Protein Myoglobin Research Articles

Salidroside and exercise performance in healthy active young adults - an exploratory, randomized, double-blind, placebo-controlled study.

Rhodiola rosea extract is purported to improve physical performance and support resilience to stress. Salidroside is considered to be one of the main constituents responsible for the ergogenic actions of R. rosea. However, R. rosea extract contains relatively little salidroside and cultivation of R. rosea is challenging as it is mainly found in high-altitude, cold regions. Additionally, the R. rosea plant is subject to conservation concerns because of its growing popularity. The purpose of this exploratory study was to evaluate the short-term effects of pure, biosynthetic salidroside supplementation on exercise performance, mood state, and markers of inflammation and muscle damage in healthy active young adults. Fifty participants (30 M, 20F; 21 ± 4 yrs; 173 ± 8 cm; 74 ± 13 kg) were randomly assigned to either salidroside (60 mg/day for 16 days) or placebo supplementation and underwent peak oxygen uptake (VO2 peak), intermittent time-to-exhaustion (TTE), and local muscular endurance assessments, along with mood state evaluations using the Profile of Mood States (POMS). Blood samples were analyzed for erythropoietin, myoglobin, creatine kinase-MM, and C-reactive protein. Salidroside supplementation enhanced overall percent predicted oxygen uptake during high-intensity intermittent exercise (p < 0.01). An increase in serum myoglobin was observed 24 hours following exercise in the placebo group (p = 0.02) compared with baseline whereas no statistically significant increase was observed for the salidroside group indicating reduced exercise-induced muscle damage. Placebo group experienced a decrease in number of intervals performed during the TTE test (p = 0.03), and a decrease in friendliness (p < 0.01) and an increase in fatigue-inertia (p < 0.01) as reported by POMS. The salidroside group exhibited stable mood states and maintained performance levels during the time-to-exhaustion test. Salidroside supplementation may enhance oxygen utilization and mitigate exercise-induced muscle damage and fatigue, warranting further research on its long-term effects and potential as an adaptogen for active individuals.

Impact of yohimbine on myoglobin stability: insights from molecular spectroscopic, and computational approaches

The prolific role of bioactive ligands in interacting with a variety of proteins has become a focal point of interest in pharmacokinetics and pharmacodynamics, thus sparking substantial enthusiasm within the realm of medicinal chemistry. The reversible binding of small molecules and proteins is a characteristic feature, and it’s essential to investigate these interactions to understand their mode and mechanism of action within the human body. Therefore, the primary objective of the present study is to understand the underlying mechanism by which yohimbine (Yoh) interacts with protein myoglobin (Mb), employing both in silico and in vitro methodologies. The emission spectroscopy studies yielded a binding constant of 105 M−1 and a binding site ratio of 1:1. The structural perturbation induced in the protein Mb by Yoh was also illustrated by circular dichroism. The results of the molecular docking investigation resulted in numerous significant interactions between Mb and Yoh, indicating a substantial binding affinity. The accuracy of the docking data was further confirmed through the use of molecular dynamics (MD) simulations, which were then followed by principal component analysis and free energy landscape investigations. The study posited that the stability of the Mb-Yoh complex remains intact throughout the simulated duration, exhibiting little alterations in its structural conformation. Therefore, the association between ligand–protein plays a key role in determining circulatory lifetimes and bioavailability. These factors, in turn, are pivotal in the rational drug design process.

Myoglobin inhibits breast cancer cell fatty acid oxidation and migration via heme-dependent oxidant production and not fatty acid binding

