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Ferulic acid ethyl ester (FAEE) is an essential raw material for the formulation of drugs for cardiovascular and cerebrovascular diseases and leukopenia. It is also used as a fixed aroma agent for food production due to its high pharmacological activity. In this study, the interaction of FAEE with Human serum albumin (HSA) and Lysozyme (LZM) was characterized by multi-spectrum and molecular dynamics simulations at four different temperatures. Additionally, the quenching mechanism of FAEE-HSA and FAEE-LZM were explored. Meanwhile, the binding constants, binding sites, thermodynamic parameters, molecular dynamics, molecular docking binding energy, and the influence of metal ions in the system were evaluated. The results of Synchronous fluorescence spectroscopy, UV-vis spectroscopy, CD, three-dimensional fluorescence spectrum, and resonance light scattering showed that the microenvironment of HSA and LZM and the protein conformation changed in the presence of FAEE. Furthermore, the effects of some common metal ions on the binding constants of FAEE-HSA and FAEE-LZM were investigated. Overall, the experimental results provide a theoretical basis for promoting the application of FAEE in the cosmetics, food, and pharmaceutical industries and significant guidance for food safety, drug design, and development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A hybrid receptor-sensor for anions originating from the merging of positively charged ammonium moieties for electrostatic attraction/stronger binding of azacrowns with directionality of calixpyrrole hydrogen bond donors for selectivity is investigated. As demonstrated this hybrid receptor-sensor shows a remarkable selectivity for orthophosphate even in the presence of other phosphates and anions found in cellular materials (K assoc H 2 PO 4 - >H 2 P 2 O 7 2- >AMP - ≫ADP 2- or ATP 3- over halides, nitrate, or hydrogen sulfate; all Na + salts in water) but also cellular polyphosphate or phospholipids. This selectivity is harnessed in a real-time monitoring of cell lysis by lysozyme, which releases orthophosphate and other phosphates and anions from the cells. This sensitive (LOD 0.4 μM) fluorescence-based microscale method compares favorably with the state-of-the-art techniques but can easily be practiced in a high-throughput screening (HTS) manner. The anion binding and selectivity in aqueous solutions were investigated by NMR and put in context with phosphate binding of the parent calix[4]pyrrole. The microscopic understanding of anion binding by the hybrid receptor was then obtained from a combination of density functional theory (DFT), classical molecular dynamics (MD) with explicit water solvation, and ab initio MD (AIMD) simulations. Correlating the NMR and fluorescence binding data with studies of solvation of the receptor, phosphate anion, and the resulting complex confirms the binding is largely driven by entropic component (TΔS) associated with receptor and anion desolvation., (© 2024 Oak Ridge National Laboratory and The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH on behalf of Chemistry Europe.)

Lysozymes have gained attention for their antiseptic properties. In silico studies have shown that the enzyme containing lysM can act as an antibacterial agent. Binding of the lysM motif of rSELys to peptidoglycan and molecular dynamics simulations showed that the protein-ligand binding is very stable. rSELys (2016 bp) is a new recombinant glycoside hydrolase from the thermophilic bacterium Cohnella sp. A01 (PTCC number: 1921). Protein expression and purification, a single band with an apparent molecular weight of ∼74 kDa was observed by SDS-PAGE. The kinetic parameters were K m 1.163 mg/ml, Vmax 670.3 U/mg, k cat 1675.75 (S -1 ), and k cat /K m 1440.88 (M -1 S -1 ). Its optimum temperature was 55 °C and pH 8. Temperature stability also showed that the temperature of 50-60 °C retained more than half of its activity after 90 min. Based on the results, rSELys demonstrated antibacterial effects on both Gram-positive and Gram-negative strains, with inhibition zones of 11 and 9 mm, respectively. SEM analysis confirmed hydrolysis activity, the MIC was determined to be 31.25 μg/ml and 3.9 μg/ml, and MBC 0.97 μg/ml, respectively. CD and fluorescence studies showed that up to a temperature of 85 °C and a pH value of 8-12 no structural changes occur, and thermal stability protein was confirmed., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Recently, buffer molecules have been known to affect intermolecular protein-protein interactions at physiological pH. However, the roles of buffer molecules and different monolayer protein concentrations remain elusive in controlling the interaction of gold nanoparticles (Au NPs) with protein molecules. Herein, for the first time taking phenylalanine functionalized gold nanoparticles (Au-Phe NPs) and lysozyme (Lyz) protein as model systems, we report that buffer molecules of different charges (at a particular pH) play diverse roles in protein-Au NPs interaction, particularly in protein induced Au NPs aggregation. Among different buffers, negatively charged buffer (citrate and phosphate) induces aggregation of both Au-Phe NPs and Lyz protein, whereas zwitterionic and positive buffer (HEPES, MOPS, and Tris) only cause the Au NPs aggregation. Taking the diverse role of buffer into account, we propose multimodal models for stability and protein induced aggregation mechanism of NPs at different monolayer (sub-, near-, and excess) concentrations of Lyz in different medium.

Mapping disulfide linkages is crucial for characterizing pharmaceutical proteins during drug development and quality control. Traditional bottom-up protein analysis workflows often suffer from incomplete mapping for tryptic peptides consisting of multiple disulfide bonds. Although the employment of a partial reduction of disulfide bonds can improve disulfide mapping, it becomes a bottleneck of analysis because individual tuning is often needed. Herein, we have developed an online disulfide-ene reaction system in which the composition of the reaction solvent can be programmed to achieve optimal partial reduction of tryptic disulfide peptides after liquid chromatography separation. By coupling this system onto a bottom-up protein analysis workflow, high coverage for sequencing (71-83%) and disulfide mapping (84-100%) was achieved for standard proteins consisting of 4-19 disulfide bonds. The analytical capability was further demonstrated by mapping 13 scrambled disulfide bonds in lysozyme and achieving compositional analysis of IgG isotypes (κ and λ) and subclasses (IgG1-IgG4) from human plasma.

Nanostructured materials represent promising substrates for biocatalyst immobilization and activation. Cellulose nanocrystals (CNCs), accessible from waste and/or renewable sources, are sustainable and biodegradable, show high specific surface area for anchoring a high number of enzymatic units, and high thermal and mechanical stability. In this work, we present a holistic enzyme-based approach to functional antibacterial materials by bioconjugation between the lysozyme from chicken egg white and enzymatic cellulose nanocrystals. The neutral CNCs were prepared by endoglucanase hydrolysis from Avicel. We explore the covalent immobilization of lysozyme on enzymatic CNCs and on their TEMPO oxidized derivatives (TO-CNCs), comparing immobilization yields, material properties, and enzymatic activities. The materials were characterized by X-ray diffractometry (XRD), attenuated total reflectance Fourier Transform infrared spectroscopy (ATR-FTIR), bicinchoninic acid (BCA) assay, field-emission scanning electron microscopy (FE-SEM) and dynamic light scattering (DLS). We demonstrate the higher overall efficiency of the immobilization process carried out on TO-CNCs, based on the success of covalent bonding and on the stability of the isolated bioconjugates., (© 2024 The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH.)

Metals have been used in medicine for centuries. However, it was not until much later that the effects of inorganic drugs could be rationalized from a mechanistic point of view. Today, thanks to the technologies available, this approach has been functionally developed and implemented. It has been found that there is probably no single biological target for the pharmacological effects of most inorganic drugs. Herein, we present an overview of some integrated and multi-technique approaches to elucidate the molecular interactions underlying the biological effects of metallodrugs. On this premise, selected examples are used to illustrate how the information obtained on metal-based drugs and their respective mechanisms can become relevant for applications in fields other than medicine. For example, some well-known metallodrugs, which have been shown to bind specific amino acid residues of proteins, can be used to solve problems related to protein structure elucidation in crystallographic studies. Diruthenium tetraacetate can be used to catalyze the conversion of hydroxylamines to nitrones with a high selectivity when bound to lysozyme. Finally, a case study is presented in which an unprecedented palladium/arsenic-mediated catalytic cycle for nitrile hydration was discovered thanks to previous studies on the solution chemistry of the anticancer compound arsenoplatin-1 (AP-1)., (© 2024 The Author(s). Chemistry - A European Journal published by Wiley-VCH GmbH.)

