Bovine Hemoglobin Research Articles

A protein structure-dependent fluorescent probe for hemoglobin monitoring and controllable imaging in living cells.

A novel protein structure-dependent non-covalent fluorescent probe, DDBM, was developed. It exhibited selective fluorescence "turn-off" responsiveness to bovine hemoglobin (BHb). This responsiveness depended on the interaction between the probe and BHb, with the methoxynaphthalene group significantly contributing to the sensitivity. Non-covalent interactions played a crucial role in stabilizing the binding of DDBM with BHb. DDBM demonstrated a robust anti-interference capability in its BHb responsiveness. Interestingly, the BHb responsiveness of DDBM could be modulated by ibuprofen. Additionally, DDBM exhibited favorable fluorescence enhancement sensitivity to bovine serum albumin (BSA), coupled with a robust anti-interference capability. These distinctive properties of DDBM enabled it to dynamically trace the metabolism of hemoglobin (Hb) and further achieve Hb-mediated precise controllable live cell imaging.

Controlled fabrication of hierarchical porous carbon nanospheres with high doped nitrogen content for high-performance adsorbent of biomacromolecule

Constructing nitrogen-doped porous carbons with high specific surface area, rapid mass transfer channels, and positive charge is a crucial requirement for high-performance adsorbents. Herein, by the kinetic self-assembly synthesis strategy, we prepared nitrogen-doped hierarchical porous carbon spheres (N-HPCS) with adjustable pore structure, high specific surface area, and high nitrogen doping content (8.88 %). By using ethylenediamine as an assisted polymerization and assembly agent, the hydrolysis and condensation rate of tetraethyl orthosilicate (TEOS) as the silica source and the condensation rate of 3-aminophenol and formaldehyde as the phenolic resin precursor were controlled by adjusting ammonia volume as the alkaline catalyst to tune kinetic self-assembly of silica and phenolic resin components, thus achieving their simultaneous or sequential nucleus and growth. After carbonization and selective silica etching, three types of carbon nanospheres with center-radial pores, hollow center-radial pores and hollow structure were obtained. High nitrogen doping content endowed the nanospheres with positive charge. Through adsorption experiments on the bovine serum albumin (BSA) and Hemoglobin (Hb) as typical biological macromolecules, hollow carbon nanospheres with center-radial pores exhibited excellent adsorption performance for BSA(622.34 mg g−1) and Hb(759.96 mg g−1). Our fabricated N-HPCS may become a potential candidate for high-performance adsorption materials.

DNA and hemoglobin binding activities: Investigation of coumarin-thiosemicarbazone hybrids

Coumarin and coumarin-thiosemicarbazone hybrids were synthesized and characterized by various techniques such as FT-IR, 1H NMR, 13C NMR, MALDI-TOF-MS spectroscopy, and single crystal X-Ray diffractometer (XRD). The photochemical and photophysical properties of the compounds, such as solvatochromism, solubility, and chemical reactivity, were analyzed using UV–vis spectroscopy in different solvents. Due to the potential biological activities of the synthesized compounds, their binding affinity and mechanisms with calf thymus DNA (ct-DNA) and bovine hemoglobin (BHb) were determined using several useful spectrophotometric and theoretical approaches such as UV–vis absorption and fluorescence spectroscopy, molecular docking, and density functional theory (DFT). The experimental results showed that the compounds exhibited strong binding interactions with DNA and BHb. Additionally, the compounds demonstrated predominantly binding modes, such as intercalation and groove binding with DNA and π–π stacking interactions with BHb.To better understand the thermodynamics of these interactions, quenching constants, binding constants, and Gibbs free energy changes (ΔG°) were calculated. Molecular docking and DFT results supported the experimental data regarding the binding affinity and mechanisms of the compounds to DNA and BHb. Overall, this comprehensive study on coumarin and coumarin-thiosemicarbazone hybrids provides valuable insights into their interaction mechanisms with critical biomolecules, highlighting their potential in therapeutic applications as multifunctional agents.

