Phase separation drives RNA virus-induced activation of the NLRP6 inflammasome.

NLRP6 is important in host defense by inducing functional outcomes including inflammasome activation and interferon production. Here, we show that NLRP6 undergoes liquid-liquid phase separation (LLPS) upon interaction with double-stranded RNA (dsRNA) in vitro and in cells, and an intrinsically disordered poly-lysine sequence (K350-354) of NLRP6 is important for multivalent interactions, phase separation, and inflammasome activation. Nlrp6-deficient or Nlrp6K350-354A mutant mice show reduced inflammasome activation upon mouse hepatitis virus or rotavirus infection, and in steady state stimulated by intestinal microbiota, implicating NLRP6 LLPS in anti-microbial immunity. Recruitment of ASC via helical assembly solidifies NLRP6 condensates, and ASC further recruits and activates caspase-1. Lipoteichoic acid, a known NLRP6 ligand, also promotes NLRP6 LLPS, and DHX15, a helicase in NLRP6-induced interferon signaling, co-forms condensates with NLRP6 and dsRNA. Thus, LLPS of NLRP6 is a common response to ligand stimulation, which serves to direct NLRP6 to distinct functional outcomes depending on the cellular context.

Aplysia CPEB can form prion-like multimers in sensory neurons that contribute to long-term facilitation.

Prions are proteins that can assume at least two distinct conformational states, one of which is dominant and self-perpetuating. Previously we found that a translation regulator CPEB from Aplysia, ApCPEB, that stabilizes activity-dependent changes in synaptic efficacy can display prion-like properties in yeast. Here we find that, when exogenously expressed in sensory neurons, ApCPEB can form an amyloidogenic self-sustaining multimer, consistent with it being a prion-like protein. In addition, we find that conversion of both the exogenous and the endogenous ApCPEB to the multimeric state is enhanced by the neurotransmitter serotonin and that an antibody that recognizes preferentially the multimeric ApCPEB blocks persistence of synaptic facilitation. These results are consistent with the idea that ApCPEB can act as a self-sustaining prion-like protein in the nervous system and thereby might allow the activity-dependent change in synaptic efficacy to persist for long periods of time.

Exploring the Conformational Landscape of Poly(l-lysine) Dendrimers Using Ion Mobility Mass Spectrometry.

Ion mobility mass spectrometry (IM-MS) measures the mass, size, and shape of ions in the same experiment, and structural information is provided via collision cross-section (CCS) values. The majority of commercially available IM-MS instrumentation relies on the use of CCS calibrants, and here, we present data from a family of poly(l-lysine) dendrimers and explore their suitability for this purpose. In order to test these compounds, we employed three different IM-MS platforms (Agilent 6560 IM-QToF, Waters Synapt G2, and a home-built variable temperature drift tube IM-MS) and used them to investigate six different generations of dendrimers in two buffer gases (helium and nitrogen). Each molecule gives a highly discrete CCS distribution suggestive of single conformers for each m/z value. The DTCCSN2 values of this series of molecules (molecular weight: 330-16,214 Da) range from 182 to 2941 Å2, which spans the CCS range that would be found by many synthetic molecules including supramolecular compounds and many biopolymers. The CCS values for each charge state were highly reproducible in day-to-day analysis on each instrument, although we found small variations in the absolute CCS values between instruments. The rigidity of each dendrimer was probed using collisionally activated and high-temperature IM-MS experiments, where no evidence for a significant CCS change ensued. Taken together, this data indicates that these polymers are candidates for CCS calibration and could also help to reconcile differences found in CCS measurements on different instrument geometries.

Disposable biosensor based on nanodiamond particles, ionic liquid and poly-l-lysine for determination of phenolic compounds.

This study describes the development of a highly sensitive amperometric biosensor for the analysis of phenolic compounds such as catechol. The biosensor architecture is based on the immobilization of tyrosinase (Tyr) on a screen-printed carbon electrode (SPE) modified with nanodiamond particles (ND), 1-butyl-3-methylimidazolium hexafluorophosphate (IL) and poly-l-lysine (PLL). Surface morphologies of the electrodes during the modification process were evaluated by scanning electron microscopy (SEM) and energy-dispersive X-ray spectroscopy (EDX). Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were used to investigate the electrochemical characteristics of the modified electrodes. Owing to the synergistic effect of the modification materials, the Tyr/PLL/ND-IL/SPE exhibited high sensitivity (328.2 µA mM-1) towards catechol with a wide linear range (5.0 × 10-8 - 1.2 × 10-5 M) and low detection limit (1.1 × 10-8 M). Furthermore, the method demonstrated good reproducibility and stability. The amperometric response of the biosensor towards other phenolic compounds such as bisphenol A, phenol, p-nitrophenol, m-cresol, p-cresol and o-cresol was also investigated. The analytical applicability of the biosensor was tested by the analysis of catechol in tap water. The results of the tap water analysis showed that the Tyr/PLL/ND-IL/SPE can be used as a practical and effective method for catechol determination.