Antifouling properties are crucial for enhancing the longevity and functionality of biomedical implants, drug delivery systems, and biosensors. Zwitterionic polymers are renowned for their exceptional surface hydration and charge neutrality, which effectively resist biomolecular adsorption and protein attachment. We propose an innovative approach to develop zwitterion-like antifouling surfaces by chelating divalent cations with anionic poly(3,4-ethylenedioxythiophene) (PEDOT) films, specifically PEDOT-PO 4 and PEDOT-COOH. The chelation behavior of these films was systematically evaluated using Na + , Mg 2+ , and Ca 2+ ions. Divalent ions, particularly Ca 2+ and Mg 2+ , exhibit a strong affinity for the anionic groups, leading to significant antifouling properties. These modified surfaces effectively repelled both negatively charged bovine serum albumin (BSA) and positively charged lysozyme (LYZ) proteins across various pH environments. This study offers valuable insights into the antifouling characteristics of charged surfaces, enhancing our understanding of how ion-mediated surface modifications influence protein adsorption and interactions.

Oligomers and amyloid fibrils formed at different stages of protein aggregation are important biomarkers for a variety of neurodegenerative diseases including Alzheimer's and Parkinson's diseases. The development of probes for the sensitive detection of oligomeric species is important for early stage diagnosis of amyloidogenic diseases. Many small molecular dyes have been developed to probe the dynamic growth of amyloid fibrils. However, there is a lack of discriminatory detection strategies to monitor the dynamics of both oligomers and amyloid fibrils based on the differential modulation of the photophysical properties of a single dye. Here we report a pyrene-based intramolecular charge transfer (ICT) dye with large Stokes shifted red-emitting aggregation induced emission (AIE) for monitoring the dynamic populations of both oligomers and fibrils during the aggregation of hen egg white lysozyme (HEWL) protein. At the early stage of protein aggregation, the accumulation of HEWL oligomers results in a rapid and substantial increase in the red AIE intensity at 660 nm. Later, as the oligomers transform into mature fibrils, the dye exhibits a distinct photophysical change. Binding of the dye to HEWL fibrils strongly suppresses the red AIE and enhances ICT emission. This is evidenced by a gradual decrease in the AIE intensity (∼660 nm) and an increase in LE (∼490 nm) and ICT (∼540 nm) emission intensities during the later stages of protein aggregation. Thus, the dye provides simultaneous measurements of the population dynamics of both HEWL oligomers and fibrils during protein aggregation based on the discriminatory modulation of AIE and ICT of the dye. The dye also enables imaging of both HEWL oligomers and fibrils simultaneously using different emission channels in super-resolution confocal fluorescence microscopy.

Misfolding and amyloid fibrillogenesis of proteins have close relationships with several neurodegenerative diseases. The present work investigates the inhibitive activities of ankaflavin (AK) and monascin (MS), two yellow pigments separated from Monascus -fermented rice, on hen egg white lysozyme (HEWL) fibrillation. The results demonstrated that AK/MS suppressed HEWL fibrillation through interfering with the nucleation period and AK was more potent. Fluorescence quenching and in silico docking studies revealed that AK/MS bond to HEWL by the formation of noncovalent forces with some critical amino acid residues that tend to form fibrils. Compared to those of AK, hydrogen bonding interactions between MS and Asn46, Trp62, and Trp63 residues in HEWL were slightly weaker. Besides, the covalent interaction between MS and HEWL with the binding site of Arg68 was found. These observations offered reasonable explanations for the difference in the mechanisms of AK and MS inhibiting HEWL fibrillogenesis. In a word, all data acquired herein indicated AK/MS as potent candidates for the improvement and treatment of neurological disorders.

Protein immobilization using water-soluble ionic metal-organic polyhedra (MOPs) acting as porous spacers has recently been demonstrated as a potent strategy for the preparation of biocatalysts. In this article, we describe a mixed-protein approach to achieve biocomposites with adjustable enzyme contents and excellent immobilization efficiencies, in a systematic and well-controlled manner. Self-assembly of either cationic or anionic MOPs with bovine serum albumin or egg white lysozyme combined with enzymes (alkaline phosphatase, laccase or cytochrome c) led to solid-state catalysts with a high retention of enzyme activity. Furthermore, for all these systems, the dilution of enzymes within the solid-state composite led to noticeably improved catalytic performances, with both higher specific activity and affinity for substrate.

Introduction: The human-derived amnion-chorion membrane (ACM) has endogenous antimicrobial properties, which are important for preventing the colonization and survival of oral bacteria on exposed membranes. This project aimed to decipher the underlying mechanism by identifying the components of ACM that confer antibacterial properties. In addition, the antimicrobial efficacy of these identified components on oral bacteria was assessed., Methods: Four antimicrobial proteins, histone H2A/H2B, cathelicidin LL-37, lactoferrin, and lysozyme, were identified via mass spectrometry in ACM. These proteins were then assessed for their efficacy in killing Streptococcus gordonii Challis. Log-phased bacterial cells were cultured with the commercially available proteins that were identified in ACM, either individually or in combination, at different concentrations. After incubation for 8 or 24 hours, the bacteria were stained with a live/dead viability kit and analyzed via confocal microscopy., Results: The combination of these proteins effectively killed S. gordonii in a dose-dependent fashion after 8 or 24 hours of incubation. When each protein was tested individually, it killed S. gordonii at a much lower efficacy relative to the combinations. The synergistic effects of the antimicrobial protein combinations were also observed in both the viable cell count recovery and minimum inhibitory concentration assays., Discussion: By shedding light on the mechanisms in the ACM's antimicrobial property, this study may raise more awareness of the potential benefit of utilization of a membrane with endogenous antimicrobial properties in regeneration surgeries., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Brummerhop, Lee, Weltman, Tribble, van der Hoeven, Chiu, Hong and Wang.)

This 60-day study aimed to examine the efficacy of a diet supplemented with mandarin peel powder (MP) in enhancing the health and survival of Oreochromis niloticus under suboptimal temperature conditions (21 ℃). One hundred and eighty Nile tilapia fish (22.51 ± 0.04 g) were randomly distributed into four experimental groups; each of 3 replicates (15 fish per replicate). The first group (CONT) received a basal diet without MP. The second (MP10%), third (MP15%), and fourth (MP20%) groups were fed diets containing 10, 15, and 20% MP powder, respectively. At the end of the feeding trail, growth performance, serum growth hormone, α-amylase enzyme, lysozyme activity, nitric oxide, protease activity, globulin, serum levels of IL-1ß, antioxidant status, and intestinal histology were measured. The results showed insignificant differences between CONT, MP15%, and MP20% groups in the final body weight and specific growth rate. The growth hormones in the MP15% and MP20% groups did not show a significant difference compared to fish fed a normal basal diet (CONT). However, the amylase enzymes were significantly greater in both groups. The MP20% and MP15% groups showed a significant increase in antioxidant, lysozyme, nitric oxide, and protease activities compared to CONT. The results also showed that fish that were fed a diet with MP had significantly less of the pro-inflammatory cytokine interleukin-1 beta, and their intestinal villi got wider, especially in the MP20% group. It could be concluded that feeding tilapia on a diet with 20% MP is an effective strategy to improve their health when the temperature is below 21 °C. This is because the fish exhibit higher levels of antioxidant activity, reduced pro-inflammatory responses, and improved intestinal health without difference in the growth performance in compared to control group., (© 2024. The Author(s).)