Electro-spinning a calcium alginate based molecular imprinted nanofiber membrane for selectively adsorbing cytochrome c

Cytochrome c (Cyt c) is a typical water-soluble heme metalloprotein with a wide variety of physiological properties. High-efficient separation of Cyt c from bio-matrix is very important to Cyt c practical use but always difficult. By coupling the electro-spinning technology and the molecular imprinting technology, a molecularly imprinted nanofiber membrane (MINM) was prepared by using calcium alginate as skeleton. The characterization indicated a well cross-linked calcium alginate nanofiber membrane was formed with good structure stability and high mechanical strength. The obtained MINM showed high capacity of 3312 mg/g on adsorbing Cyt c at the initial concentration of 2 mg/mL. The adsorption selectivity against bovine serum albumin, bovine hemoglobin and ovalbumin was as high as 36.49, 66.01 and 59.70, respectively. A good separation efficacy toward Cyt c was also observed in pig heart extracts. The obtained MINM had homogenous surface and identical adsorption sites, the adsorption of Cyt c on the membrane was monolayer, chemisorption and diffusion controlled, the molecules diffusion in the interior of the membrane was not the only rate-limiting step. The results indicated a good reusability and a promising practical application value of the obtained MINM on Cyt c separation, which further emphasized the value of the synthesis procedure employed in this study.

Engineering an Extracellular Matrix Mimic Using Hemoglobin Protein Nanofibrils.

Extracellular matrix (ECM) is essential for tissue development, providing structural support and a microenvironment that is necessary for cells. As tissue engineering advances, there is a growing demand for ECM mimics. Polycaprolactone (PCL) is a commonly used synthetic polymer for ECM mimic materials. However, its biologically inactive surface limits its direct application in tissue engineering. Our study aimed to improve the biocompatibility of PCL by incorporating hemoglobin nanofibrils (HbFs) into PCL using an electrospinning technique. HbFs were formed from bovine hemoglobin (Hb) extracted from industrial byproducts and designed to offer PCL an improved cell adhesion property. The fabricated HbFs@PCL electrospun scaffold exhibits improved fibroblast adherence, proliferation, and deeper fibroblast infiltration into the scaffold compared with the pure PCL scaffold, indicating its potential to be an ECM mimic. This study represents the pioneering utilization of Hb-sourced nanofibrils in the electrospun PCL scaffolds for tissue engineering applications.

One-step synthesis of superparamagnetic copper alginate gel microspheres (Cu2+-Alg@Fe3O4) using a compression nebulizer

For the application needs of histidine-rich protein separation, this study proposes to prepare a magnetic adsorbent enriched with copper ions and small particle size. Using a compression nebulizer, the magnetic + sodium alginate solution was broken into micrometer-sized droplets and cross-linked and cured with copper ion solution. The magnetic copper alginate microspheres (Cu2+-Alg@Fe3O4) were obtained with an average particle size of 7.1 μm, a magnetic saturation intensity of 36.9 emu·g−1, and a Cu2+ content of 149.2 mg·g−1.The adsorption equilibrium time of the Cu2+-Alg@Fe3O4 microspheres on bovine hemoglobin(BHb) was 90 min, and the maximum adsorption amount was 4318.5 mg·g−1. The Cu2+-Alg@Fe3O4 microspheres also showed stable and good adsorption performance in five cyclic adsorption experiments.

Construction and Biological Evaluation of Different Nanoshell Thickness Ni@SiO2 Nanotubes as Good Protein Separation Carriers for Bovine Hemoglobin.

