Protein Shell Research Articles

Characterization of enzyme scaffolding inside the alpha-carboxysome by cryo-electron tomography.

Roughly 20% of bacteria employ bacterial microcompartments (BMCs) to sequester enzymatic processes. In these structures, a cargo of enzymes and accessory proteins is encased within a semi-permeable protein shell that permits passage of substrates and products but restricts movement of intermediates. In addition to their importance as a component of many bacterial species’ metabolisms, BMCs have become a target of protein engineering due to their potential for enclosing cargos of choice for alternative pharmacological and biotechnological applications. The shells can be reassembled from purified proteins, and the full operons can be functionally expressed outside their native prokaryotes.

Quantifying photosynthetic metabolites permeability across bacterial microcompartments using computational microscopy

Carboxysomes are Bacterial microcompartments, found in cyanobacteria that locally concentrate carbon dioxide (CO2) to improve the efficiency of the enzyme RuBisCO, a key step towards photosynthetic carbon fixation by the Calvin-Benson-Bassham (CBB) cycle. The carboxysome shell is made up of proteins that encapsulate RuBisCO and carbonic anhydrase. Carbonic anhydrase converts soluble bicarbonate to CO2, increasing the local concentration of CO2 for RuBisCO. In addition to CO2, other metabolites such as oxygen (O2), bicarbonate (HCO3−), 3-phosphoglyceric acid (3-PGA) and ribulose-1,5-bisphosphate (RuBP) also need to permeate through the carboxysome shell to efficiently perform CO2 fixation. Quantifying the permeability of the shell for these metabolites currently remains a challenge and one of the critical design features related to the design of synthetic bacterial microcompartments for metabolic and sustainablebioengineering applications. Leveraging a high resolution cryo-electron microscopy structure of a synthetic beta-carboxysome shell and new graphics processing unit (GPU)-resident molecular simulation engines, we determine the permeability of photosynthetic metabolites across synthetic carboxysome shell through unbiased molecular simulation at all-atom resolution. We find that the carboxysome itself is not selectively permeable to bicarbonate over carbon dioxide, as originally hypothesized. Instead, the carboxysome shell proteins form a general barrier to maintain the carbon dioxide gradient generated by carbonic anhydrase activity within the carboxysome, compensating for inefficiencies of endogenous RuBisCO, specifically slow turnover rate, and photorespiration. The results from all-atom molecular simulations presented here provide a detailed mechanistic picture, using the shell permeabilities to engineer smart synthetic bacterial microcompartments and to improve photosynthetic efficiency with a wide range of applications in sustainable, metabolic, agricultural, and biomedical engineering.

A single thiolated-phage displayed nanobody-based biosensor for label-free detection of foodborne pathogen.

Rapid and sensitive detection of bacterial pathogens present in food and environmental samples is of crucial importance to ensure human health and safety. Here, we present a one-step label-free colorimetric strategy based on M13 bacteriophage-displayed nanobody (phage-Nb) derived from camelid heavy-chain antibodies specific to Vibrio parahaemolyticus (V. parahaemolyticus). The thiolation of phage-Nb (Phage-Nb-SH) on pVIII shell proteins induces the aggregation of gold nanoparticles (AuNPs), whereas the specific interaction between nanobody and bacteria prevents the aggregation of AuNPs, resulting in visible color change due to alteration of surface plasmon resonance properties. Based on this phenomenon, a simple and sensitive colorimetric immunosensor for V. parahaemolyticus was developed. The assay can be accomplished within 100min, and exhibits a visual detection limit of 104 cfu/mL and a quantitative detection limit of 103 cfu/mL, with no cross-reactivity towards other bacterial species. This strategy takes full advantages of both the high specificity of phage-Nbs and the optical properties of AuNPs, enabling simple and rapid detection of bacterial pathogens.