Multifunctional Hydrogel Enhances Inflammatory Control, Antimicrobial Activity, and Oxygenation to Promote Healing in Infectious Wounds.

Chronic infected wounds often fail to heal through normal repair mechanisms, and the persistent response of reactive oxygen species (ROS) and inflammation is a major contributing factor to the difficulty in their healing. In this context, we developed an ROS-responsive injectable hydrogel. This hydrogel is composed of ε-polylysine grafted (EPL) with caffeic acid (CA) and hyaluronic acid (HA) grafted with phenylboronic acid (PBA). Before the gelation process, a mixture CaO2@Cur-PDA (CCP) consisting of calcium peroxide (CaO2) coated with polydopamine (PDA) and curcumin (Cur) is embedded into the hydrogel. Under the conditions of chronic refractory wound environments, the hydrogel gradually dissociates. HA mimics the function of the extracellular matrix, while the released caffeic acid-grafted ε-polylysine (CE) effectively eliminates bacteria in the wound vicinity. Additionally, released CA also clears ROS and influences macrophage polarization. Subsequently, CCP further decomposes, releasing Cur, which promotes angiogenesis. This multifunctional hydrogel accelerates the repair of diabetic skin wounds infected with Staphylococcus aureus in vivo and holds promise as a candidate dressing for the healing of chronic refractory wounds.

Effect of Peptide-Polymer Host-Guest Electrostatic Interactions on Self-Assembling Peptide Hydrogels Structural and Mechanical Properties and Polymer Diffusivity.

Peptide-based supramolecular hydrogels are an attractive class of soft materials for biomedical applications when biocompatibility is a key requirement as they exploit the physical self-assembly of short self-assembling peptides avoiding the need for chemical cross-linking. Based on the knowledge developed through our previous work, we designed two novel peptides, E(FKFE)2 and K(FEFK)2, that form transparent hydrogels at pH 7. We characterized the phase behavior of these peptides and showed the clear link that exists between the charge carried by the peptides and the physical state of the samples. We subsequently demonstrate the cytocompatibility of the hydrogel and its suitability for 3D cell culture using 3T3 fibroblasts and human mesenchymal stem cells. We then loaded the hydrogels with two polymers, poly-l-lysine and dextran. When polymer and peptide fibers carry opposite charges, the size of the elemental fibril formed decreases, while the overall level of fiber aggregation and fiber bundle formation increases. This overall network topology change, and increase in cross-link stability and density, leads to an overall increase in the hydrogel mechanical properties and stability, i.e., resistance to swelling when placed in excess media. Finally, we investigate the diffusion of the polymers out of the hydrogels and show how electrostatic interactions can be used to control the release of large molecules. The work clearly shows how polymers can be used to tailor the properties of peptide hydrogels through guided intermolecular interactions and demonstrates the potential of these new soft hydrogels for use in the biomedical field in particular for delivery or large molecular payloads and cells as well as scaffolds for 3D cell culture.

Two-layer elastic models for single-yeast compressibility with flat microlevers.

Unicellular organisms such as yeast can survive in very different environments, thanks to a polysaccharide wall that reinforces their extracellular membrane. This wall is not a static structure, as it is expected to be dynamically remodeled according to growth stage, division cycle, environmental osmotic pressure and ageing. It is therefore of great interest to study the mechanics of these organisms, but they are more difficult to study than other mammalian cells, in particular because of their small size (radius of a few microns) and their lack of an adhesion machinery. Using flat cantilevers, we perform compression experiments on single yeast cells (S. cerevisiae) on poly-L-lysine-coated grooved glass plates, in the limit of small deformation using an atomic force microscope (AFM). Thanks to a careful decomposition of force-displacement curves, we extract local scaling exponents that highlight the non-stationary characteristic of the yeast behavior upon compression. Our multi-scale nonlinear analysis of the AFM force-displacement curves provides evidence for non-stationary scaling laws. We propose to model these phenomena based on a two-component elastic system, where each layer follows a different scaling law..