Gene-editing technology has become a transformative tool for the precise manipulation of biological genomes and holds great significance in the field of animal disease-resistant breeding. Mastitis, a prevalent disease in animal husbandry, imposes a substantial economic burden on the global dairy industry. In this study, a regulatory sequence gene editing breeding strategy for the successful creation of a gene-edited dairy (GED) goats with enhanced mastitis resistance using the ISDra2-TnpB system and dairy goats as the model animal is proposed. This included the targeted integration of an innate inflammatory regulatory sequence (IRS) into the promoter region of the lysozyme (LYZ) gene. Upon Escherichia Coli (E. coli) mammary gland infection, GED goats exhibited increased LYZ expression, showing robust anti-mastitis capabilities, mitigating PANoptosis activation, and alleviating blood-milk-barrier (BMB) damage. Notably, LYZ is highly expressed only in E. coli infection. This study marks the advent of anti-mastitis gene-edited animals with exogenous-free gene expression and demonstrates the feasibility of the gene-editing strategy proposed in this study. In addition, it provides a novel gene-editing blueprint for developing disease-resistant strains, focusing on disease specificity and biosafety while providing a research basis for the widespread application of the ISDra2-TnpB system., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

The objective of the present study was to compare the effectiveness of dietary supplementation of muramidase (MUR) and 2 phytogenic additives on the growth performance, intestinal morphology, bacteria load, and production of short-chain fatty acids (SCFA) of broiler chickens raised under field-like conditions. A total of 6,400 day-old Ross 308 broiler chicks were randomly selected and distributed into 32 floor pens, with 200 chicks (100 males and 100 females)/pen. The treatment groups were an unsupplemented control, and the experimental groups supplemented with MUR at 35,000 LSU(F)/kg of feed, phytogenic 1 (Phyto 1, based on thymol) at 100g/ton feed, or phytogenic 2 (Phyto 2, based on alkaloids) at 60g/ton feed, for a total period of 41 d. A 4-phase feeding program was applied (starter, grower, finisher and withdrawal). The paramenters evaluated were: growth performance, carcass yield, concentration of muranic acid in the jejunum content and excreta, liver enzyme concentration, intestinal morphology, and bacteria enumeration and short and branch chain fatty acids (SCFA and BCFA) in the cecal content. Data were analyzed by ANOVA and Tukey's test was used to separate the means. Soluble muramic acid (MurN) in the jejunum increased with the supplementation of MUR and Phyto 2 when compared to the other groups (P = 0.0001), but only the supplementation of MUR increased the concentration of MurN in the excreta. The supplementation of all feed additives improved the body weight gain and the body weight corrected feed conversion ratio when compared to the control group (P = 0.0001). MUR increased villus heigh (VH) when compared to the control or the other supplemented groups (P = 0.0001), and led to the highest concentration of most SCFA, total BCFA, and total SCFA (P < 0.05). In conclusion, the supplementation of MUR and phytogenics to the diets of broiler chickens improved the growth performance, but MUR, only, was capable of effectively degrading peptidoglycans (PGNs) in both intestinal segments, as well as to increase the abundance of beneficial bacteria and SCFA production., Competing Interests: DISCLOSURES CB, EPC, OG are employed by dsm-firmenich. The other authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The fenugreek plant ( Trigonella foenum - graecum ) is traditionally known for its anti-diabetic properties owing to its high content of furostanolic saponins, which can synergistically treat many human ailments. Non-enzymatic protein glycation leading to the formation of Advanced Glycation End products (AGE) is a common pathophysiology observed in diabetic or prediabetic individuals, which can initiate the development of neurodegenerative disorders. A potent cellular source of glycation is Methyl Glyoxal, a highly reactive dicarbonyl formed as a glycolytic byproduct. We demonstrate the in vitro glycation arresting potential of Fenfuro®, a novel patented formulation of Fenugreek seed extract with clinically proven anti-diabetic properties, in Methyl-Glyoxal (MGO) adducts of three abundant amyloidogenic cellular proteins, alpha-synuclein, Serum albumin, and Lysozyme. A 0.25% w/v Fenfuro ® was able to effectively arrest glycation by more than 50% in all three proteins, as evidenced by AGE fluorescence. Glycation-induced amyloid formation was also arrested by more than 36%, 14% and 15% for BSA, Alpha-synuclein and Lysozyme respectively. An increase in MW by attachment of MGO was also partially prevented by Fenfuro ® as confirmed by SDS-PAGE analysis. Glycation resulted in enhanced aggregation of the three proteins as revealed by Native PAGE and Dynamic Light Scattering. However, in the presence of Fenfuro ® , aggregation was arrested substantially, and the normal size distribution was restored. The results cumulatively indicated the lesser explored potential of direct inhibition of glycation by fenugreek seed in addition to its proven role in alleviating insulin resistance. Fenfuro ® boosts its therapeutic potential as an effective phytotherapeutic to arrest Type 2 diabetes.

In this study, the adsorption characteristics of novel activated biocarbons prepared from horsetail herb (a popular and troublesome weed) by physical activation (using carbon dioxide) and chemical one (using phosphoric(V) acid) in the process of simultaneous proteins immobilization in multicomponent solutions were examined. The carbon materials were characterized in terms of their porous structure, acidic-basic properties, and surface morphology. The binding mechanisms of such proteins as bovine serum albumin (BSA) and lysozyme (LSZ), differing in internal stability, were determined alone and in their blends. This was done based on the comprehensive analysis of the results of adsorption/desorption, surface, electrokinetic and stability measurements. These experiments were carried out over a wide pH range of 3-11. They included the following issues: (1) determination of the protein adsorbed/desorbed amounts on/from a surface of activated biocarbons; (2) study of the kinetics of these processes; (3) examination of the macromolecules impact on the surface charge density and zeta potential of the carbon materials; and (4) determination of the suspension stability and size of aggregates formed in the examined systems. The analysis of the obtained results indicated the differences in the binding mechanism of both proteins that is of key importance for their simultaneous immobilization on activated biocarbons surface in the soil environment., (© 2024 Wiley-VCH GmbH.)

The binding of the potential drug [V IV O(8-HQ) 2 ], where 8-HQ is 8-hydroxyquinolinato, with hen egg white lysozyme (HEWL) was evaluated through spectroscopic (electron paramagnetic resonance, EPR, and UV-visible), spectrometric (electrospray ionization-mass spectrometry, ESI-MS), crystallographic (X-ray diffraction, XRD), and computational (DFT and docking) studies. ESI-MS indicates the interaction of [V IV O(8-HQ)(H 2 O)] + and [V IV O(8-HQ) 2 (H 2 O)] species with HEWL. Room temperature EPR spectra suggest both covalent and non-covalent binding of the two different V-containing fragments. XRD analyses confirm these findings, showing that [V IV O(8-HQ)(H 2 O)] + interacts covalently with the solvent exposed Asp119, while cis-[V IV O(8-HQ) 2 (H 2 O)] non-covalently with Arg128 and Lys96 from a symmetry mate. The covalent binding of [V IV O(8-HQ)(H 2 O)] + to Asp119 is favored by a π-π contact with Trp62 and a H-bond with Asn103 of a symmetry-related molecule. Additionally, the covalent binding of V V O 2 + to Asp48 and non-covalent binding of other V-containing fragments to Arg5, Cys6, and Glu7 are revealed. Molecular docking indicates that, in the absence of the interactions occurring at the protein-protein interface close to Asp119, the covalent binding to Glu35 or Asp52 should be preferred. Such a protein-protein stabilization could be more common than what believed up today, at least in the solid state, and should be considered in the characterization of metal-protein adducts., (© 2024 Wiley-VCH GmbH.)

