Inhibition and disaggregation effect of flavonoid-derived carbonized polymer dots on protein amyloid aggregation.

In this research, four water-insoluble flavonoid compounds were utilized and reacted with arginine to prepare four carbonized polymer dots with good water-solubility in a hydrothermal reactor. Structural characterization demonstrated that the prepared carbonized polymer dots were classic core-shell structure. Effect of the prepared carbonized polymer dots on protein amyloid aggregation was further investigated using hen egg white lysozyme and human lysozyme as model protein in aqueous solution. All of the prepared carbonized polymer dots could retard the amyloid aggregation of hen egg white lysozyme and human lysozyme in a dose-depended manner. All measurements displayed that the inhibition ratio of luteolin-derived carbonized polymer dots (CPDs-1) was higher than that of the other three carbonized polymer dots under the same dosage. This result may be interpreted by the highest content of phenolic hydroxyl groups on the periphery. The inhibition ratio of CPDs-1 on hen egg white lysozyme and human lysozyme reached 88 % and 83 % at the concentration of 0.5 mg/mL, respectively. CPDs-1 also could disaggregate the formed mature amyloid fibrils into short aggregates.

Sustainable combination of ionic liquid and deep eutectic solvent for protecting and preserving of the protein structure: The synergistic interaction of enzymes and eco-friendly hybrid ionic fluids.

Hybrid ionic fluids (HIFs) are one of the emerging and fascinating sustainable solvent media, a novel environment-friendly solvent for biomolecules. The HIFs have been synthesized by combining a deep eutectic solvent (DES), an ionic liquid (IL) having a common ion. The stability and activity of hen's egg white lysozyme (Lyz) in the presence of a recently designed new class of biocompatible solvents, HIFs have been explored by UV-visible, steady-state fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) along with dynamic light scattering (DLS) measurements. This work emphasizes the effect of DES synthesized by using 1:2 choline chloride and glycerol [Glyn], ILs (1-butly-3-methylimidazolium chloride [BMIM]Cl and choline acetate [Chn][Ac]) and their corresponding HIFs on the structure and functionality of Lyz. Moving forward, we also studied the secondary structure, thermal stability and enzymatic activity and thermodynamic profile of Lyz at pH = 7 in the presence of varying concentrations (0.1 to 0.5) M of [BMIM]Cl, [Chn][Ac] ILs, [Glyn] DES and [Glyn][BMIM]Cl (hybrid ionic fluid1) as well as [Glyn][Chn][Ac] (hybrid ionic fluid2). Spectroscopic results elucidate that ILs affect the activity and structural stability of Lyz, whereas the stability and activity are increased by DES and are maintained by HIFs at all the studied concentrations. Overall, the experimental results studied elucidate expressly that the properties of Lyz are maintained in the presence of hybrid ionic fluid1 while these properties are intensified in hybrid ionic fluid2. This work has elucidated expressly biocompatible green solvents in protein stability and functionality due to the alluring properties of DES, which can counteract the negative effect of ILs in HIFs.

Photoredox C(2)-Arylation of Indole- and Tryptophan-Containing Biomolecules.

We introduce a novel and straightforward methodology for photoredox arylation of an indole scaffold using aryldiazonium salts under mild and metal-free conditions. Our approach enables the regioselective and chemoselective introduction of several aryl groups to the C(2) position of indoles and tryptophan, even in competition with other amino acids. This approach extends to the late-stage functionalization of peptides and lysozyme, heralding the unprecedented arylation of tryptophan residues in wild-type proteins and offering broad utility in chemical biology.

Integrating Explicit and Implicit Fullerene Models into UNRES Force Field for Protein Interaction Studies.