Polymeric integration of structure-switching aptamers on transistors for histamine sensing.

Aptamers that undergo large conformational rearrangements at the surface of electrolyte-gated field-effect transistor (EG-FETs)-based biosensors can overcome the Debye length limitation in physiological high ionic strength environments. For the sensitive detection of small molecules, carbon nanotubes (CNTs) that approach the dimensions of analytes of interest are promising channel materials for EG-FETs. However, functionalization of CNTs with bioreceptors using frequently reported surface modification strategies (e.g., π-π stacking), requires highly pristine CNTs deposited through methods that are incompatible with low-cost fabrication methods and flexible substrates. In this work, we explore alternative non-covalent surface chemistry to functionalize CNTs with aptamers. We harnessed the adhesive properties of poly-D-lysine (PDL), to coat the surface of CNTs and then grafted histamine-specific DNA aptamers electrostatically in close proximity to the CNT semiconducting channel. The layer-by-layer assembly was monitored by complementary techniques such as X-ray photoelectron spectroscopy, optical waveguide lightmode spectroscopy, and fluorescence microscopy. Surface characterization confirmed histamine aptamer integration into PDL-coated CNTs and revealed ∼5-fold higher aptamer surface coverage when using CNT networks with high surface areas. Specific aptamers assembled on EG-CNTFETs enabled histamine detection in undiluted high ionic strength solutions in the concentration range of 10 nM to 100 µM. Sequence specificity was demonstrated via parallel measurements with control EG-CNTFETs functionalized with scrambled DNA. Histamine aptamer-modified EG-CNTFETs showed high selectivity vs. histidine, the closest structural analog and precursor to histamine. Taken together, these results implied that target-specific aptamer conformational changes on CNTs facilitate signal transduction, which was corroborated by circular dichroism spectroscopy. Our work suggests that layer-by-layer polymer chemistry enables integration of structure-switching aptamers into flexible EG-CNTFETs for small-molecule biosensing.

Peripheral adsorption of polylysine on one leaflet of a lipid bilayer reduces the lipid diffusion of both leaflets.

Understanding polycation-lipid interaction is essential not only in molecular biology but also in the biomedical industry and pharmacology. However, the effect of the polycation-lipid interaction on the molecular properties of lipids in biomembranes remains elusive. Here, two fluorescence correlation spectroscopies (FCSs), pulse-interleaved excitation (PIE) FCS and lifetime-based FCS, were performed to elucidate the change in the lipid diffusion of a model biomembrane induced by polylysine (PLL) adsorption. The results of PIE-FCS showed that the diffusions of both anionic and zwitterionic lipids become slower in the presence of PLL but the mobility of the anionic lipids is much reduced, suggesting the preferential interaction between the PLL and the anionic lipids due to the electrostatic attraction. Furthermore, leaflet-specific lipid diffusion analysis by lifetime-based FCS clearly showed that PLL adsorption on one leaflet of the membrane reduces the lipid diffusion of both leaflets in the same manner. This clearly indicates that the interleaflet coupling is strong in the presence of PLL.

Atomistic insights into the reentrant phase-transitions in polyuracil and polylysine mixtures.

The phase separation of protein and RNA mixtures underpins the assembly and regulation of numerous membraneless organelles in cells. The ubiquity of protein-RNA condensates in cellular regulatory processes is in part due to their sensitivity to RNA concentration, which affects their physical properties and stability. Recent experiments with poly-cationic peptide-RNA mixtures have revealed closed-loop phase diagrams featuring lower and upper critical solution temperatures. These diagrams indicate reentrant phase transitions shaped by biomolecular interactions and entropic forces such as solvent and ion reorganization. We employed atomistic simulations to study mixtures with various RNA-polylysine stoichiometries and temperatures to elucidate the microscopic driving forces behind reentrant phase transitions in protein-RNA mixtures. Our findings reveal an intricate interplay between hydration, ion condensation, and specific RNA-polylysine hydrogen bonding, resulting in distinct stoichiometry-dependent phase equilibria governing stabilities and structures of the condensate phase. Our simulations show that reentrant transitions are accompanied by desolvation around the phosphate groups of RNA, with increased contacts between phosphate and lysine side chains. In RNA-rich systems at lower temperatures, RNA molecules can form an extensive pi-stacking and hydrogen bond network, leading to percolation. In protein-rich systems, no such percolation-induced transitions are observed. Furthermore, we assessed the performance of three prominent water force fields-Optimal Point Charge (OPC), TIP4P-2005, and TIP4P-D-in capturing reentrant phase transitions. OPC provided a superior balance of interactions, enabling effective capture of reentrant transitions and accurate characterization of changes in solvent reorganization. This study offers atomistic insights into the nature of reentrant phase transitions using simple model peptide and nucleotide mixtures. We believe that our results are broadly applicable to larger classes of peptide-RNA mixtures exhibiting reentrant phase transitions.