The monomeric heme protein myoglobin (Mb) is aberrantly expressed in approximately 40 % of breast tumors. Mb expression is associated with better patient prognosis, yet the molecular mechanisms underlying this effect are unclear. In muscle, Mb's heme moiety confers oxygen storage and delivery. However, prior studies demonstrate that low levels of Mb in cancer cells preclude this function. Several studies propose a fatty acid binding function for Mb via lysine residue K46. Because cancer cells can upregulate fatty acid oxidation (FAO) to fuel cell migration, we tested whether Mb-mediated fatty acid binding modulates FAO and migration. We demonstrate that stable expression of human Mb in MDA-MB-231 breast cancer cells decreases cell migration and FAO. Site-directed mutagenesis of Mb K46 disrupted fatty acid binding but did not improve FAO or migration. Conversely, cells expressing Apo-Mb (with disrupted heme binding) did not show impaired FAO or migration rates, suggesting Mb attenuates FAO and migration via a heme-dependent mechanism rather than through fatty acid binding. Mb's heme-dependent oxidant generation dysregulates migratory gene expression, which is reversed by catalase treatment. Collectively, these data demonstrate that Mb's heme-dependent oxidant production decreases breast cancer cell migration, prompting therapeutic strategies to modulate oxidant production and Mb in tumors.

(Invited) Electroanalytical Meat Science

Meat color is a crucial factor that drives consumers’ purchase decisions at the point of sale. Consumers prefer the bright-cherry red color of beef. A recent study from our research team reported that the US beef industry loses $3.7 billion annually, and 400 million pounds of beef/animal protein is discarded due to meat discoloration (brown color), raising concerns over food security, the economy, and the environment. The red color of meat is strongly associated with the ratio of oxymyoglobin to metmyoglobin. This presentation will discuss our innovative use of electrochemistry to understand the redox signals of meat discoloration covering purified myoglobin protein, various species types, and sarcoplasmic extract studies. The knowledge gained from our research is expected to allow the implementation of new electroanalytical tools useful for meat and food science fields.

Almond Consumption Modestly Improves Pain Ratings, Muscle Force Production, and Biochemical Markers of Muscle Damage Following Downhill Running in Mildly Overweight, Middle-Aged Adults: A Randomized, Crossover Trial

BackgroundAlmonds promote cardiometabolic health benefits; however, the ergogenic effect of almond supplementation on exercise recovery is less explored. ObjectivesWe evaluated the impacts of raw, shelled, almonds on pain, muscle force production, and biochemical indices of muscle damage and inflammation during recovery from eccentrically biased exercise. MethodsUsing a randomized, crossover design, 26 healthy adults (37 ± 6 y) ran downhill (–10%) for 30 min at a heart rate corresponding to 65%–70% of maximal oxygen consumption followed by 3-d recovery periods after 8-wk adaptations to either ALMOND (2 oz/d) or isocaloric pretzel (CONTROL) feedings. Volunteers consumed the study food immediately following the run and each day during recovery. Fasted blood samples were collected, and pain and muscle function were tested before the downhill run and over 72 h of recovery. ResultsDownhill running elicited moderate muscle damage (Time: P < 0.001; η2 = 0.395) with creatine kinase (CK) peaking after 24 h (CONTROL: Δ + 180% from baseline compared with ALMOND: Δ + 171% from baseline). CK was reduced after 72 h in ALMOND (Δ – 50% from peak; P < 0.05) but not CONTROL (Δ – 33% from peak; P > 0.05). Maximal torque at 120°/s of flexion was greater (Trial: P = 0.004; η2 = 0.315) in ALMOND compared with CONTROL at 24 h (Δ + 12% between trials; P < 0.05) and 72 h (Δ + 9% between trials; P < 0.05) timepoints. Pain during maximal contraction was lower (Trial: P < 0.026; η2 = 0.225) in ALMOND compared with CONTROL after 24 h (Δ – 37% between trials; P < 0.05) and 48 h (Δ – 33% between trials; P < 0.05). No differences (P > 0.05) in vertical jump force, C-reactive protein concentrations, myoglobin concentrations, and total antioxidant capacity were observed between trials. ConclusionsThis study demonstrates that 2.0 oz/d of almonds modestly reduces pain, better maintains muscle strength, and reduces the CK response to eccentric-based exercise. This apparent effect of almond ingestion on exercise recovery has the potential to promote increased exercise adherence, which should be investigated in future studies.This trial was registered at the clinicaltrials.gov as NCT04787718.

Sex similarities and divergences in systemic and muscle iron metabolism adaptations to extreme physical inactivity in rats.