Nickel nanomaterials play an important role in biological applications, but they have high toxicity and poor biocompatibility. To overcome these defects, we coated the surface of Ni nanotubes with different thicknesses of SiO2 to reduce cytotoxicity, improve biocompatibility, and broaden their biological application value. This study aimed to construct Ni nanotubes with different thicknesses of SiO2 nanoshells; investigate the effects of silicon layer thickness, incubation time, and cell line category on the cytotoxicity of the as-synthesized materials, and evaluate the biocompatibility of the materials by biological enzymes. The Ni@SiO2-NH2 was selected for use as an adsorbent for the adsorption and purification of histidine-rich proteins, such as Bovine Hemoglobin (BHb). Magnetic Ni nanotubes were prepared by the template-chemical deposition method. A modified version of the Stöber process was used for the SiO2 coating of Ni@SiO2 nanotubes, and adjusted by changing the volume of TEOS for different thicknesses of SiO2 nanoshells. Different cell lines containing tumor cells and normal cells were used in the toxicity experiment, which confirmed the low cytotoxicity and good biocompatibility of Ni@SiO2. To achieve high efficiency of immobilization and purification of histidine- rich proteins, Ni@SiO2-NH2 was obtained by introducing the amino functional group. The Ni@SiO2-NH2 was found to possess lower cytotoxicity and higher adsorption capacity compared to other synthesized materials. Besides, the Ni@SiO2-NH2 also exhibited good selectivity of histidine-rich proteins. This work has not only provided ideas for reducing the cytotoxicity and improving the biocompatibility of biological nanomaterials, but also laid a foundation for subsequent biological applications.

Efficient and rapid digestion of proteins with a dual-enzyme microreactor featuring 3-D pores formed by dopamine/polyethyleneimine/acrylamide-coated KIT-6 molecular sieve

The microreactor with two types of immobilized enzymes, exhibiting excellent orthogonal performance, represents an effective approach to counteract the reduced digestion efficiency resulting from the absence of a single enzyme cleavage site, thereby impacting protein identification. In this study, we developed a hydrophilic dual-enzyme microreactor characterized by rapid mass transfer and superior enzymatic activity. Initially, we selected KIT-6 molecular sieve as the carrier for the dual-IMER due to its three-dimensional network pore structure. Modification involved co-deposition of polyethyleneimine (PEI) and acrylamide (AM) as amine donors, along with dopamine to enhance material hydrophilicity. Remaining amino and double bond functional groups facilitated stepwise immobilization of trypsin and Glu-C. Digestion times for bovine serum albumin (BSA) and bovine hemoglobin (BHb) on the dual-IMER were significantly reduced compared to solution-based digestion (1 min vs. 36 h), resulting in improved sequence coverage (91.30% vs. 82.7% for BSA; 90.24% vs. 89.20% for BHb). Additionally, the dual-IMER demonstrated excellent durability, retaining 96.08% relative activity after 29 reuse cycles. Enhanced protein digestion efficiency can be attributed to several factors: (1) KIT-6’s large specific surface area, enabling higher enzyme loading capacity; (2) Its three-dimensional network pore structure, facilitating faster mass transfer and substance diffusion; (3) Orthogonality of trypsin and Glu-C enzyme cleavage sites; (4) The spatial effect introduced by the chain structure of PEI and glutaraldehyde’s spacing arm, reducing spatial hindrance and enhancing enzyme–substrate interactions; (5) Mild and stable enzyme immobilization. The KIT-6-based dual-IMER offers a promising technical tool for protein digestion, while the PDA/PEI/AM-KIT-6 platform holds potential for immobilizing other proteins or active substances.

Molecular mechanism of phytochemical cinnamic acid/cinnamaldehyde from the spice cinnamon binding to bovine haemoglobin

Cinnamic acid (CA) and cinnamaldehyde (CMA) are essential components of the spice cinnamon, a commonly used food oxidizer and preservative. In this work, the binding mechanism and structural information of CA/CMA to bovine haemoglobin (BHb) were investigated through multi-spectroscopic and molecular simulation experiments focusing on assessing the effect of phytochemicals in spices on BHb. Fluorescence and isothermal titration calorimetry experiments showed that CA possessed a binding constant of 105 L/mol and an electrostatic interaction with BHb resulting in the formation of stable complexes compared to CMA-BHb. Spectroscopic results (UV–Vis, CD and synchrotron fluorescence experiments) demonstrated that the addition of CA/CMA differently altered or affected the polarity of the BHb microenvironment, slightly perturbing the secondary structure of BHb. Thermodynamic analyses and molecular docking data were analyzed to confirm the dominance of van der Waals and hydrogen bonding forces in the energy contribution of the CMA-BHb system, and molecular dynamics simulations demonstrated that CA/CMA was relatively stable in the hydrophobic pocket of the protein (BHb). These findings will provide a basis for applying phytochemicals in spices in meat processing.