Folding molecular origami from ribosomal RNA.

Approximately 80 percent of the total RNA in cells is ribosomal RNA (rRNA), making it an abundant and inexpensive natural source of long, single-stranded nucleic acid, which could be used as raw material for the fabrication of molecular origami. In this study, we demonstrate efficient and robust construction of 2D and 3D origami nanostructures utilizing cellular rRNA as a scaffold and DNA oligonucleotide staples. We present calibrated protocols for the robust folding of contiguous shapes from one or two rRNA subunits that are efficient to allow folding using crude extracts of total RNA. We also show that RNA maintains stability within the folded structure. Lastly, we present a novel and comprehensive analysis and insights into the stability of RNA:DNA origami nanostructures and demonstrate their enhanced stability when coated with polylysine-polyethylene glycol in different temperatures, low Mg2+ concentrations, human serum, and in the presence of nucleases (DNase I or RNase H). Thus, laying the foundation for their potential implementation in emerging biomedical applications, where folding rRNA into stable structures outside and inside cells would be desired.

Poly-ICLC, a multi-functional immune modulator for treating cancer.

Immunotherapies have become the first line of treatment for many cancer types. Unfortunately, only a small fraction of patients benefits from these therapies. This low rate of success can be attributed to 3 main barriers: 1) low frequency of anti-tumor specific T cells; 2) lack of infiltration of the anti-tumor specific T cells into the tumor parenchyma and 3) accumulation of highly suppressive cells in the tumor mass that inhibit the effector function of the anti-tumor specific T cells. Thus, the identification of immunomodulators that can increase the frequency and/or the infiltration of antitumor specific T cells while reducing the suppressive capacity of the tumor microenvironment is necessary to ensure the effectiveness of T cell immunotherapies. In this review, we discuss the potential of poly-ICLC as a multi-functional immune modulator for treating cancer and its impact on the 3 above mentioned barriers. We describe the unique capacity of poly-ICLC in stimulating 2 separate pattern recognition receptors, TLR3 and cytosolic MDA5 and the consequences of these activations on cytokines and chemokines production. We emphasize the role of poly-ICLC as an adjuvant in the setting of peptide-based cancer vaccines and in situ tumor vaccination by mimicking natural immune responses to infections. Finally, we summarize the impact of poly-ICLC in enhancing T infiltration into the tumor parenchyma and address the implication of this finding in the clinic.

Roe-inspired stem cell microcapsules for inflammatory bowel disease treatment.

Mesenchymal stem cells (MSCs), which exert regulatory effects on various immune cells, have been a promising therapy for inflammatory bowel disease treatment. However, their therapeutic effects are limited by lack of nutritional supply, immune system attack, and low accumulation on the target site. Here, inspired by the natural incubation mechanism of roe, we present immune-isolating, wet-adhesive, and nutrient-rich microcapsules for therapeutic MSCs encapsulation. The adhesive shells were fabricated by ionic cross-linking of alginate and visible curing of epsilon-poly-L-lysine-graft-methacrylamide and dopamine methacrylamide, which encapsulated the liquid core of the MSCs and roe proteins. Due to the core-shell construction of the resultant microcapsules, the MSCs might escape from attack of the immune system while still maintaining immunomodulating functions. In addition, the roe proteins encapsulated in the core phase offered sufficient nutrient supply for MSCs' survival and proliferation. Furthermore, after intraperitoneal transplantation, the wet-adhesive radicals on the shell surface could immobilize the MSCs-encapsulating microcapsules onto the bowel. Based on these features, practical values of the roe-inspired microcapsules with MSCs encapsulation were demonstrated by applying them to treat dextran sulfate sodium (DSS)-induced colitis through increasing residence time, regulating immune imbalance, and relieving disease progression. We believe that the proposed roe-inspired microcapsules with MSCs encapsulation are potential for clinical application.

Development of collagenous scaffolds for wound healing: characterization and in vivo analysis.