Fullerenes, particularly C60, exhibit unique properties that make them promising candidates for various applications, including drug delivery and nanomedicine. However, their interactions with biomolecules, especially proteins, remain not fully understood. This study implements both explicit and implicit C60 models into the UNRES coarse-grained force field, enabling the investigation of fullerene-protein interactions without the need for restraints to stabilize protein structures. The UNRES force field offers computational efficiency, allowing for longer timescale simulations while maintaining accuracy. Five model proteins were studied: FK506 binding protein, HIV-1 protease, intestinal fatty acid binding protein, PCB-binding protein, and hen egg-white lysozyme. Molecular dynamics simulations were performed with and without C60 to assess protein stability and investigate the impact of fullerene interactions. Analysis of contact probabilities reveals distinct interaction patterns for each protein. FK506 binding protein (1FKF) shows specific binding sites, while intestinal fatty acid binding protein (1ICN) and uteroglobin (1UTR) exhibit more generalized interactions. The explicit C60 model shows good agreement with all-atom simulations in predicting protein flexibility, the position of C60 in the binding pocket, and the estimation of effective binding energies. The integration of explicit and implicit C60 models into the UNRES force field, coupled with recent advances in coarse-grained modeling and multiscale approaches, provides a powerful framework for investigating protein-nanoparticle interactions at biologically relevant scales without the need to use restraints stabilizing the protein, thus allowing for large conformational changes to occur. These computational tools, in synergy with experimental techniques, can aid in understanding the mechanisms and consequences of nanoparticle-biomolecule interactions, guiding the design of nanomaterials for biomedical applications.

Thiacalixarene Carboxylic Acid Derivatives as Inhibitors of Lysozyme Fibrillation.

Amyloid fibroproliferation leads to organ damage and is associated with a number of neurodegenerative diseases affecting populations worldwide. There are several ways to protect against fibril formation, including inhibition. A variety of organic compounds based on molecular recognition of amino acids within the protein have been proposed for the design of such inhibitors. However, the role of macrocyclic compounds, i.e., thiacalix[4]arenes, in inhibiting fibrillation is still almost unknown. In the present work, the use of water-soluble thiacalix[4]arene derivatives for the inhibition of hen egg-white lysozyme (HEWL) amyloid fibrillation is proposed for the first time. The binding of HEWL by the synthesized thiacalix[4]arenes (logKa = 5.05-5.13, 1:1 stoichiometry) leads to the formation of stable supramolecular systems capable of stabilizing the protein structure and protecting against fibrillation by 29-45%. The macrocycle conformation has little effect on protein binding strength, and the native HEWL secondary structure does not change via interaction. The synthesized compounds are non-toxic to the A549 cell line in the range of 0.5-250 µg/mL. The results obtained may be useful for further investigation of the anti-amyloidogenic role of thiacalix[4]arenes, and also open up future prospects for the creation of new ways to prevent neurodegenerative diseases.

An eminent approach towards next generation solvents for sustainable packaging and stability of enzymes: a comprehensive study of ionic liquid and deep eutectic solvent mixtures.

Hybrid ionic fluids (HIFs) are newly emerging and fascinating sustainable solvent media, which are attracting a great deal of scientific interest in protecting the native structure of proteins. For a few decades, there has been a demand to consider ionic liquids (ILs) and deep eutectic solvents (DESs) as biocompatible solvent media for enzymes; however, in some cases, these solvent media also show limitations. Therefore, this work focuses on synthesising novel HIFs to intensify the properties of existing ILs and DESs by mixing them. Herein, HIFs have been synthesised by the amalgamation of a deep eutectic solvent (DES) and an ionic liquid (IL) with a common cation or anion. Later on, the stability and activity of hen's egg white lysozyme (Lyz) in the presence of biocompatible solvent media and HIFs were studied by various techniques such as UV-vis, steady-state fluorescence, circular dichroism (CD), Fourier transform infrared spectroscopy (FT-IR) and dynamic light scattering (DLS) measurements. This work emphasises the effect of a DES (synthesised using 1 : 2 choline chloride and malonic acid) [Maline], ILs (1-butyl-3-methylimidazolium chloride [BMIM]Cl or choline acetate [Chn][Ac]) and their corresponding HIFs on the structure and functionality of Lyz. Moreover, we also studied the secondary structure, thermal stability, enzymatic activity and thermodynamic profile of Lyz at pH = 7 in the presence of varying concentrations (0.1 to 0.5 M) of [BMIM]Cl and [Chn][Ac] ILs, Maline as a DES, and Maline [BMIM]Cl (HIF1) and Maline [Chn][Ac] (HIF2). Spectroscopic results elucidate that ILs affect the activity and structural stability of Lyz. In contrast, the stability and activity are inhibited by DES and are enhanced by HIFs at all the studied concentrations. Overall, the experimental results studied explicitly elucidate that the structure and stability of Lyz are maintained in the presence of HIF1 while these properties are intensified in HIF2. This study shows various applications in biocompatible green solvents, particularly in the stability and functionality of proteins, due to their unique combination where the properties counteract the negative effect of either DESs or ILs in HIFs.