Shell proteins and microstructural analysis identify the origin of shell arts with species resolution in pearl oysters

Molluscan shells are diverse, beautiful, and mechanically robust. They were commonly used by humans during ancient times as money, decorative objects, or functional tools. Luodian, a unique shell art in China dating back 3000 years, has served as a cultural carrier between China and Europe. To understand the production processes as well as underlying cultural contexts, it is important to identify the material origin of the art. This study combined proteomics and microstructural analysis to identify the raw material origins of three Luodian samples from the Qing Dynasty with species resolution and potential maturity levels. Shell proteomics and multiple sequence alignment have identified key shell matrix proteins (N66 and Pif) from Pinctada maxima, a pearl oyster distributed in the Indo-Pacific region. Although other factors (temperature, salinity, etc.) may contribute to the thickness of nacreous tablets in the shell, the thickness is inversely proportional to the shell size in the pearl oyster. We inferred the maturity of raw materials in different Luodian samples, which are tailored to different purposes. This study provides a framework and foundation for identifying the origin of various shell arts, especially those made from pearl oysters.

Производственные испытания эффективности препарата «Битоксибациллин» в Липецкой области на птицефабрике «Новоникольская»

Bitoxybacillin is a biological insecticide whose main active ingredient is bacterial spores and cells preserved in a protein shell, endotoxin and thermostable exotoxin. The results of production experiments with Bitoxybacillin held in the Lipetsk region on the Novonikolskaya poultry farm during the period from April 25 to September 10 2021 are presented in the article. According to the results of conducted researches Bitoxybacillin preparation is effective when used in 3 % concentration when the layer thickness is up to 50 cm – 3 liters per 1 m2, when manure is accumulated when the layer thickness is more than 50 cm – 10 liters per 1 m2.

In-depth magnetometry and EPR analysis of the spin structure of human-liver ferritin: from DC to 9 GHz.

Ferritin, the major iron storage protein in organisms, stores iron in the form of iron oxyhydroxide most likely involving phosphorous as a constituent, the mineral form of which is not well understood. Therefore, the question of how the ca. 2000 iron atoms in the ferritin core are magnetically coupled is still largely open. The ferritin core, with a diameter of 5-8 nm, is encapsulated in a protein shell that also catalyzes the uptake of iron and protects the core from outside interactions. Neurodegenerative disease is associated with iron imbalance, generating specific interest in the magnetic properties of ferritin. Here we present 9 GHz continuous wave EPR and a comprehensive set of magnetometry techniques including isothermal remanent magnetization (IRM) and AC susceptibility to elucidate the magnetic properties of the core of human liver ferritin. For the analysis of the magnetometry data, a new microscopic model of the ferritin-core spin structure is derived, showing that magnetic moment is generated by surface-spin canting, rather than defects. The analysis explicitly includes the distribution of magnetic parameters, such as the distribution of the magnetic moment. This microscopic model explains some of the inconsistencies resulting from previous analysis approaches. The main findings are a mean magnetic moment of 337μB with a standard deviation of 0.947μB. In contrast to previous reports, only a relatively small contribution of paramagnetic and ferrimagnetic phases is found, in the order of maximally 3%. For EPR, the over 30 mT wide signal of the ferritin core is analyzed using the model of the giant spin system [Fittipaldi et al., Phys. Chem. Chem. Phys., 2016, 18, 3591-3597]. Two components are needed minimally, and the broadening of these components suggests a broad distribution of the magnetic resonance parameters, the zero-field splitting, D, and the spin quantum number, S. We compare parameters from EPR and magnetometry and find that EPR is particularly sensitive to the surface spins of the core, revealing the potential to use EPR as a diagnostic for surface-spin disorder.

The buckling-condensation mechanism driving gas vesicle collapse.