The development of wound dressings from biomaterials has been the subject of research due to their unique structural and functional characteristics. Proteins from animal origin, such as collagen and chitosan, act as promising materials for applications in injuries and chronic wounds, functioning as a repairing agent. This study aims to evaluate in vitro effects of scaffolds with different formulations containing bioactive compounds such as collagen, chitosan, N-acetylcysteine (NAC) and ε-poly-lysine (ε-PL). We manufactured a scaffold made of a collagen hydrogel bioconjugated with chitosan by crosslinking and addition of NAC and ε-PL. Cell viability was verified by resazurin and live/dead assays and the ultrastructure of biomaterials was evaluated by SEM. Antimicrobial sensitivity was assessed by antibiogram. The healing potential of the biomaterial was evaluated in vivo, in a model of healing of excisional wounds in mice. On the 7th day after the injury, the wounds and surrounding skin were processed for evaluation of biochemical and histological parameters associated with the inflammatory process. The results showed great cell viability and increase in porosity after crosslinking while antimicrobial action was observed in scaffolds containing NAC and ε-PL. Chitosan scaffolds bioconjugated with NAC/ε-PL showed improvement in tissue healing, with reduced lesion size and reduced inflammation. It is concluded that scaffolds crosslinked with chitosan-NAC-ε-PL have the desirable characteristics for tissue repair at low cost and could be considered promising biomaterials in the practice of regenerative medicine.

Epsilon-poly-l-lysine alleviates brown blotch disease of postharvest Agaricus bisporus mushrooms by directly inhibiting Pseudomonas tolaasii and inducing mushroom disease resistance.

The natural antimicrobial peptide, epsilon-poly-l-lysine (ε-PL), is widely acknowledged as a food preservative. However, its potential in managing bacterial brown blotch disease in postharvest edible mushrooms and the associated mechanism remain unexplored. In this study, concentrations of ε-PL ≥ 150 mg L-1 demonstrated significant inhibition effects, restraining over 80% of growth and killed over 99% of Pseudomonas tolaasii (P. tolaasii). This inhibition effect occurred in a concentration-dependent manner. The in vivo findings revealed that treatment with 150 mg L-1 ε-PL effectively inhibited P. tolaasii-caused brown blotch disease in Agaricus bisporus (A. bisporus) mushrooms. Plausible mechanisms underlying ε-PL's action against P. tolaasii in A. bisporus involve: (i) damaging the cell morphology and membrane integrity, and increasing uptake of propidium iodide and leakage of cellular components of P. tolaasii; (ii) interaction with intracellular proteins and DNA of P. tolaasii; (iii) inhibition of P. tolaasii-induced activation of polyphenol oxidase, elevation of antioxidative enzyme activities, stimulation of phenylpropanoid biosynthetic enzyme activities and metabolite production, and augmentation of pathogenesis-related protein contents in A. bisporus mushrooms. These findings suggest promising prospects for the application of ε-PL in controlling bacterial brown blotch disease in A. bisporus.

Attapulgite-enhanced ε-poly-l-lysine for heightened antibacterial efficiency against Pseudomonas syringae pv. Tomato.

ε-Poly-l-lysine (ε-PL) is an effective antimicrobial peptide for controlling fungal plant diseases, exhibiting significant antifungal activity and safety. Despite its known efficacy, the potential of ε-PL in combating plant bacterial diseases remains underexplored. This study evaluated the effectiveness of ε-PL and its nanomaterial derivative in managing tomato bacterial spot disease caused by Pseudomonas syringae pv. tomato. Results indicated that ε-PL substantially inhibited the growth of Pseudomonas syringae pv. tomato. Additionally, when ε-PL was loaded onto attapulgite (encoded as ATT@PL), its antibacterial effect was significantly enhanced. Notably, the antibacterial efficiency of ATT@PL containing 18.80 µg/mL ε-PL was even close to that of 100 µg/mL pure ε-PL. Further molecular study results showed that, ATT@PL stimulated the antioxidant system and the salicylic acid signaling pathway in tomatoes, bolstering the plants disease resistance. Importantly, the nanocomposite demonstrated no negative effects on both seed germination and plant growth, indicating its safety and aligning with sustainable agricultural practices. This study not only confirmed the effectiveness of ε-PL in controlling tomato bacterial spot disease, but also introduced an innovative high antibacterial efficiency ε-PL composite with good bio-safety. This strategy we believe can also be used in improving other bio-pesticides, and has high applicability in agriculture practice.