To overcome bacterial invasion and infection, animals have evolved various antimicrobial effectors such as antimicrobial peptides and lysozymes. Although C. elegans is exposed to a variety of microbes due to its bacterivorous lifestyle, previous work on the components of its immune system mainly based on the description of transcriptional changes during bacterial challenges. Very few effector components of its immune system have been characterized so far. To investigate the role of lysozymes in terms of antibacterial defense and digestion, we studied a member of the widely neglected family of C. elegans invertebrate lysozymes (ILYS). We focused on the so far virtually undescribed ILYS-5, which we purified from protein extracts of C. elegans tracing its peptidoglycan-degrading activity and localized the tissue expression of the gene in vivo using a translational reporter construct. We recombinantly synthesized ILYS-5 and determined the physicochemical activity optimum and the antibacterial spectrum of a lysozyme from C. elegans for the first time. With an activity optimum at low ionic strength (≤100 mM) and at acidic pH (≤ pH 4.0), ILYS-5 is likely to be involved in killing and digestion of bacteria within acidified phagolysosomes and acidic regions of the gut, presumably secreted by lysosome-like vesicles. This notion is supported by potent activity against various live Gram-positive and Gram-negative bacteria. Notably, members of the natural associated microbiome of C. elegans are substantially less susceptible to ILYS-5. Ablation of the ilys-5 gene resulted in reduction of lifespan and fertility when cultured on the standard food bacterium Escherichia coli OP50, whereas exposure of the ilys-5 knock-out mutant to the host-associated bacterium Pseudomonas lurida MYb11 did not have a clear effect. These findings indicate a role of ILYS-5 in immunity and nutrition and a co-evolved adaptation of host and bacteria to the mutualistic nature of their interaction., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Reversible and irreversible amyloids are two diverging cases of protein (mis)folding associated with the cross-β motif in the protein folding and aggregation energy landscape. Yet, the molecular origins responsible for the formation of reversible vs irreversible amyloids have remained unknown. Here we provide evidence at the atomic level of distinct folding motifs for irreversible and reversible amyloids derived from a single protein sequence: human lysozyme. We compare the 2.8 Å structure of irreversible amyloid fibrils determined by cryo-electron microscopy helical reconstructions with molecular insights gained by solid-state NMR spectroscopy on reversible amyloids. We observe a canonical cross-β-sheet structure in irreversible amyloids, whereas in reversible amyloids, there is a less-ordered coexistence of β-sheet and helical secondary structures that originate from a partially unfolded lysozyme, thus carrying a "memory" of the original folded protein precursor. We also report the structure of hen egg-white lysozyme irreversible amyloids at 3.2 Å resolution, revealing another canonical amyloid fold, and reaffirming that irreversible amyloids undergo a complete conversion of the native protein into the cross-β structure. By combining atomic force microscopy, cryo-electron microscopy and solid-state NMR, we show that a full unfolding of the native protein precursor is a requirement for establishing irreversible amyloid fibrils., (© 2024. The Author(s).)

Carbon dots (CDs) are carbon nano materials (CNMs) that find use across several biological applications because of their water solubility, biocompatible nature, eco-friendliness, and ease of synthesis. Additionally, their physiochemical properties can be chemically tuned for further optimization towards specific applications. Here, we investigate the efficacy of C70-derived Graphene Acid Quantum Dots (GAQDs) in mitigating the transformation of soluble, monomeric Hen Egg-White Lysozyme (HEWL) to mature fibrils during its amyloidogenic trajectory. Our findings reveal that GAQDs exhibit dose-dependent inhibition of HEWL fibril formation (up to 70 % at 5 mg/mL) without affecting mitochondrial membrane potential or inducing apoptosis at the same density. Furthermore, GAQDs scavenged reactive oxygen species (ROS); achieving a 50 % reduction in ROS levels at a mere 100 µg/mL when exposed to a standard free radical generator. GAQDs were not only found to be biocompatible with a human neuroblastoma-derived SHSY-5Y cell line but also rescued the cells from rotenone-induced apoptosis. The GAQD-tolerance of SHSY-5Y cells coupled with their ability to restitute cells from rotenone-dependent apoptosis, when taken in conjunction with the biocompatibility data, indicate that GAQDs possess neuroprotective potential. The data position this class of CNMs as promising candidates for resolving aberrant cellular outputs that associate with the advent and progress of multifactorial neurodegenerative disorders including Parkinson's (PD) and Alzheimer's diseases (AD) wherein environmental causes are implicated (95 % etiology). The data suggest that GAQDs are a multifunctional carbon-based sustainable nano-platform at the intersection of nanotechnology and neuroprotection for advancing green chemistry-derived, sustainable healthcare solutions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Single-domain antibodies, including variable domains of the heavy chains of heavy chain-only antibodies (VHHs) from camelids and variable domains of immunoglobulin new antigen receptors (VNARs) from cartilaginous fish, show the therapeutic potential of targeting antigens in a cytosol reducing environment. A large proportion of single-domain antibodies contain non-canonical cysteines and corresponding non-canonical disulfide bonds situated on the protein surface, rendering them vulnerable to environmental factors. Research on non-canonical disulfide bonds has been limited, with a focus solely on VHHs and utilizing only cysteine mutations rather than the reducing agent treatment. In this study, we examined an anti-lysozyme VNAR and an anti-BC2-tag VHH, including their non-canonical disulfide bond reduced counterparts and non-canonical cysteine mutants. Both the affinity and stability of the VNARs and VHHs decreased in the non-canonical cysteine mutants, whereas the reduced-state samples exhibited decreased thermal stability, with their affinity remaining almost unchanged regardless of the presence of reducing agents. Molecular dynamics simulations suggested that the decrease in affinity of the mutants resulted from increased flexibility of the CDRs, the disappearance of non-canonical cysteine-antigen interactions, and the perturbation of other antigen-interacting residues caused by mutations. These findings highlight the significance of non-canonical cysteines for the affinity of single-domain antibodies and demonstrate that the mutation of non-canonical cysteines is not equivalent to the disruption of non-canonical disulfide bonds with a reducing agent when assessing the function of non-canonical disulfide bonds.

Here we used a 3D human hepatic tumour cell culture model to assess the in vitro efficacy of "active" metformin-loaded nanoparticles (NPs) as anticancer therapeutics. The metformin nanocarrier design was repurposed from previous studies targeting bacterial and fungal biofilms with antimicrobials loaded in protease-coated nanoparticles. These active nanocarriers were constructed with shellac cores loaded with metformin as the anticancer agent and featured a surface coating of the cationic protease lysozyme. The lysozyme's role as a nanocarrier surface coating is to partially digest the extracellular matrix (ECM) of the 3D tumour cell culture which increases its porosity and the nanocarrier penetration. Hep-G2 hepatic 3D clusteroids were formed using a water-in-water (w/w) Pickering emulsion based on an aqueous two-phase system (ATPS). Our specific metformin nano-formulation, comprising 0.25 wt% lysozyme-coated, 0.4 wt% metformin-loaded, 0.2 wt% shellac NPs sterically stabilized with 0.25 wt% Poloxamer 407, demonstrated significantly enhanced anticancer efficiency on 3D hepatic tumour cell clusteroids. We examined the role of the lysozyme surface functionality of the metformin nanocarriers in their ability to kill both 2D and 3D hepatic tumour cell cultures. The anticancer efficiency at high metformin payloads was compared with that at a high concentration of nanocarriers with a lower metformin payload. It was discovered that the high metformin payload NPs were more efficient than the lower metformin payload NPs with a higher nanocarrier concentration. This study introduces a reliable in vitro model for potential targeting of solid tumours with smart nano-therapeutics, presenting a viable alternative to animal testing for evaluating anticancer nanotechnologies.

The partition behavior of single and double-point mutants of bacteriophage T4 lysozyme (T4 lysozyme) and staphylococcal nuclease A was examined in different aqueous two-phase systems (ATPSs) and studied by Solvent Interaction Analysis (SIA). Additionally, the solvent accessible surface area (SASA) of modeled mutants of both proteins was calculated. The in silico calculations and the in vitro analyses of the staphylococcal nuclease and T4 lysozyme mutants correlate, indicating that the partition analysis in ATPSs provides a valid descriptor (SIA signature) covering various protein features, such as structure, structural dynamics, and conformational stability.