Gas vesicles (GVs) are proteinaceous cylindrical shells found within bacteria or archea growing in aqueous environments and are composed primarily of two proteins, gas vesicle protein A and C (GvpA and GvpC). GVs exhibit strong performance as next-generation ultrasound contrast agents due to their gas-filled interior, tunable collapse pressure, stability in vivo and functionalizable exterior. However, the exact mechanism leading to GV collapse remains inconclusive, which leads to difficulty in predicting collapse pressures for different species of GVs and in extending favorable nonlinear response regimes. Here, we propose a two stage mechanism leading to GV loss of echogenicity and rupture under hydrostatic pressure: elastic buckling of the cylindrical shell coupled with condensation driven weakening of the GV membrane. Our goal is to therefore test whether the final fracture of the GV membrane occurs by the interplay of both mechanisms or purely through buckling failure as previously believed. To do so, we (1) compare the theoretical condensation and buckling pressures with that for experimental GV collapse and (2) describe how condensation can lead to plastic buckling failure. GV shell properties that are necessary input to this theoretical description, such as the elastic moduli and wettability of GvpA, are determined using molecular dynamics simulations of a novel structural model of GvpA that better represents the hydrophobic core. For GVs that are not reinforced by GvpC, this analytical framework shows that the experimentally observed pressures resulting in loss of echogenicity coincide with both the elastic buckling and condensation pressure regimes. We also found that the stress strain curve for GvpA wetted on both the interior and exterior exhibits a loss of mechanical stability compared to GvpA only wetted on the exterior by the bulk solution. We identify a pressure vs. vesicle size regime where condensation can occur prior to buckling, which may preclude nonlinear shell buckling responses in contrast imaging.

Close packings of identical proteins in small spherical capsids and similar proteinaceous shells.

Understanding the principles governing protein arrangement in viral capsids and structurally similar protein shells can enable the development of new antiviral strategies and the design of artificial protein cages for various applications. We study these principles within the context of the close packing problem, by analyzing dozens of small spherical shells assembled from a single type of protein. First, we use icosahedral spherical close packings containing 60T identical disks, where T ≤ 4, to rationalize the protein arrangement in twenty real icosahedral shells both satisfying and violating the paradigmatic Caspar-Klug model. We uncover a striking correspondence between the protein mass centers in the considered shells and the centers of disks in the close packings. To generalize the packing model, we consider proteins with a weak shape anisotropy and propose an interaction energy, minimization of which allows us to obtain spherical dense packings of slightly anisotropic structural units. In the case of strong anisotropy, we model the proteins as sequences of overlapping discs of different sizes, with minimum energy configuration not only resulting in packings, accurately reproducing locations and orientations of individual proteins, but also revealing that icosahedral packings that display the handedness of real capsids are energetically more favorable. Finally, by introducing effective disc charges, we rationalize the formation of inter-protein bonds in protein shells.

Hidden symmetry of the flavivirus protein shell and pH-controlled reconstruction of the viral surface.

Using recent Zika virus structural data we reveal a hidden symmetry of protein order in immature and mature flavivirus shells, violating the Caspar-Klug paradigmatic model of capsid structures. We show that proteins of the outer immature shell layer exhibit trihexagonal tiling, while proteins from inner and outer layers conjointly form a double-shelled close-packed structure, based on a common triangular spherical lattice. Within the proposed structural model, we furthermore rationalize the structural organization of misassembled non-infectious subviral particles that have no inner capsid. We consider a pH-controlled structural reconstruction of the outer shell from the trimeric to the dimeric state, and demonstrate that this transition, occurring during the virus maturation, can be induced by changes in protein charges at lower pH, leading to a decrease in the electrostatic interaction free energy. This transition could also be assisted by electrostatic attraction of shell proteins to the interposed lipid membrane substrate separating the shells.

Micromagnetic and morphological characterization of heteropolymer human ferritin cores.