Enhanced ε­poly­L­lysine production in Streptomyces species by combining interspecific hybridization with multiple antibiotic resistance.

To improve the ε-PL production in wild-type strains of Streptomyces. albulus, Streptomyces. noursei, Streptomyces. rochei and Streptomyces. yunnanensis, the interspecific hybridization based on protoplast fusion was first performed. Two-species hybridizations failed to obtain hybrids with significant increase in ε-PL production, but four-species hybridizations succeed in acquiring many high-yield hybrids. 16S rDNA homology alignment and RAPD confirmed that the hybrid HX17 was restructured by integrating gene fragments from S. albulus and S. rochei with S. noursei as the carrier. S. noursei HX17 was subsequently suffered from mutagenesis and genome shuffling combining with multiple antibiotic resistance, and a mutant S. noursei GX6 was obtained with ε-PL yield of 2.23 g/L in shake-flask fermentation. In fed-batch fermentation, the ε-PL production of GX6 reached 47.2 g/L, which was increased by 95.6% to 136.8% over the wild parents. Ribosomal genes associated with antibiotics were sequenced and majority of mutant strains had mutations at different sites, indicating that the increase of antibiotic resistance was strongly associated with them. This research proved that combining interspecific hybridization with multiple antibiotic resistance was as an effective approach to rapidly improve the ε-PL production in Streptomyces species.

A Blood Test for the Diagnosis of Multiple Sclerosis.

Multiple sclerosis (MS) is an autoimmune chronic disease characterized by inflammation and demyelination of the central nervous system (CNS). Despite numerous studies conducted, valid biomarkers enabling a definitive diagnosis of MS are not yet available. The aim of our study was to identify a marker from a blood sample to ease the diagnosis of MS. In this study, since there is evidence connecting the serotonin pathway to MS, we used an ELISA (Enzyme-Linked Immunosorbent Assay) to detect serum MS-specific auto-antibodies (auto-Ab) against the extracellular loop 1 (ECL-1) of the 5-hydroxytryptamine (5-HT) receptor subtype 2A (5-HT2A). We utilized an ELISA format employing poly-D-lysine as a pre-coating agent. The binding of 208 serum samples from controls, both healthy and pathological, and of 104 serum samples from relapsing-remitting MS (RRMS) patients was tested. We observed that the serum-binding activity in control cohort sera, including those with autoimmune and neurological diseases, was ten times lower compared to the RRMS patient cohort (p = 1.2 × 10-47), with a sensitivity and a specificity of 98% and 100%, respectively. These results show that in the serum of patients with MS there are auto-Ab against the serotonin receptor type 2A which can be successfully used in the diagnosis of MS due to their high sensitivity and specificity.

Effects of dietary supplement of ε-polylysine hydrochloride on laying performance, egg quality, serum parameters, organ index, intestinal morphology, gut microbiota and volatile fatty acids in laying hens.

BACKGROUND: ε-polylysine hydrochloride (ε-PLH) is a naturally occurring antimicrobial peptide extensively utilized in the food and medical industries. However, its impact on animal husbandry remains to be further explored. Therefore, the present study aimed to determine the effect of ε-PLH on laying hens' health and laying performance. RESULTS: Dietary supplementation with ε-PLH to the diet significantly increased average egg weight during weeks 1-8. Meanwhile, compared with the control group, supplementation with ε-PLH decreased the feed egg ratio during weeks 9-12 and egg breakage rate during weeks 9-16 ，whereas it increased eggshell strength during weeks 1-4 and 13-16 . The ε-PLH 0.05% group increased yolk percentage during weeks 5-8 and yolk color during weeks 1-4 . Furthermore, ε-PLH supplementation significantly increased the concentrations of total protein, albumin, globulin and reproductive hormones estradiol, as well as decreased interleukin-1 beta and malondialdehyde in the serum. Compared with the control group, supplementation with 0.05% ε-PLH significantly increased the relative abundance of Cyanobacteria and Gastranaerophilales and decreased the abundance of Desulfovibrio and Streptococcus in the cecum microbiota. In addition, ε-PLH 0.1% supplementation also increased acetic acid content in the cecum. CONCLUSION: Dietary supplementation with ε-PLH has a positive impact on both productive performance and egg quality in laying hens. Furthermore, ε-PLH can also relieve inflammation by promoting the immunity and reducing oxidative damage during egg production. ε-PLH has been shown to improve intestinal morphology, gut microbial diversity and intestinal health. © 2023 Society of Chemical Industry.