Tannic acid (TA)-derived carbon dots (TACDs) were synthesized for the first time via a solvothermal method using TA as one of the raw materials, which may effectively inhibit amyloid fibril aggregation and disaggregate mature fibril. The fluorescent property of TACDs were modulated by adjusting the ratio of TA to o-phenylenediamine (oPD), and TACDs fabricated with the precursor ratio as 1:1 showed the best fluorescent property. Circular dichroism spectra (CD) showed that the structure of β-sheet decreased as the concentration of TACDs increased. The inhibition efficiency, as confirmed by thioflavin T (ThT) and transmission electron microscopy (TEM), is extraordinary at 98.16%, whereas disaggregation efficiency is noteworthy at 97.97%, and the disaggregated lysozyme fibrils did not reaggregate after 7 days. More critically, TACDs can also alleviate the cellular toxicity caused by Aβ fibrils and improve cell viability. This work offers a new perspective on the design of scavengers for amyloid plaques., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Lysozymes are hydrolytic enzymes, and they are ubiquitous among all living organisms. They are mostly associated with antibacterial properties through their muramidase activity, while other properties such as iso-peptidase activity are also common. Invertebrate-type (i-type) lysozymes include the enzyme Destabilase, which is present in the salivary secretions of the medicinal leach Hirundo medicinalis. Destabilase has the ability to hydrolyse the ε-(γ-glutamyl)-lysine iso-peptide bonds formed by transglutaminase in fibrin of vertebrate blood, thereby destabilising blood clots. We have identified an i-type lysozyme from the hemocytes of the freshwater crayfish Pacifastacus leniusculus, which was found to be upregulated at the protein level in response to an injection of the β-1,3-glucan laminarin. Based on its sequence we predicted that this lysozyme would lack muramidase activity, and therefore we decided to determine its putative immune function. The P. leniusculus i-type lysozyme (Pl-ilys), is a protein with 159 amino acid residues, including a 29 residue signal peptide, with a predicted molecular weight of 16 kDa and a predicted pI of 5.6. It is expressed primarily in the hemocytes and to a lesser extent in the hematopoietic tissue. A recombinant mature Pl-ilys using an E. coli expression system was produced, and we could ascertain that this enzyme was deficient of muramidase activity. Moreover, no iso-peptidase activity could be detected against the substrate l-γ-glutamine-p-nitroanilide. Analysis of the conserved domains in Pl-ilys showed a putative destabilase domain, and thus we tested the clot dissolving activity of this enzyme. We could show that the purified P. leniusculus clotting protein which had been coagulated and clotted with transglutaminase was dissolved by the addition of Pl-ilys. Taken together our results indicate that Pl-ilys has a clot dissolving or destabilising activity in crustacean blood., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The present work explores the specificity of supramolecular assemblies comprising dialkylaminostyrylhetarene dye molecules incorporated into phosphatidylcholine (PC) or phosphatidylserine (PS) aggregates. In PS-based assemblies, the dyes demonstrate a concentration-dependent fluorescent response, distinguishing anionic proteins such as bovine serum albumin (BSA) and pepsin from lysozyme (LYZ) in aqueous solutions. Conversely, no significant response is observed when the dyes are incorporated into the well-organized bilayers of neutral PC. The fluorescent response arises from the binding of dyes to proteins, leading to the detachment of dye molecules from the assemblies, rather than from the binding of proteins to the assemblies, although the latter process is facilitated by electrostatic attraction. Thus, both the poor ordering of PS molecules and the interfacial arrangement of the dyes are prerequisites for the fluorescent response of dye-PS aggregates. The structure of the dyes significantly impacts the spectral features of dye-PS and dye-protein assemblies. An optimal dye structure has been identified for the recognition of BSA, with a limit of detection (LOD) of 10.8 nM., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Lysozyme, a well-known bacteriolytic enzyme, exhibits a fascinating yet complex behavior when it comes to protein aggregation. Under certain conditions, this enzyme undergoes flexible transformation, transitioning from partially unfolded intermediate units of native conformers into complex cross-β-rich nano fibrillar amyloid architectures. Formation of such lysozyme amyloids has been implicated in a multitude of pathological and medical severities, like hepatic dysfunction, hepatomegaly, splenic rupture as well as spleen dysfunction, nephropathy, sicca syndrome, renal dysfunction, renal amyloidosis, and systemic amyloidosis. In this comprehensive review, we have attempted to provide in-depth insights into the aggregating behavior of lysozyme across a spectrum of variables, including concentrations, temperatures, pH levels, and mutations. Our objective is to elucidate the underlying mechanisms that govern lysozyme's aggregation process and to unravel the complex interplay between its structural attributes. Moreover, this work has critically examined the latest advancements in the field, focusing specifically on novel strategies and systems, that have been implemented to delay or inhibit the lysozyme amyloidogenesis. Apart from this, we have tried to explore and advance our fundamental understanding of the complex processes involved in lysozyme aggregation. This will help the research community to lay a robust foundation for screening, designing, and formulating targeted anti-amyloid therapeutics offering improved treatment modalities and interventions not only for lysozyme-linked amyloidopathy but for a wide range of amyloid-related disorders., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2023. Published by Elsevier B.V.)

1 H- 1 H nuclear cross-relaxation experiments have been carried out with lysozyme in variable glycerol viscosity to study intramolecular motion, self-diffusion, and isotropic rigid-body rotational tumbling at 298 K, pH 3.8. Dynamics of intramolecular 1 H- 1 H cross-relaxation rates, the increase in internuclear spatial distances, and lateral and rotational diffusion coefficients all show fractional viscosity dependence with a power law exponent κ in the 0.17-0.83 range. The diffusion coefficient of glycerol D s with the bulk viscosity itself is non-Stokesian, having a fractional viscosity dependence on the medium viscosity (D s  ∼ η -κ , κ ≈ 0.71). The concurrence and close similarity of the fractional viscosity dependence of glycerol diffusion on the one hand, and diffusion and intramolecular cross-relaxation rates of the protein on the other lead to infer that relaxation of glycerol slaves protein relaxations. Glycerol-transformed native lysozyme to a quasi-native state does not affect the conclusion that both global and internal fluctuations are slaved to glycerol relaxation., Competing Interests: Declaration of competing interest There is no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The interactions of anionic sodium dodecyl sulphate (SDS), cationic cetyltrimethylammonium bromide (CTAB) and nonionic triton X-100 (TX-100) surfactants with lysozyme at pH = 2.4 have been studied individually as well as in combination with 2,2,2-trifluoroetanol (TFE). Urea has also been used in combination with surfactants. By using these combinations, efforts have been made to obtain partially folded conformations of the protein in the presence of surfactants and effect of α-helix inducer 2,2,2-trifluoroethanol on these intermediate states. Thermodynamic analysis of all these interactions has been done employing a combination of UV-visible, fluorescence and circular dichroism spectroscopies. The results have been correlated with each other and characterized qualitatively as well as quantitatively. At lower concentration of surfactant, the thermodynamic parameters indicated the destabilizing effect of SDS, stabilizing effect of CTAB and unappreciable destabilizing impact of TX-100 on lysozyme. The enhancement in destabilization effect or reduction in stabilization effect of surfactants on lysozyme in the presence of TFE and urea has also been indicated.Communicated by Ramaswamy H. Sarma.