The physical properties of in vitro iron-reconstituted and genetically engineered human heteropolymer ferritins were investigated. High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM), electron energy-loss spectroscopy (EELS), and 57Fe Mössbauer spectroscopy were employed to ascertain (1) the microstructural, electronic, and micromagnetic properties of the nanosized iron cores, and (2) the effect of the H and L ferritin subunit ratios on these properties. Mössbauer spectroscopic signatures indicate that all iron within the core is in the high spin ferric state. Variable temperature Mössbauer spectroscopy for H-rich (H21/L3) and L-rich (H2/L22) ferritins reconstituted at 1000 57Fe/protein indicates superparamagnetic behavior with blocking temperatures of 19 K and 28 K, while HAADF-STEM measurements give average core diameters of (3.7 ± 0.6) nm and (5.9 ± 1.0) nm, respectively. Most significantly, H-rich proteins reveal elongated, dumbbell, and crescent-shaped cores, while L-rich proteins present spherical cores, pointing to a correlation between core shape and protein shell composition. Assuming an attempt time for spin reversal of τ 0 = 10-11 s, the Néel-Brown formula for spin-relaxation time predicts effective magnetic anisotropy energy densities of 6.83 × 104 J m-3 and 2.75 × 104 J m-3 for H-rich and L-rich proteins, respectively, due to differences in surface and shape contributions to magnetic anisotropy in the two heteropolymers. The observed differences in shape, size, and effective magnetic anisotropies of the derived biomineral cores are discussed in terms of the iron nucleation sites within the interior surface of the heteropolymer shells for H-rich and L-rich proteins. Overall, our results imply that site-directed nucleation and core growth within the protein cavity play a determinant role in the resulting core morphology. Our findings have relevance to iron biomineralization processes in nature and the growth of designer's magnetic nanoparticles within recombinant apoferritin nano-templates for nanotechnology.

Modification of bacterial microcompartments with target biomolecules via post-translational SpyTagging.

Bacterial microcompartments (BMCs) are proteinaceous organelle-like structures formed within bacteria, often encapsulating enzymes and cellular processes, in particular, allowing toxic intermediates to be shielded from the general cellular environment. Outside of their biological role they are of interest, through surface modification, as potential drug carriers and polyvalent antigen display scaffolds. Here we use a post-translational modification approach, using copper free click chemistry, to attach a SpyTag to a target protein molecule for attachment to a specific SpyCatcher modified BMC shell protein. We demonstrate that a post-translationally SpyTagged material can react with a SpyCatcher modified BMC and show its presence on the surface of BMCs, enabling future investigation of these structures as polyvalent antigen display scaffolds for vaccine development. This post-translational 'click' methodology overcomes the necessity to genetically encode the SpyTag, avoids any potential reduction in expression yield and expands the scope of SpyTag/SpyCatcher vaccine scaffolds to form peptide epitope vaccines and small molecule delivery agents.

Abstract 1466: In Vitro Assembly of a Microcompartment from a Single Carboxysome Shell Protein

Abstract 1466: In Vitro Assembly of a Microcompartment from a Single Carboxysome Shell Protein

Screening of anti-liver fibrosis peptides from turtle shell protein using two-enzyme hydrolysis by molecular docking.

Turtle shell as a food residue of Pelodiscus sinensis (a type of edible aquatic animal) is widely used in Traditional Chinese Medicine for hepatic fibrosis therapy. Previous studies have demonstrated that the peptides (<6 kDa) derived from turtle shells are considered effective components. The protein of turtle shells has important potential as a source of bioactive peptides which may play a role as ingredients in functional foods. In the present study, the protein of turtle shell was hydrolyzed using a two-enzyme combination. It was found that the hydrolysates obtained by a combination of pepsin and trypsin showed the highest anti-liver fibrosis activity relative to other combinations in a cell viability assay. The hydrolysates were separated and purified by ultra-filtration (<6 kDa), gel filtration chromatography (GFC) and high-performance liquid chromatography (HPLC). Subsequently, the sequences of purified peptides were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). Molecular docking was used to analyze the interaction of these peptides with the transforming growth factor-β1 (TGF-β1) receptor. Two (GPPGVPGPGPL, TSLPVPAPV) of these novel peptides displayed lower binding energies to the TGF-β1 receptor (-8.18 kcal mol-1, -8 kcal mol-1). Finally, the above two peptides were synthesized chemically and their in vitro anti-liver fibrosis activity was verified by MTT assay. Among them, GPPGVPGPGPL showed a better in vitro anti-liver fibrosis activity (IC50: 80.13 μM). We established a method to obtain anti-liver fibrosis peptides from turtle shells by using bioactivity-guided isolation with molecular docking. Turtle shell protein is an excellent source of anti-liver fibrosis peptides which can offer therapeutic and commercial benefits as an ingredient in functional foods.