Previous data in humans suggest that extreme physical inactivity (EPI) affects iron metabolism differently between sexes. Our objective was to deepen the underlying mechanisms by studying rats of both sexes exposed to hindlimb unloading (HU), the reference experimental model mimicking EPI. Eight-week-old male and female Wistar rats were assigned to control (CTL) or hindlimb unloading (HU) conditions (n=12/group). After 7days of HU, serum, liver, spleen, and soleus muscle were removed. Iron parameters were measured in serum samples, and ICP-MS was used to quantify iron in tissues. Iron metabolism genes and proteins were analysed by RT-qPCR and Western blot. Compared with control males, control females exhibited higher iron concentrations in serum (+43.3%, p<0.001), liver (LIC; +198%, P<0.001), spleen (SIC; +76.1%, P<0.001), and transferrin saturation (TS) in serum (+53.3%, P<0.001), contrasting with previous observations in humans. HU rat males, but not females, exhibited an increase of LIC (+54% P<0.001) and SIC (+30.1%, P=0.023), along with a rise of H-ferritin protein levels (+60.9% and +134%, respectively, in liver and spleen; P<0.05) and a decrease of TFRC protein levels (-36%; -50%, respectively, P<0.05). HU males also exhibited an increase of splenic HO-1 and NRF2 mRNA levels, (p<0.001), as well as HU females (P<0.001). Concomitantly to muscle atrophy observed in HU animals, the iron concentration increased in soleus in females (+26.7, P=0.004) while only a trend is observed in males (+17.5%, P=0.088). In addition, the H-ferritin and myoglobin protein levels in soleus were increased in males (+748%, P<0.001, +22%, P=0.011, respectively) and in females (+369%, P<0.001, +21.9%, P=0.007, respectively), whereas TFRC and ferroportin (FPN) protein levels were reduced in males (-68.9%, P<0.001, -76.8%, P<0.001, respectively) and females (-75.9%, P<0.001, -62.9%, P<0.001, respectively). Interestingly, in both sexes, heme exporter FLVCR1 mRNA increased in soleus, while protein levels decreased (-39.9% for males P=0.010 and -49.1% for females P<0.001). Taken together, these data support that, in rats (1) extreme physical inactivity differently impacts the distribution of iron in both sexes, (2) splenic erythrophagocytosis could play a role in this iron misdistribution. The higher iron concentrations in atrophied soleus from both sexes are associated with a decoupling between the increase in iron storage proteins (i.e., ferritin and myoglobin) and the decrease in levels of iron export proteins (i.e., FPN and FLVCR1), thus supporting an iron sequestration in skeletal muscle under extreme physical inactivity.

An Improved Diabatization Scheme for Computing the Electronic Circular Dichroism of Proteins.

We advance the quality of first-principles calculations of protein electronic circular dichroism (CD) through an amelioration of a key deficiency of a previous procedure that involved diabatization of electronic states on the amide chromophore (to obtain interamide couplings) in a β-strand conformation of a diamide. This yields substantially improved calculated far-ultraviolet (far-UV) electronic circular dichroism (CD) spectra for β-sheet conformations. The interamide couplings from the diabatization procedure for 13 secondary structural elements (13 diamide structures) are applied to compute the CD spectra for seven example proteins: myoglobin (α helix), jacalin (β strand), concanavalin A (β type I), elastase (β type II), papain (α + β), 310-helix bundle (310-helix) and snow flea antifreeze protein (polyproline). In all cases, except concanavalin A and papain, the CD spectra computed using the interamide couplings from the diabatization procedure yield improved agreement with experiment with respect to previous first-principles calculations.