Zinc has been proposed as a topical therapeutic agent for the prevention and treatment of various oral diseases. The purpose of this scoping review was to investigate the effects of zinc on the enzymatic activities of lysozyme, peroxidase, and α-amylase from the perspective of developing oral health care products and therapeutic agents for oral diseases. A comprehensive review of the scientific literature was conducted on the direct interactions of zinc with lysozyme, peroxidase, and α-amylase from various sources. Most of the reports on the effects of zinc on the enzymatic activities of lysozyme, peroxidase, and α-amylase involved enzymes derived from bacteria, fungi, animals, and plants. Studies of human salivary enzymes were scarce. Zinc was found to inhibit the enzymatic activities of lysozyme, peroxidase, and α-amylase under diverse experimental conditions. The suggested mechanism was ionic interactions between zinc and enzyme molecules. The possibility that zinc causes structural changes to enzyme molecules has also been suggested. In conclusion, for zinc to be used as an effective topical therapeutic agent for oral health, further studies on the activity of human salivary enzymes are warranted, and additional information regarding the type and concentration of effective zinc compounds is also required., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The emergence of antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) has become a global health challenge due to the overuse of antibiotics. Natural substances including enzymes and essential oils have shown great potential as alternative treatment options. However, the combinational use of these natural agents remains challenging due to the denaturation of enzymes upon direct contact with oil. In this study, we report the design of a Pickering emulsion containing two natural antibacterial agents, lysozyme and tea tree oil, stabilized by fractal silica nanoparticles. In this design, the enzyme activity is kept and the volatility problem of tea tree oil is mitigated. Due to synergistic bacterial cell wall digestion and membrane disruption functions, potent bactericidal efficacy in vitro against drug-resistant bacteria is achieved. The therapeutic potential is further demonstrated in a wound healing model with drug-resistant bacteria infection, better than a synthetic antibiotic, Ampicillin. This study opens new avenues for the development of natural product-based antimicrobial treatments with promising application potential., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Anaerobic digestion (AD) is widely employed for sludge stabilization and waste reduction. However, the slow hydrolysis process hinders methane production and leads to prolonged sludge issues. In this study, an efficient and eco-friendly lysozyme pre-treatment method was utilized to address these challenges. By optimizing lysozyme dosage, hydrolysis and cell lysis were maximized. Furthermore, lysozyme combined with hydrothermal pretreatment enhanced overall efficiency. Results indicate that: (1) When lysozyme dosage reached 90 mg/g TS after 240 min of pretreatment, SCOD, soluble polysaccharides, and protein content reached their maxima at 855.00, 44.09, and 204.86 mg/L, respectively. This represented an increase of 85.87%, 365.58%, and 259.21% compared to the untreated sludge. Three-dimensional fluorescence spectroscopy revealed the highest fluorescence intensity in the IV region (soluble microbial product), promoting microbial metabolic activity. (2) Lysozyme combined with hydrothermal pretreatment significantly increased SCOD, soluble proteins, and polysaccharide release from sludge, reducing SCOD release time. Orthogonal experiments identified Group 3 as the most effective for SCOD and soluble polysaccharide release, while Group 9 released the most soluble proteins. The significance order of factors influencing SCOD, soluble proteins, and polysaccharide release is hydrothermal temperature > hydrothermal time > enzymatic digestion time.(3) The lysozyme-assisted hydrothermal pretreatment group exhibited the fastest release and the highest SCOD concentration of 8,135.00 mg/L during anaerobic digestion. Maximum SCOD consumption and cumulative gas production increased by 95.89% and 130.58%, respectively, compared to the control group, allowing gas production to conclude 3 days earlier., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Apoptosis associated speck like protein containing a card (ASC), the key adaptor protein of the assembly and activation of canonical inflammasomes, has been found to play a significant role in neuroinflammation after spinal cord injury (SCI). The previous studies indicated that widely block or knockout ASC can ameliorate SCI. However, ASC is ubiquitously expressed in infiltrated macrophages and local microglia, so further exploration is needed on which type of cell playing the key role. In this study, using the LysM cre ;Asc flox/flox mice with macrophage-specifc ASC conditional knockout (CKO) and contusive SCI model, we focus on evaluating the specific role of ASC in lysozyme 2 (LysM) + myeloid cells (mainly infiltrated macrophages) in this pathology. The results revealed that macrophage-specifc Asc CKO exhibited the follow effects: (1) A significant reduction in the numbers of infiltrated macrophages in the all phases of SCI, and activated microglia in the acute and subacute phases. (2) A significant reduction in ASC, caspase-1, interleukin (IL)-1β, and IL-18 compared to control mice. (3) In the acute and subacute phases of SCI, M1 subset differentiation was inhibited, and M2 differentiation was increased. (4) Histology and hindlimb motor recoveries were improved. In conclusion, this study elucidates that macrophage-specific ASC CKO can improve nerve function recovery after SCI by regulating M1/M2 polarization through inhibiting ASC-dependent inflammasome signaling axis. This indicates that ASC in peripheral infiltrated macrophages may play an important role in SCI pathology, at least in mice, could be a potential target for treatment., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Water-in-water (W/W) emulsions provide bio-compatible all-aqueous compartments for artificial patterning and assembly of living cells. Successful entrapment of cells within a W/W emulsion via the formation of semipermeable capsules is a prerequisite for regulating on the size, shape, and architecture of cell aggregates. However, the high permeability and instability of the W/W interface, restricting the assembly of stable capsules, pose a fundamental challenge for cell entrapment. The current study addresses this problem by synthesizing multi-armed protein fibrils and controlling their assembly at the W/W interface. The multi-armed protein fibrils, also known as 'fibril clusters', were prepared by cross-linking lysozyme fibrils with multi-arm polyethylene glycol (PEG) via click chemistry. Compared to linear-structured fibrils, fibril clusters are strongly adsorbed at the W/W interface, forming an interconnected meshwork that better stabilizes the W/W emulsion. Moreover, when fibril clusters are complexed with alginate, the hybrid microcapsules demonstrate excellent mechanical robustness, semi-permeability, cytocompatibility and biodegradability. These advantages enable the encapsulation, entrapment and long-term culture of tumor spheroids, with great promise for applications for anti-cancer drug screening, tumor disease modeling, and tissue repair engineering., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Hexosomes (HEXs) are nanoparticles formed by dispersing a lipid reverse hexagonal phase in water. Although they have attracted a great interest in the development of delivery systems, few lipids have been employed in their production. Galactolipids, especially monogalactosyldiacylglycerol (MGDG), are the main lipid constituents of plants and can be obtained from vegetal biomass, making them good candidates for the obtention of HEXs. In this work, the aqueous phase behavior of MGDG from sweet potato leaves was investigated and the resulting hexagonal phase was downsized into HEXs with the aid of stabilizer decaglycerol monooleate (DGMO), a food-grade emulsifier from vegetable oils. The nanoparticles presented enhanced long-term colloidal stability in different storage conditions and their inner liquid crystalline structure could be tuned by the amount of DGMO employed. Moreover, by adding sodium oleate (NaO) HEXs displayed enhanced loading efficiency of lysozyme, an edible protein with biological properties. Finally, the sustained release of incorporated protein could be finely tuned by changing HEXs composition. Collectively, the results demonstrate, for the first time, the viability of producing biobased, renewable sourced galactolipid hexosomes with potential applications in the development of functional foods, also contributing to a sustainable management of biomass waste., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study aimed to investigate the effects of zinc compounds on the enzymatic activities of lysozyme, peroxidase, and the glucose oxidase-mediated peroxidase (GO-PO) system and their antifungal activities. Four different zinc compounds (zinc chloride, gluconate, lactate, and sulfate) were incubated with hen egg-white lysozyme (HEWL), bovine lactoperoxidase (bLPO), the GO-PO system, and human unstimulated whole saliva in solution and on a hydroxyapatite surface. Enzymatic activities of lysozyme, peroxidase, and the GO-PO system were measured through the hydrolysis of Micrococcus lysodeikticus, oxidation of fluorogenic 2',7'-dichlorofluorescin, and glucose assay, respectively. Interactions between zinc and enzymes were analyzed by surface plasmon resonance (SPR). The minimum inhibitory concentration (MIC) and candidacidal activities of zinc compounds were examined against three Candida albicans strains. Zinc gluconate and sulfate significantly increased the enzymatic activities of salivary lysozyme in the solution assay and of HEWL and salivary lysozyme on the hydroxyapatite surface. However, all examined zinc compounds significantly decreased the enzymatic activities of bLPO and salivary peroxidase in solution and on the surface. SPR analyses revealed binding of zinc to lysozyme and peroxidase, with affinity differing according to the zinc compounds. The MIC of zinc compounds against C. albicans was 1.0-2.4 mM. Candidacidal activities were 17.7-38.8% and 23.7-47.0% at 1.0 and 10 mM concentrations, respectively. In conclusion, zinc compounds enhanced lysozyme activity but inhibited peroxidase activity. Zinc compounds exhibited concentration-dependent candidacidal activity against C. albicans. Zinc compounds are potential therapeutic agents for oral health, especially for geriatric patients., (© 2024. The Author(s).)

The unique bacterial infection microenvironment (IME) usually requires complicated design of nanomaterials to adapt to IME for enhancing antibacterial therapy. Here, an alternative IME adaptative nitrite reductase-mimicking nanozyme is constructed by in situ growth of ultrasmall copper sulfide clusters on the surface of a nanofibrillar lysozyme assembly (NFLA/CuS NHs), which can temporally regulate nitric oxide (NO) gradient concentration to kill bacteria initially and promote tissue regeneration subsequently. Benefiting from a copper nitrite reductase (CuNIR)-inspired structure with CuS cluster as active center and NFLA as skeleton, NFLA/CuS NHs efficiently boost the catalytic reduction of nitrite to NO. The inherent supramolecular fibrillar networks displays excellent bacterial capture capability, facilitating initial high-concentration NO attacks on the bacteria. The subsequent catalytic release of low-concentration NO by NFLA/CuS NHs-mediated nitrite reduction remarkably promotes cell migration and angiogenesis. This work paves the way for dynamically eliminating MDR bacterial infection and promoting tissue regeneration in a simple and smart way through CuNIR-mimicking catalysis.