Crossover Dynamics of Rotavirus Disease under Fractional Piecewise Derivative with Vaccination Effects: Simulations with Real Data from Thailand, West Africa, and the US

Many diseases are caused by viruses of different symmetrical shapes. Rotavirus particles are approximately 75 nm in diameter. They have icosahedral symmetry and particles that possess two concentric protein shells, or capsids. In this research, using a piecewise derivative framework with singular and non-singular kernels, we investigate the evolution of rotavirus with regard to the effect of vaccination. For the considered model, the existence of a solution of the piecewise rotavirus model is investigated via fixed-point results. The Adam–Bashforth numerical method along with the Newton polynomial is implemented to deduce the numerical solution of the considered model. Various versions of the stability of the solution of the piecewise rotavirus model are presented using the Ulam–Hyres concept and nonlinear analysis. We use MATLAB to perform the numerical simulation for a few fractional orders to study the crossover dynamics and evolution and effect of vaccination on rotavirus disease. To check the validity of the proposed approach, we compared our simulated results with real data from various countries.

Molecular characterization of accripin11, a soluble shell protein with an acidic C-terminus, identified in the prismatic layer of the Mediterranean fan mussel Pinna nobilis (Bivalvia, Pteriomorphia).

We have identified a novel shell protein, accripin11, as a major soluble component of the calcitic prisms of the fan mussel Pinna nobilis. Initially retrieved from a cDNA library, its full sequence is confirmed here by transcriptomic and proteomic approaches. The sequence of the mature protein is 103 residues with a theoretical molecular weight of 11 kDa and is moderately acidic (pI 6.74) except for its C-terminus which is highly enriched in aspartic acid. The protein exhibits a peculiar cysteine pattern in its central domain. The full sequence shares similarity with six other uncharacterized molluscan shell proteins from the orders Ostreida, Pteriida and Mytilida, all of which are pteriomorphids and produce a phylogenetically restricted pattern of nacro-prismatic shell microstructures. This suggests that accripin11 is a member of a family of clade-specific shell proteins. A 3D model of accripin11 was predicted with AlphaFold2, indicating that it possesses three short alpha helices and a disordered C-terminus. Recombinant accripin11 was tested in vitro for its ability to influence the crystallization of CaCO3 , while a polyclonal antibody was able to locate accripin11 to prismatic extracts, particularly in the acetic acid-soluble matrix. The putative functions of accripin11 are further discussed in relation to shell biomineralization.

Biosynthetic Gas Vesicles Combined with Focused Ultrasound for Blood-Brain Barrier Opening.

Focused ultrasound (FUS) combined with microbubbles (MBs) has emerged as a potential approach for opening the blood-brain barrier (BBB) for delivering drugs into the brain. However, MBs range in size of microns and thus can hardly extravasate into the brain parenchyma. Recently, growing attention has been paid to gas vesicles (GVs), which are genetically encoded gas-filled nanostructures with protein shells, due to their potential for extravascular targeting in ultrasound imaging and therapy. However, the use of GVs as agents for BBB opening has not yet been investigated. In this study, GVs were extracted and purified from Halobacterium NRC-1. Ultrasound imaging performance of GVs was assessed in vitro and in vivo. Then, FUS/GVs-mediated BBB opening for small molecular Evans blue or large molecular liposome delivery across the BBB was examined. The results showed a good contrast performance of GVs for brain perfusion ultrasound imaging in vivo. At the acoustic negative pressure of 1.5 MPa, FUS/GVs opened the BBB safely, and effectively enhanced Evans blue and 200-nm liposome delivery into the brain parenchyma. Our study suggests that biosynthetic GVs hold great potential to serve as local BBB-opening agents in the development of new targeted drug delivery strategies for central nervous system disorders.