Self-sacrificing template synthesis of ultralight porous hydroxyapatite microtube-based ceramics for hemoglobin adsorption

Strong and porous hydroxyapatite ceramics are highly desired for biomedical materials. However, 1D hydroxyapatite crystals with enhanced bioactivity, densification, and sinterability are limited in practical application for self-agglomeration and unfavorable 3D morphology. Herein, a sacrificed template approach is developed to prepare ultralight porous hydroxyapatite microtube-based ceramics (HMTC). First, hydroxyapatite microtubes (HMTs) are self-assembled proceeding from nanowires to microtubes through a solvothermal reaction. Then, HMTs are shaped into 3D columns by using chitosan aerogel as a template and sintered at 1300 °C to obtain ultralight porous ceramics. The chitosan template considerably contributes to constructing the macro-meso-microporous architecture of HMTC, thereby benefiting the loading capacity, biomolecule migration, cell proliferation, and other biomedical applications. HMTC exhibits ultralight weight but excellent compressive resistance to bear objects weighing hundreds of times more. HMTC exhibits a high adsorption capacity (311 mg g−1) towards bovine hemoglobin (BHb) at the pH near pI by the driving force of ionic interaction and hydrophobic interaction. The adsorption kinetics and fixed-bed adsorption confirm that the physisorption process is controlled by the intraparticle diffusion of BHb. The excellent protein adsorption performance and good recyclability demonstrate the biological availability of HMTC.

Screening of liquid–liquid phase separation conditions for proteins with a mixed solution kit of biomacromolecular crowding agents

Protein liquid–liquid phase separation (LLPS) is a significant process in biology and material sciences. Finding suitable conditions for protein LLPS is essential for its in-depth and systematic study. Here, we provide a mixed solution screening kit based on biomacromolecule crowding agents to find protein LLPS conditions. The screening kit consists of 96 chemical formulations, in which the biomacromolecule crowding agents are used as the key components. The aqueous solution of the target protein is mixed with each chemical agent in the kit and then placed in an incubator for some time. The LLPS conditions for the target protein can be found by examining the LLPS phenomena in the droplets. We used nine proteins as model target proteins—lysozyme, bovine serum albumin, bovine hemoglobin, ovalbumin, catalase, FUS, Aβ, Tau, and α-Synuclein—to evaluate the performance of the screening kit. We found that utilization of the screening kit is efficient and useful to find different conditions for LLPS of proteins. The screening results reveal two types of conditions: those that promote and those that inhibit LLPS. Both conditions are extremely valuable because those that promote LLPS can be used to do extensive studies on the LLPS processes of the proteins of interest, whereas those that inhibit LLPS can be utilized as leads for developing novel medications to treat LLPS-related disorders. Furthermore, novel applications for the kit were investigated by utilizing the screening products.

Isolation and characterization of a Mannheimiahaemolytica secreted serine protease that degrades sheep and bovine fibrinogen

Mannheimiahaemolytica is an opportunistic agent of the respiratory tract of bovines, a member of the Pasteurellaceae family, and the causal agent of fibrinous pleuropneumonia. This bacterium possesses different virulence factors, allowing it to colonize and infect its host. The present work describes the isolation and characterization of a serine protease secreted by M. haemolytica serotype 1. This protease was isolated from M. haemolytica cultured media by precipitation with 50 % methanol and ion exchange chromatography on DEAE-cellulose. It is a 70-kDa protease able to degrade sheep and bovine fibrinogen or porcine gelatin but not bovine IgG, hemoglobin, or casein. Mass spectrometric analysis indicates its identity with protease IV of M. haemolytica. The proteolytic activity was active between pH 5 and 9, with an optimal pH of 8. It was stable at 50 °C for 10 min but inactivated at 60 °C. The sera of bovines with chronic or acute pneumonia recognized this protease. Still, it showed no cross-reactivity with rabbit hyperimmune serum against the secreted metalloprotease from Actinobacilluspleuropneumoniae, another member of the Pasteurellaceae family. M. haemolytica secreted proteases could contribute to the pathogenesis of this bacterium through fibrinogen degradation, a characteristic of this fibrinous pleuropneumonia.