Interfacial Interactions between Nanoplastics and Biological Systems: toward an Atomic and Molecular Understanding of Plastics-Driven Biological Dyshomeostasis.

Micro- and nano-plastics (NPs) are found in human milk, blood, tissues, and organs and associate with aberrant health outcomes including inflammation, genotoxicity, developmental disorders, onset of chronic diseases, and autoimmune disorders. Yet, interfacial interactions between plastics and biomolecular systems remain underexplored. Here, we have examined experimentally, in vitro, in vivo, and by computation, the impact of polystyrene (PS) NPs on a host of biomolecular systems and assemblies. Our results reveal that PS NPs essentially abolished the helix-content of the milk protein ß-lactoglobulin (BLG) in a dose-dependent manner. Helix loss is corelated with the near stoichiometric formation of ß-sheet elements in the protein. Structural alterations in BLG are also likely responsible for the nanoparticle-dependent attrition in binding affinity and weaker on-rate constant of retinol, its physiological ligand (compromising its nutritional role). PS NP-driven helix-to-sheet conversion was also observed in the amyloid-forming trajectory of hen egg-white lysozyme (accelerated fibril formation and reduced helical content in fibrils). Caenorhabditis elegans exposed to PS NPs exhibited a decrease in the fluorescence of green fluorescent protein-tagged dopaminergic neurons and locomotory deficits (akin to the neurotoxin paraquat exposure). Finally, in silico analyses revealed that the most favorable PS/BLG docking score and binding energies corresponded to a pose near the hydrophobic ligand binding pocket (calyx) of the protein where the NP fragment was found to make nonpolar contacts with side-chain residues via the hydrophobic effect and van der Waals forces, compromising side chain/retinol contacts. Binding energetics indicate that PS/BLG interactions destabilize the binding of retinol to the protein and can potentially displace retinol from the calyx region of BLG, thereby impairing its biological function. Collectively, the experimental and high-resolution in silico data provide new insights into the mechanism(s) by which PS NPs corrupt the bimolecular structure and function, induce amyloidosis and onset neuronal injury, and drive aberrant physiological and behavioral outcomes.

Dynamic Personality of Proteins and Effect of the Molecular Environment.

Protein dynamics display distinct traits that are linked to their specific biological function. However, the interplay between intrinsic dynamics and the molecular environment on protein stability remains poorly understood. In this study, we investigate, by incoherent neutron scattering, the subnanosecond time scale dynamics of three model proteins: the mesophilic lysozyme, the thermophilic thermolysin, and the intrinsically disordered ß-casein. Moreover, we address the influence of water, glycerol, and glucose, which create progressively more viscous matrices around the protein surface. By comparing the protein thermal fluctuations, we find that the internal dynamics of thermolysin are less affected by the environment compared to lysozyme and ß-casein. We ascribe this behavior to the protein dynamic personality, i.e., to the stiffer dynamics of the thermophilic protein that contrasts the influence of the environment. Remarkably, lysozyme and thermolysin in all molecular environments reach a critical common flexibility when approaching the calorimetric melting temperature.

Picoplatin binding to proteins: X-ray structures and mass spectrometry data on the adducts with lysozyme and ribonuclease A.

The reactivity of the anticancer drug picoplatin (cis-amminedichlorido(2-methylpyridine)platinum(II) complex) with the model proteins hen egg white lysozyme (HEWL) and bovine pancreatic ribonuclease (RNase A) was investigated by electrospray ionisation mass spectrometry (ESI MS) and X-ray crystallography. The data were compared with those previously obtained for the adducts of these proteins with cisplatin, carboplatin and oxaliplatin under the same experimental conditions. ESI-MS data show binding of Pt to both proteins, with fragments retaining the 2-methylpyridine ligand and, possibly, a chloride ion. X-ray crystallography identifies different binding sites on the two proteins, highlighting a different behaviour of picoplatin in the absence or presence of dimethyl sulfoxide (DMSO). Metal-containing fragments bind to HEWL close to the side chains of His15, Asp18, Asp119 and both Lys1 and Glu7, whereas they bind to RNase A on the side chain of His12, Met29, His48, Asp53, Met79, His105 and His119. The data suggest that the presence of DMSO favours the loss of 2-methylpyridine and alters the ability of the Pt compound to bind to the two proteins. With both proteins, picoplatin appears to behave similarly to cisplatin and carboplatin when dissolved in DMSO, whereas it behaves more like oxaliplatin in the absence of the coordinating solvent. This study provides important insights into the pharmacological profile of picoplatin and supports the conclusion that coordinating solvents should not be used to evaluate the biological activities of Pt-based drugs.