A flexible linker of 8-amino acids between the membrane binding segment and the FMN domain of cytochrome P450 reductase is necessary for optimal activity

The diflavin NADPH-cytochrome P450 reductase (CYPOR) plays a critical role in human cytochrome P450 (CYP) activity by sequentially delivering two electrons from NADPH to CYP enzymes during catalysis. Although electron transfer to forty-eight human CYP enzymes by the FMN hydroquinone of CYPOR is well-known, the role of the linker between the NH2-terminus membrane-binding domain (MBD) and FMN domain in supporting the activity of P450 enzymes remains poorly understood. Here we demonstrate that a linker with at least eight residues is required to form a functional CYPOR-CYP2B4 complex. The linker has been shortened in two amino-acid increments from Phe44 to Ile57 using site directed mutagenesis. The ability of the deletion mutants to support cytochrome P450 2B4 (CYP2B4) catalysis and reduce ferric CYP2B4 was determined using an in vitro assay and stopped-flow spectrophotometry. Steady-state enzyme kinetics showed that shortening the linker by 8–14 amino acids inhibited (63–99%) the ability of CYPOR to support CYP2B4 activity and significantly increased the Km of CYPOR for CYP2B4. In addition, the reductase mutants decreased the rate of reduction of ferric CYP2B4 (46–95%) compared to wildtype when the linker was shortened by 8–14 residues. These results indicate that a linker with a minimum length of eight residues is necessary to enable the FMN domain of reductase to interact with CYP2B4 to form a catalytically competent complex. Our study provides evidence that the length of the MBD-FMN domain linker is a major determinant of the ability of CYPOR to support CYP catalysis and drug metabolism by P450 enzymes. PreambleThis manuscript is dedicated in memory of Dr. James R. Kincaid who was the doctoral advisor to Dr. Freeborn Rwere and a longtime collaborator and friend of Dr. Lucy Waskell. Dr. James R. Kincaid was a distinguished professor of chemistry specializing in resonance Raman (rR) studies of heme proteins. He inspired Dr. Rwere (a Zimbabwean native) and three other Zimbabweans (Dr. Remigio Usai, Dr. Daniel Kaluka and Ms. Munyaradzi E. Manyumwa) to use lasers to document subtle changes occurring at heme active site of globin proteins (myoglobin and hemoglobin) and cytochrome P450 enzymes. Dr. Rwere appreciate his contributions to the development of talented Black scientists from Africa.

Association between Serum Biomarkers of Muscle Damage after Isokinetic Eccentric Exercise in Trained Males and Females

Eccentric exercise is well-known to induce muscle damage, and biomarkers may be related to the magnitude of impact on muscle force and muscle soreness after exercise. However, this phenomenon still needs to be investigated in more detail. The present study aimed to analyze if a correlation between muscle force, soreness, and biomarkers after eccentric exercise exists in healthy, trained 11 males and females. Eleven volunteers with previous resistance training experience performed 6 x 10 maximal isokinetic eccentric contractions of the elbow flexors in the muscle performance laboratory up to 72 hours after exercise. Peak torque was measured by maximal voluntary isometric contractions (MVIC) and muscle soreness by visual analogic scale (VAS) before, 24h, 48h, and 72h after the isokinetic eccentric exercise protocol. A fluorescence immunoassay analyzer determined Myoglobin and C-reactive protein levels. The MVIC significantly decreased while muscle soreness increased 24h, 48h, and 72h post eccentric exercise. Myoglobin significantly increased 48h and 72h after exercise. Muscle soreness was inversely correlated with MVIC 24h, 48h, and 72h and positively correlated with myoglobin 48h and 72h after eccentric exercise. The myoglobin was not correlated with muscle force. No significant correlation between C-reactive protein and muscle soreness and MVIC was found. In conclusion, our data suggest that myoglobin could be a valuable biomarker to be assessed alongside exercise-induced muscle soreness.

Not All Binding Sites Are Equal: Site Determination and Folding State Analysis of Gas-Phase Protein-Metallodrug Adducts.