Investigation of the Interaction of Monoamine Oxidase (MAO) Inhibitor Biscoumarins with Hemoglobin and Albumin Using Multi-Spectroscopic Techniques and Molecular Docking

Coumarin and their derivatives, such as biscoumarins, which are found as active ingredients in a variety of drugs, possess a wide range of biological activities. In this paper, the inhibitory effects of biscoumarin derivatives B1 and B2 on monoamine oxidase (MAO) enzyme activity were studied. Their binding mechanism to bovine serum hemoglobin (BHb) and bovine serum albumin (BSA) was investigated using molecular docking and multi-spectroscopic methods such as absorption, fluorescence, and synchronized fluorescence spectroscopy. The fluorescence quenching processes were analyzed using the Stern-Volmer, Lineweaver-Burk, and Hill methods. In this regard, Stern-Volmer quenching constants (Ksv), Lineweaver-Burk constants (KLB), and binding constants (Ka) were calculated at three temperatures (298, 310 and 322 K). The fluorescence studies showed that the binding of biscoumarins to BHb or BSA was a driven by static quenching. The Ka values of B2 to BHb and BSA (2179.11 and 18453.98 x 105 M-1) was larger than B1 at corresponding temperature (62.75 and 599.90 x105 M-1 for BHb and BSA, respectively), which indicates that the affinity of B2 toward the proteins were higher than that of B1. Moreover, thermodynamic parameters explained that hydrogen bonds were the main binding force stabilizing the protein-ligand complexes. Additionally, molecular docking studies have revealed binding energies, ligand efficiency values, and interactions with amino acid residues of proteins. The results of these investigations may be helpful in analyzing drug-protein binding of biscoumarins for potential future applications.

Efficient Secretory Expression for Mammalian Hemoglobins in Pichia pastoris

Mammalian hemoglobins (HB) are a kind of heme-binding proteins that play crucial physiological roles in various organisms. The traditional techniques employed for the extraction of HB are expensive and time-consuming, while the yields of mammalian HB in previous reports were quite low. The industrial Pichia pastoris is a highly effective platform for the secretory expression of heterologous proteins. To achieve efficient secretory expression of HB in P. pastoris, multiple strategies were applied, including the selection of a suitable host, the screening of optimal endogenous signal peptides, the knockout of VPS10, VTH1, and PEP5, and the co-expression of Alpha-Hemoglobin Stabilizing Protein (AHSP). In addition, the conditions for producing HB were optimized at shaking-flask level (BMMY medium with 100 mg/L of hemin, 2% methanol, and 24 °C). Based on these conditions, the higher titers of bovine hemoglobin (bHB, 376.9 ± 13.3 mg/L), porcine hemoglobin (pHB, 119.2 ± 7.3 mg/L), and human hemoglobin (hHB, 101.1 ± 6.7 mg/L) were achieved at fermenter level. The engineered P. pastoris strain and comprehensive strategies can also be applied to facilitate the synthesis of other high-value-added hemoproteins or hemoenzymes.

A comprehensive study on synthesis and crystal structures of Cu(II) and Ni(II) complexes: In vitro and in silico evaluation of biomolecular interactions, antiproliferative activities and molecular docking