Dietary restriction rescues 5-fluorouracil-induced lethal intestinal toxicity in old mice by blocking translocation of opportunistic pathogens.

Chemotherapy remains a major treatment for malignant tumors, yet the application of standard dose intensity chemotherapy is limited due to the side effects of cytotoxic drugs, especially in old populations. The underlying mechanisms of cytotoxicity and strategies to increase the safety and tolerance of chemotherapy remain to be explored. Using 5-fluorouracil (5-FU), a cornerstone chemotherapeutic drug, we demonstrate that the main cause of death in ad libitum (AL) fed mice after 5-FU chemotherapy was infection caused by translocation of intestinal opportunistic pathogens. We show that these opportunistic pathogens greatly increase in the intestine after chemotherapy, which was closely related to loss of intestinal lysozyme. Of note, two weeks of dietary restriction (DR) prior to chemotherapy significantly protected the loss of lysozyme and increased the content of the beneficial Lactobacillus genera, resulting in a substantial inhibition of intestinal opportunistic pathogens and their translocation. The rescue effect of DR could be mimicked by Lysozyme or Lactobacillus gavage. Our study provides the first evidence that DR achieved a comprehensive protection of the intestinal physical, biological and chemical barriers, which significantly improved the overall survival of 5-FU-treated mice. Importantly, the above findings were more prominent in old mice. Furthermore, we show that patients over 65 years old have enriched opportunistic pathogens in their gut microbiota, especially after 5-FU based chemotherapy. Our study reveals important mechanisms for the poor chemotherapy tolerance of the elderly population, which can be significantly improved by short-term DR. This study generates new insights into methods for improving the chemotherapeutic prognosis by increasing the chemotherapy tolerance and safety of patients with malignant tumors.

Synergistic effects of ε-poly-l-lysine and lysozyme against Pseudomonas aeruginosa and Listeria monocytogenes biofilms on beef and food contact surfaces.

This study investigated the synergistic effects of ε-poly- L -lysine (ε-PL) and lysozyme against P. aeruginosa and L. monocytogenes biofilms. Single-culture biofilms of two bacteria were formed on silicone rubber (SR), stainless steel (SS), and beef surfaces and then treated with lysozyme (0.05-5 mg/mL) and ε-PL at minimum inhibitory concentrations (MICs) of 1 to 4 separately or in combination. On the SR surface, P. aeruginosa biofilm was reduced by 1.4 and 1.9 log CFU/cm2 within 2 h when treated with lysozyme (5 mg/mL) and ε-PL (4 MIC), respectively, but this reduction increased significantly to 4.1 log CFU/cm2 (P < 0.05) with the combined treatment. On beef surface, P. aeruginosa and L. monocytogenes biofilm was reduced by 4.2-5.0, and 3.3-4.2 log CFU/g when lysozyme was combined with 1, 2, and 4 MIC of ε-PL at 25 °C, respectively. Compared to 5 mg/mL lysozyme alone, the combined treatment with 1, 2, and 4 MIC of ε-PL on beef surface achieved additional reduction against P. aeruginosa biofilm of 0.5, 0.8, and 0.7 log CFU/g, respectively, at 25 °C. In addition, 0.25 mg/mL lysozyme and 0.5 MIC of ε-PL significantly (P < 0.05) suppressed the quorum-sensing (agrA) and virulence-associated (hlyA and prfA) genes of L. monocytogenes.

Comparison of Sucrose and Trehalose for Protein Stabilization Using Differential Scanning Calorimetry.