Modern approaches in metallodrug research focus on compounds that bind protein targets rather than DNA. However, the identification of protein targets and binding sites is challenging. Using intact mass spectrometry and proteomics, we investigated the binding of the antimetastatic agent RAPTA-C to the model proteins ubiquitin, cytochrome c, lysozyme, and myoglobin. Binding to cytochrome c and lysozyme was negligible. However, ubiquitin bound up to three Ru moieties, two of which were localized at Met1 and His68 as [Ru(cym)], and [Ru(cym)] or [Ru(cym)(PTA)] adducts, respectively. Myoglobin bound up to four [Ru(cym)(PTA)] moieties and five sites were identified at His24, His36, His64, His81/82 and His113. Collision-induced unfolding (CIU) studies via ion-mobility mass spectrometry allowed measuring protein folding as a function of collisional activation. CIU of protein-RAPTA-C adducts showed binding of [Ru(cym)] to Met1 caused a significant compaction of ubiquitin, likely from N-terminal S-Ru-N chelation, while binding of [Ru(cym)(PTA)] to His residues of ubiquitin or myoglobin induced a smaller effect. Interestingly, the folded state of ubiquitin formed by His functionalization was more stable than Met1 metalation. The data suggests that selective metalation of amino acids at different positions on the protein impacts the conformation and potentially the biological activity of anticancer compounds.

Reduction in systemic muscle stress markers after exercise-induced muscle damage following concurrent training and supplementation with specific collagen peptides - a randomized controlled trial.

Collagen peptide supplementation in conjunction with exercise has been shown to improve structural and functional adaptations of both muscles and the extracellular matrix. This study aimed to explore whether specific collagen peptide (SCP) supplementation combined with a concurrent training intervention can improve muscular stress after exercise-induced muscle damage, verified by reliable blood markers. 55 sedentary to moderately active males participating in a concurrent training (CT) intervention (3x/week) for 12 weeks were administered either 15 g of SCP or placebo (PLA) daily. Before (T1) and after the intervention (T2), 150 muscle-damaging drop jumps were performed. Blood samples were collected to measure creatine kinase (CK), lactate dehydrogenase (LDH), myoglobin (MYO) and high-sensitivity C-reactive protein (hsCRP) before, after, and at 2 h, 24 h and 48 h post exercise. A combination of concurrent training and SCP administration showed statistically significant interaction effects, implying a lower increase in the area under the curve (AUC) of MYO (p = 0.004, ηp2 = 0.184), CK (p = 0.01, ηp2 = 0.145) and LDH (p = 0.016, ηp2 = 0.133) in the SCP group. On closer examination, the absolute mean differences (ΔAUCs) showed statistical significance in MYO (p = 0.017, d = 0.771), CK (p = 0.039, d = 0.633) and LDH (p = 0.016, d = 0.764) by SCP supplementation. In conclusion, 12 weeks of 15 g SCP supplementation combined with CT intervention reduced acute markers of exercise-induced muscle damage and improved post-exercise regenerative capacity, as evidenced by the altered post-exercise time course. The current findings indicate that SCP supplementation had a positive effect on the early phase of muscular recovery by either improving the structural integrity of the muscle and extracellular matrix during the training period or by accelerating membrane and cytoskeletal protein repair. https://www.clinicaltrials.gov/study/NCT05220371?cond=NCT05220371&rank=1, NCT05220371.

Molecular insights into the interaction between myoglobin and Imidacloprid: Multi-spectral experiments and computational simulations

Imidacloprid (IMI) belongs to the neonicotinoid insecticides, specifically the imidazole chemical group, which exhibits a high level of contact, digestive, and systemic effects on the central nervous system of insects. In this study, we investigated the impact of IMI on the conformation and structural stability of myoglobin (Mb) protein using spectroscopic techniques combined with molecular docking and simulation approaches. Additionally, we examined the thermal stability of the resulting complex. The UV–Visible results demonstrated the interaction between IMI and Mb protein. The intrinsic fluorescence of Mb was effectively quenched by IMI, indicating successful binding and the formation of a stable complex. Thermodynamic analysis revealed that the spontaneous bonding process was primarily governed by van der Waals forces and hydrogen bonds. FT-IR analysis indicated that IMI induced conformational changes in Mb, providing further evidence of its potential toxicity and structural damage to Mb. Furthermore, the interaction significantly enhanced the thermal stability of Mb compared to Mb alone. Molecular docking revealed the preferred binding site of IMI within the Mb structure. Molecular dynamics (MD) simulation results further supported the findings by demonstrating a decrease in compactness and an increase in the stability of the Mb-IMI complex. These findings offer valuable insights into the potential toxicity risks of IMI to human health and shed light on the binding mechanism between IMI and Mb. The results of this study serve as a useful model for gaining a deeper understanding of the effects of these pesticides on proteins and body health.