Vast shotgun metagenomics data remain an underutilized resource for novel enzymes. Artificial intelligence (AI) has increasingly been applied to protein mining, but its conventional performance evaluation is interpolative in nature, and these trained models often struggle to extrapolate effectively when challenged with unknown data. In this study, we present a framework (DeepMineLys [deep mining of phage lysins from human microbiome]) based on the convolutional neural network (CNN) to identify phage lysins from three human microbiome datasets. When validated with an independent dataset, our method achieved an F1-score of 84.00%, surpassing existing methods by 20.84%. We expressed 16 lysin candidates from the top 100 sequences in E. coli, confirming 11 as active. The best one displayed an activity 6.2-fold that of lysozyme derived from hen egg white, establishing it as the most potent lysin from the human microbiome. Our study also underscores several important issues when applying AI to biology questions. This framework should be applicable for mining other proteins., Competing Interests: Declaration of interests Z. Lin, X.Y., Y.F., J.L., and S.Y. are filing both patent and computer software applications on aspects of this work. The patent applications pertain to lysin sequences, engineered lysins, and methods to generate them (patent number: ZL 2023 1 1738785.8). The copyright pertains to the DeepMineLys method and its corresponding source code (registration number: 2023SR1807560)., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mechanical energy, specifically in the form of ultrasound, can induce pressure variations and temperature fluctuations when applied to an aqueous media. These conditions can both positively and negatively affect protein complexes, consequently altering their stability, folding patterns, and self-assembling behavior. Despite much scientific progress, our current understanding of the effects of ultrasound on the self-assembly of amyloidogenic proteins remains limited. In the present study, we demonstrate that when the amplitude of the delivered ultrasonic energy is sufficiently low, it can induce refolding of specific motifs in protein monomers, which is sufficient for primary nucleation; this has been revealed by MD. These ultrasound-induced structural changes are initiated by pressure perturbations and are accelerated by a temperature factor. Furthermore, the prolonged action of low-amplitude ultrasound enables the elongation of amyloid protein nanofibrils directly from natively folded monomeric lysozyme protein, in a controlled manner, until it reaches a critical length. Using solution X-ray scattering, we determined that nanofibrillar assemblies, formed either under the action of sound or from natively fibrillated lysozyme, share identical structural characteristics. Thus, these results provide insights into the effects of ultrasound on fibrillar protein self-assembly and lay the foundation for the potential use of sound energy in protein chemistry., Competing Interests: Competing interests statement:The authors declare no competing interest.

Background: Macrophages are key players in obesity-associated cardiovascular diseases, which are marked by inflammatory and immune alterations. However, the pathophysiological mechanisms underlying macrophage's role in obesity-induced cardiac inflammation are incompletely understood. Our study aimed to identify the key macrophage population involved in obesity-induced cardiac dysfunction and investigate the molecular mechanism that contributes to the inflammatory response., Methods: In this study, we used single-cell RNA-sequencing analysis of Cd45 + CD11b + F4/80 + cardiac macrophages to explore the heterogeneity of cardiac macrophages. The CCR2 + (C-C chemokine receptor 2) macrophages were specifically removed by a dual recombinase approach, and the macrophage CCR2 was deleted to investigate their functions. We also performed cleavage under target and tagmentation analysis, chromatin immunoprecipitation-polymerase chain reaction, luciferase assay, and macrophage-specific lentivirus transfection to define the impact of lysozyme C in macrophages on obesity-induced inflammation., Results: We find that the Ccr2 cluster undergoes a functional transition from homeostatic maintenance to proinflammation. Our data highlight specific changes in macrophage behavior during cardiac dysfunction under metabolic challenge. Consistently, inducible ablation of CCR2 + CX3CR1 + macrophages or selective deletion of macrophage CCR2 prevents obesity-induced cardiac dysfunction. At the mechanistic level, we demonstrate that the obesity-induced functional shift of CCR2-expressing macrophages is mediated by the CCR2/activating transcription factor 3/lysozyme 1/NF-κB (nuclear factor kappa B) signaling. Finally, we uncover a noncanonical role for lysozyme 1 as a transcription activator, binding to the RelA promoter, driving NF-κB signaling, and strongly promoting inflammation and cardiac dysfunction in obesity., Conclusions: Our findings suggest that lysozyme 1 may represent a potential target for the diagnosis of obesity-induced inflammation and the treatment of obesity-induced heart disease., Competing Interests: None.

The unique high isoelectric point of lysozyme (LYZ) restricts its application in composite antibacterial coating due to the unfavorable liability to electrostatic interaction with other components. In this work, the antibacterial activity of a dispersible LYZ-carboxymethyl konjac glucomannan (CMKGM) polyelectrolyte complex was evaluated. Kinetic analysis revealed that, compared with free LYZ, the complexed enzyme exhibited decreased affinity (K m ) but markedly increased V max against Micrococcus lysodeikticus, and QCM and dynamic light scattering analysis confirmed that the complex could bind with the substrate but in a much lower ratio. The complexation with CMKGM did not alter the antibacterial spectrum of LYZ, and the complex exerted antibacterial function by delaying the logarithmic growth phase and impairing the cell integrity of Staphylococcus aureus. Since the LYZ-CMKGM complex is dispersible in water and could be assembled easily, it has great potential as an edible coating in food preservation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jun-Xia Xiao reports financial support was provided by National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Conjugative type 4 secretion systems (T4SSs) are the main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. To deliver the DNA substrate to recipient cells, it must cross the cell envelopes of both donor and recipient bacteria. In the T4SS from the enterococcal conjugative plasmid pCF10, PrgK is known to be the active cell wall degrading enzyme. It has three predicted extracellular hydrolase domains: metallo-peptidase (LytM), soluble lytic transglycosylase (SLT), and cysteine, histidine-dependent amidohydrolases/peptidases (CHAP). Here, we report the structure of the LytM domain and show that its active site is degenerate and lacks the active site metal. Furthermore, we show that only the predicted SLT domain is functional in vitro and that it unexpectedly has a muramidase instead of a lytic transglycosylase activity. While we did not observe any peptidoglycan hydrolytic activity for the LytM or CHAP domain, we found that these domains downregulated the SLT muramidase activity. The CHAP domain was also found to be involved in PrgK dimer formation. Furthermore, we show that PrgK interacts with PrgL, which likely targets PrgK to the rest of the T4SS. The presented data provides important information for understanding the function of Gram-positive T4SSs.IMPORTANCEAntibiotic resistance is a large threat to human health and is getting more prevalent. One of the major contributors to the spread of antibiotic resistance among different bacteria is type 4 secretion systems (T4SS). However, mainly T4SSs from Gram-negative bacteria have been studied in detail. T4SSs from Gram-positive bacteria, which stand for more than half of all hospital-acquired infections, are much less understood. The significance of our research is in identifying the function and regulation of a cell wall hydrolase, a key component of the pCF10 T4SS from Enterococcus faecalis . This system is one of the best-studied Gram-positive T4SSs, and this added knowledge aids in our understanding of horizontal gene transfer in E. faecalis as well as other medically relevant Gram-positive bacteria., Competing Interests: The authors declare no conflict of interest.