The disaccharide trehalose is generally acknowledged as a superior stabilizer of proteins and other biomolecules in aqueous environments. Despite many theories aiming to explain this, the stabilization mechanism is still far from being fully understood. This study compares the stabilizing properties of trehalose with those of the structurally similar disaccharide sucrose. The stability has been evaluated for the two proteins, lysozyme and myoglobin, at both low and high temperatures by determining the glass transition temperature, Tg, and the denaturation temperature, Tden. The results show that the sucrose-containing samples exhibit higher Tden than the corresponding trehalose-containing samples, particularly at low water contents. The better stabilizing effect of sucrose at high temperatures may be explained by the fact that sucrose, to a greater extent, binds directly to the protein surface compared to trehalose. Both sugars show Tden elevation with an increasing sugar-to-protein ratio, which allows for a more complete sugar shell around the protein molecules. Finally, no synergistic effects were found by combining trehalose and sucrose. Conclusively, the exact mechanism of protein stabilization may vary with the temperature, as influenced by temperature-dependent interactions between the protein, sugar, and water. This variability can make trehalose to a superior stabilizer under some conditions and sucrose under others.

Chitosan-coated liposome with lysozyme-responsive properties for on-demand release of levofloxacin.

As an anti-infection antibiotic delivery route, a drug-controlled release system based on a specific condition stimulus response can enhance drug stability and bioavailability, reduce antibiotic resistance, achieve on-demand release and improve targeting and utilization efficiency. In this study, chitosan-coated liposomes containing levofloxacin (Lef@Lip@CS) were prepared with lysozyme in body fluids serving as an intelligent "switch" to enable accurate delivery of antibiotics through the catalytic degradation ability of chitosan. Good liposome encapsulation efficacy (64.89 ± 1.86 %) and loading capacity (5.28 ± 0.18 %) were achieved. The controlled-release behavior and morphological characterization before and after enzymatic hydrolysis confirmed that the levofloxacin release rate depended on the lysozyme concentration and the degrees of deacetylation of chitosan. In vitro bacteriostatic experiments showed significant differences in the effects of Lef@Lip@CS before and after enzyme addition, with 6-h inhibition rate of 72.46 % and 100 %, and biofilm removal rates of 51 % and 71 %, respectively. These findings show that chitosan-coated liposomes are a feasible drug delivery system responsive to lysozyme stimulation.

Epigallocatechin-3-gallate attenuates the sulfamethoxazole-induced immunotoxicity and reduces SMZ residues in Procambarus clarkii.

Sulfamethoxazole (SMZ) is a commonly used antibiotic in aquaculture, and its residues in water bodies pose a significant threat to aquatic organisms in the water environment. In the present study, epigallocatechin-3-gallate (EGCG), a catecholamine, was used to mitigate the immunotoxicity caused by SMZ exposure in Procambarus clarkii. EGCG reduced the apoptosis rate, which was elevated by SMZ exposure, and increased the total hemocyte count. Simultaneously, EGCG enhanced the activities of enzymes related to antibacterial and antioxidant activities, such as superoxide dismutase (SOD), catalase (CAT), lysozyme (LZM), acid phosphatase (ACP), and GSH, which were decreased following SMZ exposure. Hepatopancreatic histology confirmed that EGCG ameliorated SMZ-induced tissue damage caused by SMZ exposure. In addition to EGCG attenuating SMZ-induced immunotoxicity in crayfish, we determined that EGCG can effectively reduce SMZ residues in crayfish exposed to SMZ. In addition, at the genetic level, the expression levels of genes related to the immune response in hemocytes were disrupted after SMZ exposure, and EGCG promoted their recovery and stimulated an increase in the expression levels of metabolism-related transcripts in hemocytes. The transcriptome analysis was conducted, and "phagosome" and "apoptosis" pathways were shown to be highlighted using Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. To the best of our knowledge, this is the first study to confirm that EGCG attenuates SMZ-induced immunotoxicity in aquatic animals and reduces SMZ residues in aquatic animals exposed to SMZ. Our study contributes to the understanding of the mechanisms by which EGCG reduces the immunotoxicity of antibiotic residues in aquatic animals.

Mix and Shake: A Mild Way to Drug-Loaded Lysozyme Nanoparticles.