Mechanistics of pH-Dependent Sulfmyoglobin Formation: Spin Control and His64 Proton Relay

The chemistry of hydrogen sulfide (H2S) has been directed towards physiologically relevant hemeproteins, including myoglobin, hemoglobin, and other similar proteins. Despite substantial efforts, there remains a need to elucidate the mechanism and identify the species involved in the reaction between oxy-hemeproteins and H2S. Here, we summarize both our experimental data and computational modeling results revealing the mechanisms by which sulfmyoglobin (sulfMb) and sulfhemoglobin (sulfHb) are formed. Our experimental data at pH 7.4 reveal differences in intensity between sulfMb and sulfHb chromophores in the 620 nm charge transfer region. This behavior could be attributed to the incomplete reaction of tetrameric oxy-Hb with H2S, where not all heme groups form sulfheme. The data also show that, for the reaction of oxy-myoglobin (oxy-Mb) and H2S, the 622 nm charge transfer band increases in intensity from a pH of 6.6 to 5.0. This increase is attributed to the presence of the heme pocket distal His64εδ, which is positively charged, resulting in an elevated yield of sulfMb formation compared to the mono-protonated tautomer, His64ε. Computational hybrid QM/MM methods support the conclusion, indicating that oxy-Mb His64εδ (pH 5.0) reacts with H2S in the triplet state, favored by −31.0 kcal/mol over the singlet His64ε (pH 6.6) species. The phenomenon is facilitated by a hydrogen bonding network within the heme pocket, between His64εδ, heme Fe(II)O2, and H2S. The results establish an energetically favored quantitative mechanism to produce sulfMb (−69.1 kcal/mol) from the reactions of oxy-Mb and H2S. Curiously, the mechanism between met-aquo Mb, H2O2, and H2S shows similar reaction pathways and leads to sulfheme formation (−135.3 kcal/mol). The energetic barrier towards intermediate Cpd-0 is the limiting step in sulfheme formation for both systems. ‬Both mechanisms show that the thiyl radical, HS•, is the species attacking the β-β double bond of heme pyrrole B, leading to the sulfheme structure.

Myoglobin-Catalyzed Azide Reduction Proceeds via an Anionic Metal Amide Intermediate.

Nitrene transfer reactions catalyzed by heme proteins have broad potential for the stereoselective formation of carbon-nitrogen bonds. However, competition between productive nitrene transfer and the undesirable reduction of nitrene precursors limits the broad implementation of such biocatalytic methods. Here, we investigated the reduction of azides by the model heme protein myoglobin to gain mechanistic insights into the factors that control the fate of key reaction intermediates. In this system, the reaction proceeds via a proposed nitrene intermediate that is rapidly reduced and protonated to give a reactive ferrous amide species, which we characterized by UV/vis and Mössbauer spectroscopies, quantum mechanical calculations, and X-ray crystallography. Rate-limiting protonation of the ferrous amide to produce the corresponding amine is the final step in the catalytic cycle. These findings contribute to our understanding of the heme protein-catalyzed reduction of azides and provide a guide for future enzyme engineering campaigns to create more efficient nitrene transferases. Moreover, harnessing the reduction reaction in a chemoenzymatic cascade provided a potentially practical route to substituted pyrroles.

Methylglyoxal-induced modification of myoglobin: An insight into glycation mediated protein aggregation.