Cross-Species Permissivity: Structure of a Goat Adeno-Associated Virus and Its Complex with the Human Receptor AAVR.

Adeno-associated virus (AAV) is a small ssDNA satellite virus of high interest (in recombinant form) as a safe and effective gene therapy vector. AAV's human cell entry receptor (AAVR) contains polycystic kidney disease (PKD) domains bound by AAV. Seeking understanding of the spectrum of interactions, goat AAVGo.1 is investigated, because its host is the species most distant from human with reciprocal cross-species cell susceptibility. The structure of AAVGo.1, solved by cryo-EM to 2.9 Å resolution, is most similar to AAV5. Through ELISA (enzyme-linked immunosorbent assay) studies, it is shown that AAVGo.1 binds to human AAVR more strongly than do AAV2 or AAV5, and that it joins AAV5 in a class that binds exclusively to PKD domain 1 (PKD1), in contrast to other AAVs that interact primarily with PKD2. The AAVGo.1 cryo-EM structure of a complex with a PKD12 fragment of AAVR at 2.4 Å resolution shows PKD1 bound with minimal change in virus structure. There are only minor conformational adaptations in AAVR, but there is a near-rigid rotation of PKD1 with maximal displacement of the receptor domain by ~1 Å compared to PKD1 bound to AAV5. AAVGo.1 joins AAV5 as the second member of an emerging class of AAVs whose mode of receptor-binding is completely different from other AAVs, typified by AAV2. IMPORTANCE Adeno-associated virus (AAV) is a small ssDNA satellite parvovirus. As a recombinant vector with a protein shell encapsidating a transgene, recombinant AAV (rAAV) is a leading delivery vehicle for gene therapy, with two FDA-approved treatments and 150 clinical trials for 30 diseases. The human entry receptor AAVR has five PKD domains. To date, all serotypes, except AAV5, have interacted primarily with the second PKD domain, PKD2. Goat is the AAV host most distant from human with cross-species cell infectivity. AAVGo.1 is similar in structure to AAV5, the two forming a class with a distinct mode of receptor-binding. Within the two classes, binding interactions are mostly conserved, giving an indication of the latitude available in modulating delivery vectors.

Role of carboxysomes in cyanobacterial CO2 assimilation: CO2 concentrating mechanisms and metabolon implications.

Many carbon-fixing organisms have evolved CO2 concentrating mechanisms (CCMs) to enhance the delivery of CO2 to RuBisCO, while minimizing reactions with the competitive inhibitor, molecular O2 . These distinct types of CCMs have been extensively studied using genetics, biochemistry, cell imaging, mass spectrometry, and metabolic flux analysis. Highlighted in this paper, the cyanobacterial CCM features a bacterial microcompartment (BMC) called 'carboxysome' in which RuBisCO is co-encapsulated with the enzyme carbonic anhydrase (CA) within a semi-permeable protein shell. The cyanobacterial CCM is capable of increasing CO2 around RuBisCO, leading to one of the most efficient processes known for fixing ambient CO2 . The carboxysome life cycle is dynamic and creates a unique subcellular environment that promotes activity of the Calvin-Benson (CB) cycle. The carboxysome may function within a larger cellular metabolon, physical association of functionally coupled proteins, to enhance metabolite channelling and carbon flux. In light of CCMs, synthetic biology approaches have been used to improve enzyme complex for CO2 fixations. Research on CCM-associated metabolons has also inspired biologists to engineer multi-step pathways by providing anchoring points for enzyme cascades to channel intermediate metabolites towards valuable products.

Effects of Using Processed Amaranth Grain with and without Enzyme on Performance, Egg Quality, Antioxidant Status and Lipid Profile of Blood and Yolk Cholesterol in Laying Hens.

