyamine synthesis, and redox homeostasis. While all methyltransferases consume SAM, only a subset of highly tissue specific methyltransferases regulates SAM homeostasis. Among them, glycine N-methyltransferase (GNMT) is enriched in the liver and its dysregulated activity has been linked to compromised liver function. GNMT is inhibited by the methyl carrier 5-methyltetrahydrofolate (5mTHF), suggesting a negative-feedback mechanism regulating its activity. Here, we identify the GNMT N-terminal tail, and specifically phosphorylation at serine 9 (S9ph), as a regulatory modification linking folate-dependent feedback inhibition to SAM homeostasis. Structural and biochemical analyses and molecular dynamics simulations revealed that the N-terminal tail is required for catalytic turnover of SAM and for 5mTHF binding. Phosphoproteomic analysis showed that GNMT S9ph is abundant in mouse liver and further enriched in aged mice. Consistent with loss of folate-dependent negative feedback, both distal N-terminal truncation (residues 1-8) and a phosphomimetic substitution abolished 5mTHF binding while maintaining catalytic activity. In hepatocyte cell lines lacking endogenous GNMT, lentiviral overexpression of constitutively active GNMT mutants depleted SAM, increased SAH, disrupted protein methylation, impaired growth, and induced transcriptional responses consistent with methyl-donor stress. Together, these findings identify the GNMT N-terminus as a tunable phosphoregulatory domain that dynamically regulates GNMT activity and cellular methylation potential.

Riboflavin is an essential water-soluble vitamin that serves as a precursor for the biosynthesis of the flavin cofactors FMN and FAD, which play pivotal roles in numerous redox and energy metabolism reactions. With the growing global demand for sustainable vitamin production, microbial fermentation has become an attractive alternative to chemical synthesis due to its environmental and economic advantages. Among microbial hosts, Bacillus subtilis has emerged as a leading cell factory for riboflavin production owing to its GRAS status, well-characterized genetics, and efficient protein secretion system. This review provides a comprehensive overview of recent advances in metabolic engineering strategies to enhance riboflavin biosynthesis in B. subtilis. Key topics include strengthening biosynthetic and precursor pathways, relieving feedback inhibition, balancing metabolic flux and cell growth, employing adaptive laboratory evolution, and utilizing omics-guided optimization and 13C metabolic flux analysis. Moreover, the integration of synthetic biology tools such as riboswitch engineering, regulatory element design, and high-throughput screening has significantly accelerated strain improvement. Despite remarkable progress, challenges remain in achieving precise regulatory control, optimizing multi-gene expression, and enhancing genome integration efficiency. Future research combining multi-omics data, synthetic regulatory design, and machine learning-driven predictive modeling is expected to further advance the development of intelligent B. subtilis cell factories. However, the practical implementation of these systems remains constrained by the metabolic burden of overproduction and the lack of universal regulatory models that can predict strain performance across varying industrial scales.

Human milk oligosaccharides (HMOs) are key functional components of human milk. This study reports a modular systems engineering framework in Escherichia coli MG1655 for the efficient synthesis of neutral core HMOs, including lacto-N-triose II (LNTri II), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT). The strategy involved the sequential optimization of three modules: glycosyltransferase expression, nucleotide sugar donor supply (via gene integration and feedback inhibition removal), and transport engineering. First applied to construct a high-yield LNTri II platform, the same modular logic was then adapted by swapping the terminal glycosyltransferase to generate LNT or LNnT producers. All strains were constructed via stable chromosomal integration, ensuring plasmid-free, inducer-free operation. In 5-L bioreactors, the engineered strains achieved high titers of 81.96 g/L LNTri II, 78.52 g/L LNT, and 43.17 g/L LNnT. This work provides a streamlined and adaptable paradigm for developing microbial cell factories for complex oligosaccharide synthesis.

Visual motion information is essential to guiding the movements of many animals. The establishment of direction-selective signals, a hallmark of motion detection, is considered a core neural computation and has been characterized extensively in primates, mice, and fruit flies. In flies, the circuits that produce direction-selective signals rely on feedforward visual pathways that connect peripheral visual inputs to the dendrites of the ON and OFF-direction-selective cells. Here, we describe a novel role for feedback inhibition in motion computation. Two GABAergic neurons, C2 and C3, connect to neurons upstream of the direction-selective T4 and T5 cells, and blocking C2 and C3 affects direction selectivity in T4/T5. In the ON pathway, this is likely achieved by C2-mediated suppression of responses in the major T4 input neuron Mi1. Together, C2 and C3 suppress responses to non-preferred stimuli in both T4 and T5. At the behavioral level, feedback inhibition temporally sharpens responses to ON-moving stimuli, enhancing the fly's ability to discriminate visual stimuli that occur in quick succession. GABAergic inhibitory feedback neurons thus constitute an essential component within the circuitry that computes visual motion.