Biocompatible nanoparticles as drug carriers can improve the therapeutic efficiency of hydrophobic drugs. However, the synthesis of biocompatible and biodegradable polymeric nanoparticles can be time-consuming and often involves toxic solvents. Here, a simple method for protein-based stable drug-loaded particles with a narrow polydispersity is introduced. In this process, lysozyme is mixed with hydrophobic drugs (curcumin, ellipticine, and dasatinib) and fructose to prepare lysozyme-based drug particles of around 150 nm in size. Fructose is mixed with the drug to generate nanoparticles that serve as templates for the lysozyme coating. The effect of lysozyme on the physicochemical properties of these nanoparticles is studied by transmission electron microscopy (TEM) and scattering techniques (e.g., dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS)). We observed that lysozyme significantly stabilized the curcumin fructose particles for 7 days. Moreover, additional drugs, such as ellipticine and dasatinib, can be loaded to form dual-drug particles with narrow polydispersity and spherical morphology. The results also reveal that lysozyme dual ellipticine/dasatinib curcumin particles enhance the cytotoxicity and uptake on MCF-7 cells, RAW 264.7 cells, and U-87 MG cells due to the larger and rigid hydrophobic core. In summary, lysozyme in combination with fructose and curcumin can serve as a powerful combination to form protein-based stable particles for the delivery of hydrophobic drugs.

Detection and disaggregation of amyloid fibrils by luminescent amphiphilic platinum(II) complexes.

Cyclometallated Pt(II) complexes possessing hydrophobic 2-phenylpyridine (ppy) ligands and hydrophilic acetonylacetone (acac) ligands have been investigated for their ability to detect amyloid fibrils via luminescence response. Using hen egg-white lysozyme (HEWL) as a model amyloid protein, Pt(II) complexes featuring benzanilide-substituted ppy ligands and ethylene glycol-functionalized acac ligands demonstrated enhanced luminescence in the presence of HEWL fibrils, whereas Pt(II) complexes lacking complementary hydrophobic/hydrophilic ligand sets displayed little to no emission enhancement. An amphiphilic Pt(II) complex incorporating a bis(ethylene glycol)-derivatized acac ligand was additionally found to trigger restructuring of HEWL fibrils into smaller spherical aggregates. Amphiphilic Pt(II) complexes were generally non-toxic to SH-SY5Y neuroblastoma cells, and several complexes also exhibited enhanced luminescence in the presence of Aß42 fibrils associated with Alzheimer's disease. This study demonstrates that easily prepared and robust (ppy)PtII(acac) complexes show promising reactivity toward amyloid fibrils and represent attractive molecular scaffolds for design of small-molecule probes targeting amyloid assemblies.

Starch-Based Polysaccharide Systems with Bioactive Substances: Physicochemical and Wettability Characteristics.

Polysaccharide-based systems have very good emulsifying and stabilizing properties, and starch plays a leading role. Their modifications should add new quality features to the product to such an extent that preserves the structure-forming properties of native starch. The aim of this manuscript was to examine the physicochemical characteristics of the combinations of starch with phospholipids or lysozymes and determine the effect of starch modification (surface hydrophobization or biological additives) and preparation temperature (before and after gelatinization). Changes in electrokinetic potential (zeta), effective diameter, and size distribution as a function of time were analyzed using the dynamic light scattering and microelectrophoresis techniques. The wettability of starch-coated glass plates before and after modification was checked by the advancing and receding contact angle measurements, as well as the angle hysteresis, using the settle drop method as a complement to profilometry and FTIR. It can be generalized that starch dispersions are more stable than analogous n-alkane/starch emulsions at room and physiological temperatures. On the other hand, the contact angle hysteresis values usually decrease with temperature increase, pointing to a more homogeneous surface, and the hydrophobization effect decreases vs. the thickness of the substrate. Surface hydrophobization of starch carried out using an n-alkane film does not change its bulk properties and leads to improvement of its mechanical and functional properties. The obtained specific starch-based hybrid systems, characterized in detail by switchable wettability, give the possibility to determine the energetic state of the starch surface and understand the strength and specificity of interactions with substances of different polarities in biological processes and their applicability for multidirectional use.

Effects of high dietary inclusion of Arthrospira platensis, either extruded or supplemented with a super-dosing multi-enzyme mixture, on broiler growth performance and major meat quality parameters.