The objective of this study was to investigate the effects of feeding Amaranthus hybridus chlorostachys grain (AG) with (+E) and without enzyme (-E) on performance, egg quality, antioxidant status and lipid profile of blood serum and yolk cholesterol in laying hens. A total of 960 white leghorn (Hy-line W-36) commercial layers (56 weeks) were divided into 10 groups with 8 replicates per group (12 birds per replicate, including 3 adjacent cages with 4 birds each). A completely randomized design was implemented with a 5 × 2 factorial arrangement of treatments consisting of five levels of AG (0, 100, 200, 300 and 400 g/kg) and two levels of multienzyme complex addition (0 -E and 0.25 +E g/kg) fed to the hens for 12 weeks (2 wk. adaptation + 10 wk. main experiment). Feed intake (FI) and percentage of hen day production (HDP) were not affected by main effect of the AG level, but egg mass (EM) and egg weight (EW) were decreased (p &lt; 0.01), and the feed conversion ratio (FCR) was impaired (p &lt; 0.01). EM, EW and FCR were improved by enzyme addition (p &lt; 0.01). EM, EW and FCR were affected (p &lt; 0.01) by the interaction of AG and enzyme addition. The highest value of EM and the lowest value of FCR were observed in hens on the diet containing 200 g/kg AG with enzyme addition. Egg yolk cholesterol content was reduced (p &lt; 0.05) by up to 10% with increasing levels AG in experimental diets. The egg quality traits, including Haugh units of protein quality, strength and shell thickness, were not affected by the main effects or interaction of AG and enzyme consumption. Amaranth feeding led to a decrease (p &lt; 0.05) in triglyceride (TG) and low-density lipoprotein (LDL) while also promoting increases (p &lt; 0.05) in the high-density lipoprotein (HDL) and total antioxidant capacity (TAC) of the blood. A comparison of the effects of contrasts showed that functional parameters (except FI), yolk cholesterol, antioxidant parameters (except MDA) and blood lipid profile had differed significantly (p &lt; 0.05) between the hens fed amaranth versus those not fed amaranth. These findings indicate that feeding a diet containing up to 200 g/kg of AG with enzyme addition can improve EW, EM and FCR. Feeding laying hens diets containing AG also positively influenced blood traits and antioxidant status in laying hens while reducing egg yolk cholesterol content.

Dietary supplementation with protein hydrolysates from the shell of red swamp crayfish (Procambarus clarkii) affects growth, muscle antioxidant capacity and circadian clock genes expression of zebrafish (Danio rerio)

Protein hydrolysates are rich in amino acids and bioactive peptides, beneficial for the organism health. This study analyzed not only the effects of dietary supplementation with P. clarkii shell protein hydrolysates (PCSPHs) on the growth performance, muscle biochemical indices and histological characteristics of zebrafish, but also the changes of muscle antioxidant capacity and circadian clock genes expression were also evaluated. After feeding, the final body weight, weight gain rate and specific growth rate of zebrafish fed with PCSPHs supplementation were significantly higher than normal diet, and reduced contents of muscle triglyceride and aspartate aminotransferase. Meanwhile, dietary supplementation with PCSPHs improved total amino acid contents and myofiber structure of zebrafish. Antioxidant enzyme activities of total superoxide dismutase (T-SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) content, and the expression of antioxidant related genes increased with PCSPHs supplementation. Furthermore, the expression of Nrf2 protein showed a significantly up-regulated trend with the increasing of dietary PCSPHs levels. However, it was interesting that the expression of circadian rhythm related genes was also affected by dietary PCSPHs levels, and showed a significant up-regulated and down-regulated trend. The above results showed that dietary supplementation with PCSPHs could promote the growth performance, muscle amino acid contents and myofiber structure of zebrafish, but also enhance the muscle antioxidant capacity and regulates the expression of circadian clock genes. It is hypothesized that this may be related to the expression of antioxidant signaling pathway Nrf2/Keap1. Our study will provide a theoretical basis for improving the muscle quality and health of aquatic animals.