BackgroundBacillus methanolicus MGA3 is a thermotolerant methylotroph that utilizes methanol, a renewable C₁ substrate, as its sole carbon and energy source. The strain naturally overproduces and secretes L-glutamate, making it a promising platform for engineering pathways toward L-glutamate-derived amino acids such as L-proline, which has applications in nutrition, stress protection, and industry.ResultsHeterologous expression of an osmotic stress–responsive L-proline biosynthetic operon from the mesophile Bacillus licheniformis in the thermotolerant B. methanolicus strain MGA3 did not increase L-proline levels but instead led to accumulation of L-citrulline. This was likely due to heat sensitivity of pyrroline-5-carboxylate reductase (ProH), the last enzyme of the osmoregulatory L-proline biosynthetic route, and metabolic crosstalk between L-proline and L-arginine pathways operating in Bacilli. To overcome these limitations, a synthetic operon containing the native anabolic proBA and proI L-proline biosynthetic genes from B. methanolicus MGA3 was engineered to remove transcriptional T-box regulation and biochemical feedback inhibition of ProB enzyme activity. Expression of this engineered operon enhanced L-proline synthesis and triggered its secretion during methanol-based growth of B. methanolicus MGA3 at 50° C. In fed-batch fermentation with methanol as carbon and energy source, extracellular L-proline levels reached 262 ± 20 mg L⁻1 after 40 h. During the fermentation process, a stepwise increase in medium osmolarity was observed, likely due to large-scale L-glutamate excretion, which impaired cellular growth.ConclusionsThis study links osmolarity dynamics to methanol-based fermentation in B. methanolicus MGA3 and demonstrates its potential as a cell factory for L-proline and L-citrulline production. These findings support further strain optimization for producing value-added amino acids and highlight the relevance of methylotrophic thermophiles in sustainable biotechnology.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12934-026-03032-8.

Motion is an important feature of visual scenes and retinal neuronal circuits selectively signal different motion features. It has been shown that the retina can extrapolate the position of a moving object, thereby compensating sensory transmission delays and enabling signal processing in real-time. Amacrine cells, the inhibitory interneurons of the retina, play essential roles in such computations although their precise function remain unclear. Here, we computationally explore the potential effects of two different inhibitory connectivity motifs on the retina's response to moving objects, in a simplified model of the retina: feed-forward and recurrent feed-back inhibition. In this model, both motifs can account for motion anticipation with two different mechanisms. Feed-forward inhibition truncates motion responses and shifts peak responses forward via subtractive inhibition, whereas recurrent feed-back coupling evokes excitatory and inhibitory waves with different phases that interfere and shift the response peak. A key difference between the two mechanisms is how the anticipatory peak shift scales with the speed of a moving object. Motion prediction with feed-forward circuits monotonically decreases with increasing speeds, while recurrent feed-back coupling induces tuning curves that exhibit a preferred speed for which motion prediction is maximal.

Aspergillus niger, a generally recognized as safe (GRAS) filamentous fungus, is a promising chassis for edible mycelia but lacks meat-like sensory properties. Here, modular metabolic engineering including relieving feedback inhibition (ΔHRM), overexpressing rate-limiting enzymes, and enhancing iron uptake (ΔsreA) was employed to enhance heme biosynthesis, endowing mycelia with a meat-red color. Untargeted metabolomics uncovered a heme tolerance mechanism via precursor efflux mediated by ABC transporters. Heterologous expression of hemoglobins and a P450 enzyme further improved heme utilization, and genome-wide variations analysis uncovered adaptive responses to oxidative and endoplasmic reticulum (ER) folding stress under high-heme pressure. Notably, this enhanced heme supply substantially boosted functional metabolite production, increasing monacolin J yield by 9.99-fold and ergothioneine yield by 1.11-fold. The engineered mycelium closely resembled commercial plant-based meat in color. This study provides a feasible approach to developing A. niger as a sustainable chassis for functional meat alternatives.

2-Oxo acid dehydrogenase complexes include four members to oxidatively decarboxylate pyruvate, 2-oxoglutarate, 2-oxoadipate, and branched-chain 2-oxo acids. The complexes produce CO2, acyl-coenzyme As (acyl-CoAs), and NADH through consecutive action of three enzymes with their coenzymes: 2-oxo acid dehydrogenase with thiamine diphosphate, dihydrolipoyllysine-residue acyltransferase with the lipoyllysine prosthetic group, and dihydrolipoyl dehydrogenase with FAD. Some of the complexes include additional regulatory proteins. Producing energy in the form of NADH and acyl-CoAs, which are feedback inhibitors, the complexes mediate the Ca2+ activation of mitochondrial function, couple metabolism of glucose and amino acids, are essential for biosynthesis of the signaling molecules acetylcholine and glutamate, regulate metabolism through posttranslational acylations including histones acylations, and sense the metabolic imbalance as nonoptimal ratios of their substrates and products. The complexes signal the imbalance by the generation of reactive species: O2*-, thiyl radicals, thiamine-dependent carbon radicals, and/or peracids, potentially involved in DNA damage and development of diseases.