We develop a multiscale coarse-grain model of the NIST Monoclonal Antibody Reference Material 8671 (NISTmAb) to enable systematic computational investigations of high-concentration physical instabilities such as phase separation, clustering, and aggregation. Our multiscale coarse-graining strategy captures atomic-resolution interactions with a computational approach that is orders of magnitude more efficient than atomistic models, assuming the biomolecule can be decomposed into one or more rigid bodies with known, fixed structures. This method reduces interactions between tens of thousands of atoms to a single anisotropic interaction site. The anisotropic interaction between unique pairs of rigid bodies is precomputed over a discrete set of relative orientations and stored, allowing interactions between arbitrarily oriented rigid bodies to be interpolated from the precomputed table during coarse-grained Monte Carlo simulations. We present this approach for lysozyme and lactoferrin as a single rigid body and for the NISTmAb as three rigid bodies bound by a flexible hinge with an implicit solvent model. This coarse-graining strategy predicts experimentally measured radius of gyration and second osmotic virial coefficient data, enabling routine Monte Carlo simulation of medically relevant concentrations of interacting proteins while retaining atomistic detail. All methodologies used in this work are available in the open-source software Free Energy and Advanced Sampling Simulation Toolkit.

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.

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.

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.

Antimicrobial resistance represents a global health emergency, necessitating the introduction of novel antimicrobial agents. In the present study, lysozyme and holin from Shigella flexneri 1.1868 phage SGF2, named LysSGF2 and HolSGF2, respectively, were cloned, expressed, and characterized. LysSGF2 and HolSGF2 showed lytic activities against S. flexneri 1.1868 cells at 4-55 °C and pH 3.1-10.3. LysSGF2 exhibited antimicrobial activity against five gram-negative and two gram-positive bacteria. HolSGF2 showed antimicrobial activity against four gram-negative and one gram-positive species. The antibacterial activities of LysSGF2 and HolSGF2 were determined in liquid beverages, including bottled water and milk. The relative lytic activity of LysSGF2 combined with HolSGF2 against the tested bacteria was approximately 46-77 % in water. Furthermore, the combination markedly decreased the viable counts of tested bacteria by approximately 3-5 log CFU/mL. LysSGF2 and HolSGF2 could efficiently remove biofilms on polystyrene, glass, and stainless-steel. The efficacy of the LysSGF2 and HolSGF2 combination against the tested bacteria on polystyrene was 58-71 %. Combination treatment effectively killed biofilm cells formed on stainless-steel and glass by 1-4 log CFU/mL. ese results indicate that LysSGF2 and HolSGF2 can successfully control both the planktonic and biofilm cells of common pathogenic bacteria, suggesting that the combined or single use of LysSGF2 and HolSGF2 may be of great value in food processing.

Quantifying the formation and decomposition of amyloid is a crucial issue in the development of new drugs and therapies for treating amyloidosis. The current technologies for grasping amyloid formation and decomposition include fluorescence analysis using thioflavin-T, secondary structure analysis using circular dichroism, and image analysis using atomic force microscopy or transmission electron microscopy. These technologies typically require spectroscopic devices or expensive nanoscale imaging equipment and involve lengthy analysis, which limits the rapid screening of amyloid-degrading drugs. In this study, we introduce a technology for rapidly assessing amyloid decomposition using capillary flow-based paper (CFP). Amyloid solutions exhibit gel-like physical properties due to insoluble denatured polymers, resulting in a shorter flow distance on CFP compared to pure water. Experimental conditions were established to consistently control the flow distance based on a hen-egg-white lysozyme amyloid solution. It was confirmed that as amyloid is decomposed by trypsin, the flow distance increases on the CFP. Our method is highly useful for detecting changes in the gel properties of amyloid solutions within a minute, and we anticipate its use in the rapid, large-scale screening of anti-amyloid agents in the future.

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.

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.

BACKGROUND: Children with moderate or severe wasting are at particularly high risk of recurrent or persistent diarrhoea, nutritional deterioration and death following a diarrhoeal episode. Lactoferrin and lysozyme are nutritional supplements that may reduce the risk of recurrent diarrhoeal episodes and accelerate nutritional recovery by treating or preventing underlying enteric infections and/or improving enteric function.
METHODS: In this factorial, blinded, placebo-controlled randomised trial, we aim to determine the efficacy of lactoferrin and lysozyme supplementation in decreasing diarrhoea incidence and improving nutritional recovery in Kenyan children convalescing from comorbid diarrhoea and wasting. Six hundred children aged 6-24 months with mid-upper arm circumference <12.5 cm who are returning home after an outpatient visit or inpatient hospital stay for diarrhoea will be enrolled. Children will be randomised to 16 weeks of lactoferrin, lysozyme, a combination of the two, or placebo and followed for 24 weeks, with biweekly home visits by community health workers and clinic visits at 4, 10, 16 and 24 weeks. The primary analysis will compare the incidence of moderate-to-severe diarrhoea and time to nutritional recovery between each intervention arm and placebo. The trial will also test whether these interventions reduce enteric pathogen carriage, decrease enteric permeability and/or increase haemoglobin concentration in enrolled children. Finally, we will evaluate the acceptability, adherence and cost-effectiveness of lactoferrin and/or lysozyme.
BACKGROUND: The trial has been approved by the institutional review boards of the Kenya Medical Research Institute, the University of Washington, the Kenyan Pharmacy and Poisons Board, and the Kenyan National Commission on Science, Technology and Innovation. The results of this trial will be shared with local and international stakeholders and published in peer-reviewed journals, and the key findings will be presented at relevant conferences.
BACKGROUND: NCT05519254, PACTR202108480098476.

UNASSIGNED: Endolysins are phage-encoded lytic enzymes that degrade bacterial peptidoglycan at the end of phage lytic cycles to release new phage particles. These enzymes are being explored as an alternative to small-molecule antibiotics.
UNASSIGNED: The crystal structure of KTN6 Gp46 was determined and compared with a ColabFold model. Cleavage specificity was examined using a peptidoglycan digest and reversed-phase high-performance liquid chromatography coupled to mass spectrometry (HPLC/MS).
UNASSIGNED: The structure of KTN6 Gp46 could be determined at 1.4 Å resolution, and key differences in loops of the putative peptidoglycan binding domain were identified in comparison with its closest known homologue, the endolysin of phage SPN1S. Reversed-phase HPLC/MS analysis of the reaction products following peptidoglycan digestion confirmed the muramidase activity of Gp46, consistent with structural predictions.
UNASSIGNED: These insights into the structure and function of endolysins further expand the toolbox for endolysin engineering and explore their potential in enzyme-based antibacterial design strategies.