Post-translational modification of proteins by Maillard reaction, known as glycation, is thought to be the root cause of different complications, particularly in diabetes mellitus and age-related disorders. Methylglyoxal (MG), a reactive α-oxoaldehyde, increases in diabetic condition and reacts with the proteins to form advanced glycation end products (AGEs) following a Maillard-like reaction. In a time-dependent reaction study of MG with the heme protein myoglobin (Mb), MG was found to induce significant structural alterations of the heme protein, such as heme loss, changes in tryptophan fluorescence, and decrease of α-helicity with increased β-sheet content. These changes were found to occur gradually with increasing period of incubation. Incubation of Mb with MG induced the formation of several AGE adducts, including, carboxyethyllysine at Lys-16, carboxymethyllysine at Lys-87, carboxyethyllysine or pyrraline-carboxymethyllysine at Lys-133, carboxyethyllysine at Lys-42 and hydroimidazolone or argpyrimidine at Arg-31 and Arg-139. MG induced amyloid-like aggregation of Mb was detected at a longer period of incubation. MG-derived AGEs, therefore, appear to have an important role as the precursors of protein aggregation, which, in turn, may be associated with pathophysiological complications.

Effect of linoleic acid-induced oxidation on the water retention of golden pompano: Myoglobin and myofibrillar protein oxidation

This study investigated the interrelationship among changes in myoglobin (Mb) morphology, myofibrillar protein (MP) oxidation, and water-holding capacity (WHC) of golden pompano under oxidized linoleic acid (OLA). Results showed that the WHC, springiness, and chewiness of golden pompano were increased at low OLA concentrations. Compared with the control, centrifugal and cooking losses were reduced by 11.78% and 6.22%, respectively. By contrast, high concentrations of OLA showed a decreasing trend in muscle WHC. The porphyrin ring of Mb was disrupted, and the contents of heme and heme iron were significantly decreased (P < 0.05) during oxidation. The carbonyl content gradually increased, whereas total sulfhydryl decreased with the increase in OLA concentration. The α-helix contents of MP decreased and the random coil increased. The surface hydrophobicity of MP increased (≤10 mmol/L), and then gradually decreased with the increase of OLA concentrations. The Pearson analysis showed that WHC was closely related to protein oxidation. Mb would be pre-oxidized before MP due to linoleic acid oxidation, which may further promote oxidation and cause structural changes in MP, affecting WHC in golden pompano. Muscle WHC would be increased at low OLA concentrations (≤10 mmol/L), while reduced at high OLA concentrations (≥20 mmol/L).

A Single Dose of Microencapsulated Cocoa Supplementation Does Not Speed up Muscle Force Recovery after Eccentric Exercise-Induced Muscle Damage: A Placebo-Controlled, Double-Blind, Crossover Study

Exercise-induced muscle damage is associated with symptoms such as inflammation, delayed-onset muscle soreness, and impaired muscle performance. The intake of cocoa polyphenols has been suggested to improve muscle recovery due to their antioxidant and anti-inflammatory capacity. However, their bioavailability presents a challenge. Therefore, food microencapsulation may be an alternative to protect polyphenols, ensuring their biological effects. This study aimed to investigate the effect of a single dose of microencapsulated cocoa on the changes in muscle damage markers after eccentric exercise. In this randomized, double-blind, crossover study, fourteen healthy volunteers with previous resistance training experience performed 6 × 10 maximal isokinetic eccentric contractions of their elbow flexors using an isokinetic dynamometer after ingesting 25 g of microencapsulated cocoa or placebo. Peak isometric torque was measured using maximal voluntary isometric contractions, and pain was measured using a visual analogic scale both before and 24 h, 48 h, and 72 h after the damage protocol. Plasma glutathione and malondialdehyde levels were measured using high-performance liquid chromatography, and concentrations of myoglobin and C-reactive protein were determined using a fluorescence immunoassay analyzer. Significant decreases were seen in the peak isometric torque and pain measures from pre- to 72 h post-eccentric exercise. A significant main effect for time was found only for plasma myoglobin at 2 h, 48 h, and 72 h, and for C-reactive protein at 2 h, compared to the pre-eccentric exercise values. No significant time-treatment effects were observed (all p &gt; 0.05). This study demonstrated that microencapsulated cocoa cannot improve muscle recovery after eccentric exercise, at least when a single dose is consumed.