Lysozyme, an antimicrobial agent, is extensively employed in the food and healthcare sectors to facilitate the breakdown of peptidoglycan. However, the methods to improve its catalytic activity and secretory expression still need to be studied. In the present study, twelve lysozymes from different origins were heterologously expressed using the Komagataella phaffii expression system. Among them, the lysozyme from the European flat oyster Ostrea edulis (oeLYZ) showed the highest activity. Via a semi-rational approach to reduce the structural free energy, the double mutant Y15A/S39R (oeLYZ dm ) with the catalytic activity 1.8-fold greater than that of the wild type was generated. Subsequently, different N-terminal fusion tags were employed to enhance oeLYZ dm expression. The fusion with peptide tag 6×Glu resulted in a remarkable increase in the recombinant oeLYZdm expression, from 2.81 × 10 3 U mL -1 to 2.11 × 10 4 U mL -1 in shake flask culture, and eventually reaching 2.05 × 10 5 U mL -1 in a 3-L fermenter. The work produced the greatest amount of heterologous oeLYZ expression in microbial systems that are known to exist. Reducing the structural free energy and employing the N-terminal fusion tags are effective strategies to improve the catalytic activity and secretory expression of lysozyme., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Human lysozyme undergoes a phase-separation process to form insoluble amyloid-architects that cause several pathologies including systemic amyloidosis. Here we have tailored 6-gingerol by extending its molecular framework with active functional groups to specifically target lysozyme phase-transition events. Aggregation assay revealed that tailored 6-gingerol with 4-aromatic moieties (MTV4) substantially suppressed the conversion of the lysozyme low-density liquid phase (LDLP) to solid-phase structured amyloids. The data obtained from biophysical, computational, and microscopic imaging tools suggest direct intervention of MTV4 with the liquid-liquid phase separation. The CD data suggest that MTV4 was able to retain the native conformation of lysozyme. Both biomolecular and computational data reveal the interference of MTV4 with the aggregation-prone hydrophobic stretches within the lysozyme, thereby retaining the native structure and reversing the misfolded intermediates to active monomers. Also, MTV4 was able to induce rapid dissolution of preformed-toxic amyloid fibrils. These results reinforce the importance of the aromatic-aromatic interaction in preventing human lysozyme phase separation.

In biomedical and biotechnological domains, liquid protein formulations are vital tools, offering versatility across various fields. However, maintaining protein stability in a liquid form presents challenges due to environmental factors, driving research to refine formulations for broader applications. In our recent study, we investigated the relationship between deep eutectic solvents (DESs) and the natural presence of osmolytes in specific combinations, showcasing the effectiveness of a bioinspired osmolyte-based DES in stabilizing a model protein. Recognizing the need for a more nuanced understanding of osmolyte-based DES stabilization capabilities under different storage conditions, here we broadened the scope of our osmolyte-based DES experimental screening, and delved deeper into structural changes in the enzyme under these conditions. We subjected lysozyme solutions in DESs based on various kosmotropic osmolytes (TMAO, betaine, sarcosine, DMSP, ectoine, GPC, proline, sorbitol and taurine) paired either with another kosmotropic (glycerol) or with chaotropic osmolyte urea to rigorous conditions: heat shock (at 80 °C) and repetitive freeze-thaw cycles (at -20 and -80 °C). Changes in enzyme activity, colloidal stability, and conformational alterations were then monitored using bioassays, aggregation tests, and spectroscopic techniques (FT-IR and CD). Our results demonstrate the remarkable effectiveness of osmolyte-based DES in stabilizing lysozyme under stress conditions, with sarcosine- and betaine-based DESs containing glycerol as a hydrogen bond donor showing the highest efficacy, even at high enzyme loadings up to 200 mg ml -1 . Investigation of the individual and combined effects of the DES components on enzyme stability confirmed the synergistic behavior of the kosmotrope-urea mixtures and the cumulative effects in kosmotrope-glycerol mixtures. Additionally, we have shown that the interplay between the enzyme's active and stable (but inactive) states is highly influenced by the water content in DESs. Finally, toxicity assessments of osmolyte-based DESs using cell lines (Caco-2, HaCaT, and HeLa) revealed no risks to human health.

The dynamics of lysozyme is probed by attaching -SCN to all alanine residues. The one-dimensional infrared spectra exhibit frequency shifts in the position of the maximum absorption of 4 cm-1, which is consistent with experiments in different solvents and indicates moderately strong interactions of the vibrational probe with its environment. Isotopic substitution 12C → 13C leads to a redshift by -47 cm-1, which agrees quantitatively with experiments for CN-substituted copper complexes in solution. The low-frequency, far-infrared part of the protein spectra contains label-specific information in the difference spectra when compared with the wild type protein. Depending on the position of the labels, local structural changes are observed. For example, introducing the -SCN label at Ala129 leads to breaking of the α-helical structure with concomitant change in the far-infrared spectrum. Finally, changes in the local hydration of SCN-labeled alanine residues as a function of time can be related to the reorientation of the label. It is concluded that -SCN is potentially useful for probing protein dynamics, both in the high-frequency part (CN-stretch) and in the far-infrared part of the spectrum., (© 2024 Author(s). Published under an exclusive license by AIP Publishing.)

As a common pathological hallmark, protein aggregation into amyloids is a highly complicated phenomenon, attracting extensive research interest for elucidating its structural details and formation mechanisms. Membrane deposition and disulfide-driven protein misfolding play critical roles in amyloid-type aggregation, yet the underlying molecular process remains unclear. Here, we employed sum frequency generation (SFG) vibrational spectroscopy to comprehensively investigate the remodeling process of lysozyme, as the model protein, into amyloid-type aggregates at the cell membrane interface. It was discovered that disulfide reduction concurrently induced the transition of membrane-bound lysozyme from predominantly α-helical to antiparallel β-sheet structures, under a mode switch of membrane interaction from electrostatic to hydrophobic, and subsequent oligomeric aggregation. These findings shed light on the systematic understanding of dynamic molecular mechanisms underlying membrane-interactive amyloid oligomer formation.

The current research aimed to shed light on the efficacy of Escherichia coli strain Nissle 1917 (EcN) on goldfish (……) growth, gut immunity, morphology, bacterial nutritional enzyme activity and resistance to Aeromonas hydrophila infection. Fish fed with EcN at 10 6 , 10 7 and 10 8  CFU/g feed for 80 days showed an enhancement in growth better than control fish. The gut innate immunity in terms of lysozyme activity, immunoglobulin and total protein levels was increased in the treatment fish with the best result being observed in fish fed EcN at 10 8  CFU/ g. In addition, an increase was noted in the upregulation of immune-relevant genes, namely lysozyme, interleukin-1β, inducible nitric oxide synthase and tumor necrosis factor α of fish intestine. A marked surge in the number of proteolytic and heterotrophic bacteria was noted in the gut of fish nourished with the probiotic. Histological studies exhibited an improvement in the intestinal absorption surface area, intraepithelial lymphocyte count and goblet cell density. Significantly higher survival rate was obtained in fish fed EcN at 10 8  CFU/g compared with the fish fed with the basal diet. These data exhibited the beneficial effect of EcN on goldfish growth, digestive enzymes, intestine heterotrophic bacteria and resistance against Aeromonas hydrophila challenge. This study confirmed the favorable outcomes resulting from the administration of EcN at10 8 CFU/g., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Biopolymeric implantable patches are popular scaffolds for myocardial regeneration applications. Besides being biocompatible, they can be tailored to have required properties and functionalities for this application. Recently, fibrillar biobased nanostructures prove to be valuable in the development of functional biomaterials for tissue regeneration applications. Here, periodate-oxidized nanofibrillated cellulose (OxNFC) is blended with lysozyme amyloid nanofibrils (LNFs) to prepare a self-crosslinkable patch for myocardial implantation. The OxNFC:LNFs patch shows superior wet mechanical properties (60 MPa for Young's modulus and 1.5 MPa for tensile stress at tensile strength), antioxidant activity (70% scavenging activity under 24 h), and bioresorbability ratio (80% under 91 days), when compared to the patches composed solely of NFC or OxNFC. These improvements are achieved while preserving the morphology, required thermal stability for sterilization, and biocompatibility toward rat cardiomyoblast cells. Additionally, both OxNFC and OxNFC:LNFs patches reveal the ability to act as efficient vehicles to deliver spermine modified acetalated dextran nanoparticles, loaded with small interfering RNA, with 80% of delivery after 5 days. This study highlights the value of simply blending OxNFC and LNFs, synergistically combining their key properties and functionalities, resulting in a biopolymeric patch that comprises valuable characteristics for myocardial regeneration applications., (© 2024 The Author(s). Macromolecular Rapid Communications published by Wiley‐VCH GmbH.)