BACKGROUND: This investigation assessed the effects of high dietary inclusion of Spirulina (Arthrospira platensis) on broiler chicken growth performance, meat quality and nutritional attributes. For this, 120 male broiler chicks were housed in 40 battery brooders (three birds per brooder). Initially, for 14 days, a standard corn and soybean meal diet was administered. Subsequently, from days 14 to 35, chicks were assigned to one of the four dietary treatments (n = 10 per treatment): (1) control diet (CTR); (2) diet with 15% Spirulina (SP); (3) diet with 15% extruded Spirulina (SPE); and (4) diet with 15% Spirulina plus a super-dosing enzymes supplement (0.20% pancreatin extract and 0.01% lysozyme) (SPM). RESULTS: Throughout the experimental period, both SP and SPM diets resulted in decreased final body weight and body weight gain compared to control (p < 0.001), with the SPE diet showing comparable results to CTR. The SPE diet prompted an increase in average daily feed intake (p = 0.026). However, all microalga treatments increased the feed conversion ratio compared to CTR. Dietary inclusion of Spirulina notably increased intestinal content viscosity (p < 0.010), which was mitigated by the SPM diet. Spirulina supplementation led to lower pH levels in breast meat 24 h post-mortem and heightened the b* colour value in both breast and thigh meats (p < 0.010). Furthermore, Spirulina contributed to an increased accumulation of total carotenoids, n-3 polyunsaturated fatty acids (PUFA), and saturated fatty acids (SFA), while diminishing n-6 PUFA, thus altering the n-6/n-3 and PUFA/SFA ratios favourably (p < 0.001). However, it also reduced zinc concentration in breast meat (p < 0.001). CONCLUSIONS: The findings indicate that high Spirulina levels in broiler diets impair growth due to increased intestinal viscosity, and that extrusion pre-treatment mitigates this effect. Despite reducing digesta viscosity, a super-dosing enzyme mix did not improve growth. Data also indicates that Spirulina enriches meat with antioxidants and n-3 PUFA but reduces α-tocopherol and increases saturated fats. Reduced zinc content in meat suggests the need for Spirulina biofortification to maintain its nutritional value.

Turn-off enzyme activity of histidine-rich peptides for the detection of lysozyme.

An assay that integrates histidine-rich peptides (HisRPs) with high-affinity aptamers was developed enabling the specific and sensitive determination of the target lysozyme. The enzyme-like activity of HisRP is inhibited by its interaction with a target recognized by an aptamer. In the presence of the target, lysozyme molecules progressively assemble on the surface of HisRP in a concentration-dependent manner, resulting in the gradual suppression of enzyme-like activity. This inhibition of HisRP's enzyme-like activity can be visually observed through color changes in the reaction product or quantified using UV-visible absorption spectroscopy. Under optimal conditions, the proposed colorimetric assay for lysozyme had a detection limit as low as 1 nM and exhibited excellent selectivity against other nonspecific interferents. Furthermore, subsequent research validated the practical applicability of the developed colorimetric approach to saliva samples, indicating that the assay holds significant potential for the detection of lysozymes in samples derived from humans.

The Effect of Hyperbaric Storage on the Nutritional Value and Retention of Certain Bioactive Proteins in Human Milk.

Human milk (HM) contains the essential macronutrients and bioactive compounds necessary for the normal growth and development of newborns. The milk collected by human milk banks is stored frozen and pasteurized, reducing its nutritional and biological value. The purpose of this study was to determine the effect of hyperbaric storage at subzero temperatures (HS-ST) on the macronutrients and bioactive proteins in HM. As control samples, HM was stored at the same temperatures under 0.1 MPa. A Miris HM analyzer was used to determine the macronutrients and the energy value. The lactoferrin (LF), lysozyme (LYZ) and α-lactalbumin (α-LAC) content was checked using high-performance liquid chromatography, and an ELISA test was used to quantify secretory immunoglobulin A (sIgA). The results showed that the macronutrient content did not change significantly after 90 days of storage at 60 MPa/-5 °C, 78 MPa/-7 °C, 111 MPa/-10 °C or 130 MPa/-12 °C. Retention higher than 90% of LYZ, α-LAC, LF and sIgA was observed in the HM stored at conditions of up to 111 MPa/-10 °C. However, at 130 MPa/-12 °C, there was a reduction in LYZ and LF, by 39 and 89%, respectively. The storage of HM at subzero temperatures at 0.1 MPa did not affect the content of carbohydrates or crude and true protein. For fat and the energy value, significant decreases were observed at -5 °C after 90 days of storage.