Cu(II) and Ni(II) complexes, [Cu(NNN)2](NO3)2 (1) and [Ni(NNN)2](NO3)2 (2) (NNN-donor ligand: 2,2′:6′,2″-terpyridine), have been synthesized and characterized by electronic absorption spectroscopy, CHN analysis, FTIR, ESI-MS and X-ray crystallography techniques. Interaction of the complexes 1 and 2 with biomolecules (calf thymus DNA (CT-DNA) and bovine serum albumin (BSA)) has been investigated by electronic absorption and fluorescence spectroscopy. The results show that the complexes 1 and 2 can bind to CT-DNA via a moderate intercalation mode. Moreover, the fluorescence quenching mechanism between the complexes 1 and 2 and BSA is a static quenching process. The antiproliferative activities of the complexes 1 and 2 against human breast cancer (MCF-7) and lung cancer (A549 and H1299) cell lines were investigated. The complex 1 was found to have promising antiproliferative activity in selected cell line, with lower IC50 values than cisplatin. Molecular docking studies conducted across the complexes 1 and 2 have provided their potential as prospective chemotherapeutic agents against tumors. The complexes 1 and 2 demonstrated spontaneous binding with targets including anaplastic lymphoma kinase (ALK), heat shock protein 90 (Hsp90), epidermal growth factor receptor (EGFR), bovine hemoglobin (BHb), human epidermal growth factor receptor 2 (HER2), and B-DNA. These interactions are primarily driven by van der Waals forces and π-π interactions. Determining binding constants and the multiplicity of binding sites enhances understanding of these molecular interactions.

(43) - A Newly Produced Polymerized Bovine Hemoglobin and Hemoglobin-Based Heart Preservation Solution Prolonged Preservation Duration of Donor Heart in Cold Static Storage and Reduced Myocardial Injury After Allograft Transplantation

(43) - A Newly Produced Polymerized Bovine Hemoglobin and Hemoglobin-Based Heart Preservation Solution Prolonged Preservation Duration of Donor Heart in Cold Static Storage and Reduced Myocardial Injury After Allograft Transplantation

Evaluation of protein and lipid ingredients through in vitro digestibility for tropical gar (Atractosteus tropicus) juveniles

Tropical gar (Atractosteus tropicus) is an ecologically, culturally, economically, and scientifically important species. Therefore, it is essential to know the available resources to design feeds for the culture of this species. This study evaluated the in vitro digestibility of meals and oils commonly used in aquaculture, using crude extract of the stomach and intestines of A. tropicus juveniles. We used the pH stat titration system to determine the degree of hydrolysis (DH%), the concentration of amino acids released (mg g of meal-1), and the speed of hydrolysis (SH%) of various animal and vegetable ingredients. The relative digestibility of each protein ingredient was calculated using bovine hemoglobin for acid digestion and Hammersten bovine casein for the alkaline phase as reference ingredients (100% digestibility). For lipid ingredients, fish oil was used as a reference. The highest relative digestibility for protein ingredients was observed with fish, crustaceans, beef, poultry meat and offal, and beef blood meals. The best lipid SH was observed with soybean lecithin and cod liver, soybean, olive, maize, and canola oils. It is possible to use a wide variety of terrestrial animal proteins and vegetable oils for manufacturing a diet for A. tropicus rearing.

Partial purification and characterization of protease extracted from kinema

Proteases are large group of highly demanded enzymes having huge application in food and pharmaceutical industries. Numerous sources, including plants, microorganisms, and animals, can be used to obtain protease. Due to its affordability and safety consideration, fermented foods have recently attracted more attention as a source of microbial protease. The present study aimed to extract protease from kinema, partially purify the extracted protease following dialysis after precipitation with ammonium sulfate, and determine general characteristics of protease. The kinema having highest proteolysis activity after three days of control fermentation (Temperature 30±2 °C, RH 66 ± 2%) was taken for the study. About 2.45 fold of purification with overall recovery of 63.21% was achieved after precipitation with ammonium sulfate at 30–70% saturation level followed by dialysis of crude extracted protease. The dialysed kinema protease had specific activity of 7.90 U/mg. The enzyme remained actively functional across a wider pH (5–9) and temperature (40-60 °C) range. SDS-PAGE and Zymogram confirmed the presence of three major active bands respectively of 29.04 kDa, 36.09 kDa and 46.35 kDa in the kinema protease extract. The enzyme kinetics data on casein, fitted to Mechaelis Mentens’ plots showed the protease had Vmax of 1.001 U/ml with corresponding Km value of 0.825 mg/ml. Metal ions such as iron, mercury and aluminium showed the inhibition effect whereas presence of sodium, zinc, and calcium shows the activation effect on protease performance. The enzyme was active over various natural substrates; showing maximal activity on casein, and subsequent to bovine serum albumin, gelatin, hemoglobin and whey protein respectively. Furthermore, molecular weight distribution of the protease extract and activity inhibition with ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride, suggesting the protease from kinema could be a metal dependent serine protease or mixture of them.