Design of an Oxygen‐Tolerant Photo‐RAFT System for Protein‐Polymer Conjugation Achieving High Bioactivity

AbstractProtein‐polymer conjugates have significant potential in pharmaceutical and biomedical applications. To enable their widespread use, robust conjugation techniques are crucial. This study introduces a photo‐initiated reversible addition‐fragmentation chain‐transfer (Photo‐RAFT) polymerization system that exhibits excellent oxygen tolerance. This system allows for the synthesis of protein‐polymer conjugates with high bioactivity under mild and aerobic conditions. Three photocatalytic systems utilizing Eosin Y (EY) as the photocatalyst with two different cocatalysts (ascorbic acid and triethanolamine) were investigated, each generating distinct reactive oxygen species (ROS) such as singlet oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals. The impact of these ROS on three model proteins (lysozyme, albumin, and myoglobin) was evaluated, demonstrating varying bioactivities based on the ROS produced. The EY/TEOA system was identified as the optimal photo‐RAFT initiating system, enabling the preparation of protein‐polymer conjugates under aerobic conditions while maintaining high protein enzymatic activity. To showcase the potential of this approach, lysozyme‐poly(dimethylaminoethyl acrylate) conjugates were successfully prepared and exhibited enhanced antimicrobial property against Gram‐positive and Gram‐negative bacteria.

Design of oxygen-tolerant Photo-RAFT system for protein-polymer conjugation achieving high bioactivity.

Protein-polymer conjugates have significant potential in pharmaceutical and biomedical applications. To enable their widespread use, robust conjugation techniques are crucial. This study introduces a photo-initiated reversible addition-fragmentation chain-transfer (Photo-RAFT) polymerization system that exhibits excellent oxygen tolerance. This system allows for the synthesis of protein-polymer conjugates with high bioactivity under mild and aerobic conditions. Three photocatalytic systems utilizing Eosin Y (EY) as the photocatalyst with two different cocatalysts (ascorbic acid and triethanolamine) were investigated, each generating distinct reactive oxygen species (ROS) such as singlet oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals. The impact of these ROS on three model proteins (lysozyme, albumin, and myoglobin) was evaluated, demonstrating varying bioactivities based on the ROS produced. The EY/TEOA system was identified as the optimal photo-RAFT initiating system, enabling the preparation of protein-polymer conjugates under aerobic conditions while maintaining high protein enzymatic activity. To showcase the potential of this approach, lysozyme-poly(dimethylaminoethyl acrylate) conjugates were successfully prepared and exhibited enhanced antimicrobial property against gram-positive and gram-negative bacteria.

Comparative analysis of the myoglobin gene in whales and humans reveals evolutionary changes in regulatory elements and expression levels.

Cetacea and other diving mammals have undergone numerous adaptations to their aquatic environment, among them high levels of the oxygen-carrying intracellular hemoprotein myoglobin in skeletal muscles. Hypotheses regarding the mechanisms leading to these high myoglobin levels often invoke the induction of gene expression by exercise, hypoxia, and other physiological gene regulatory pathways. Here we explore an alternative hypothesis: that cetacean myoglobin genes have evolved high levels of transcription driven by the intrinsic developmental mechanisms that drive muscle cell differentiation. We have used luciferase assays in differentiated C2C12 cells to test this hypothesis. Contrary to our hypothesis, we find that the myoglobin gene from the minke whale, Balaenoptera acutorostrata, shows a low level of expression, only about 8% that of humans. This low expression level is broadly shared among cetaceans and artiodactylans. Previous work on regulation of the human gene has identified a core muscle-specific enhancer comprised of two regions, the "AT element" and a C-rich sequence 5' of the AT element termed the "CCAC-box". Analysis of the minke whale gene supports the importance of the AT element, but the minke whale CCAC-box ortholog has little effect. Instead, critical positive input has been identified in a G-rich region 3' of the AT element. Also, a conserved E-box in exon 1 positively affects expression, despite having been assigned a repressive role in the human gene. Last, a novel region 5' of the core enhancer has been identified, which we hypothesize may function as a boundary element. These results illustrate regulatory flexibility during evolution. We discuss the possibility that low transcription levels are actually beneficial, and that evolution of the myoglobin protein toward enhanced stability is a critical factor in the accumulation of high myoglobin levels in adult cetacean muscle tissue.