First report of infectious spleen and kidney necrosis virus (ISKNV) in two native cichlids cultured in Brazil

Peacock bass (syn.: tucunaré, Cichla ocellaris) and the pearl cichlids (syn.: acará, Geophagus brasiliensis) are South American cichlids that are highly valued in both the ornamental and sport fish industries. Since 2017, a number of outbreaks of infectious spleen and kidney necrosis virus (ISKNV) have been reported on Brazilian food and ornamental fish farms. In this study, we detected ISKNV in farmed peacock bass and pearl cichlid by PCR and sequence analysis of the partial major capsid protein (MCP) gene. Moribund peacock bass (n=10) and pearl cichlids (2) from a farm experiencing elevated mortality among juveniles and adults of these species, were submitted for bacteriological and molecular diagnostics. Spleen, liver, brain, and kidney tissues were cultured on 5% sheep blood agar and cystine heart agar with 1% glucose and bovine haemoglobin. No bacteria were isolated from the 12 fish. Additionally, DNA extracts from the liver and spleen of all animals were tested for ISKNV using two conventional polymerase chain reaction (cPCR) assays and two nested PCR (nPCR) assays. ISKNV DNA was amplified in all 12 fish DNA extracts tested, in two or more of the PCR assays. Selected ISKNV amplicons were confirmed by Sanger sequencing. The nucleotide sequences derived from these animals were identical to ISKNV strains previously detected in food (e.g., tilapia and carp) and ornamental species, including strains previously detected in fish from Brazil. To the authors’ knowledge, this is the first report of ISKNV in these native Brazilian cichlids.

Identification of novel antioxidant and anti-inflammatory peptides from bovine hemoglobin by computer simulation of enzymolysis, molecular docking and molecular dynamics

Due to the structural diversity and complex mechanisms of action of bioactive peptides, screening for specific functional peptides is often challenging. To efficiently screen bioactive peptides with antioxidant and anti-inflammatory effects from bovine hemoglobin, we employed bioinformatics methods to perform virtual enzymatic hydrolysis using online tools and predicted the bioactivity, toxicity, and sensitization scores of the resulting peptides. Molecular docking and molecular dynamics simulations with Keap1 and TLR4 were subsequently conducted to screen for antioxidant and anti-inflammatory peptides. Finally, peptides ARRF and ARNF were synthesized using the Fmoc solid-phase method. The oxidative stress and inflammation model in RAW264.7 cells was induced using lipopolysaccharide (LPS), followed by treatment with peptides ARRF and ARNF to verify their antioxidant and anti-inflammatory activities. The results demonstrated that 529 bovine hemoglobin oligopeptides were produced following virtual enzymatic hydrolysis, of which nine were identified as eligible based on predictions of biological activity, toxicity, solubility, and sensitization. Molecular docking results indicated that the oligopeptides ARNF, QADF, and ARRF exhibited favorable interactions with Keap1, while ARNF, RRF, and ARRF showed strong interactions with TLR4. The primary active sites binding to the Keap1 receptor included Val465, Thr560, and Gly464. The main active sites binding to the TLR4 receptor were Asn309, Asn305, and Glu286. Hydrogen bonding, electrostatic interactions, and hydrophobic interactions were identified as the primary modes of interaction between the oligopeptides and the Keap1 and TLR4 receptors. Molecular dynamics simulations further confirmed that the selected bovine hemoglobin peptides could stably bind to Keap1 and TLR4 receptors. Cell experiments demonstrated that ARRF and ARNF effectively ameliorated LPS-induced oxidative stress and inflammation in RAW264.7 cells. ConclusionCompared to traditional methods, this study promptly screens bovine hemoglobin antioxidant and anti-inflammatory peptides, offering a novel approach for rapidly identifying food-derived bioactive peptides.