Effects of Different Na+ Concentrations on cAMP-Dependent Protein Kinase Activity in Postmortem Meat.

cAMP-dependent protein kinase (PKA) activity regulates protein phosphorylation, with Na+ playing a crucial role in PKA activity. The aim of this study was to investigate the effects of different Na+ concentrations on PKA activity and protein phosphorylation level in postmortem muscle. The study consisted of two experiments: (1) NaCl of 0, 20, 100, 200 and 400 mM was added to a muscle homogenate incubation model to analyze the effect of Na+ concentration on PKA activity, and (2) the same concentrations were added to pure PKA in vitro incubation models at 4 °C to verify the effect of Na+ on PKA activity. The PKA activity of the muscle homogenate model increased with storage time in groups with different Na+ concentrations. High concentrations of Na+ inhibited sarcoplasmic protein phosphorylation. The PKA activity at 24 h of storage and the sarcoplasmic protein phosphorylation level at 12 h of storage in the group with 200 mM Na+ was lower than that of the other groups. After 1 h incubation, the PKA activity of samples in the 200 mM Na+ group was inhibited and lower than that in the other Na+ groups in the in vitro incubation model. These results suggest that the Na+ concentration at 200 mM could better inhibit PKA activity. This study provided valuable insights for enhancing curing efficiency and improving meat quality.

Acetylation and Phosphorylation Regulate the Role of Pyruvate Kinase as a Glycolytic Enzyme or a Protein Kinase in Lamb.

Protein post-translational modifications (PTMs) play an essential role in meat quality development. However, the effect of specific PTM sites on meat proteins has not been investigated yet. The characteristics of pyruvate kinase M (PKM) were found to exhibit a close correlation with final meat quality, and thus, serine 99 (S99) and lysine 137 (K137) in PKM were mutated to study their effect on PKM function. The structural and functional properties of five lamb PKM variants, including wild-type PKM (wtPKM), PKM_S99D (S99 phosphorylation), PKM_S99A (PKM S99 dephosphorylation), PKM_K137Q (PKM K137 acetylation), and PKM_K137R (PKM K137 deacetylation), were evaluated. The results showed that the secondary structure, tertiary structure, and polymer formation were affected among different PKM variants. In addition, the glycolytic activity of PKM_K137Q was decreased because of its weakened binding with phosphoenolpyruvate. In the PKM_K137R variant, the actin phosphorylation level exhibited a decrease, suggesting a low kinase activity of PKM_K137R. The results of molecular simulation showed a 42% reduction in the interface area between PKM_K137R and actin, in contrast to wtPKM and actin. These findings are significant for revealing the mechanism of how PTMs regulate PKM function and provide a theoretical foundation for the development of precise meat quality preservation technology.

Comparative effects of phosphorylation and acetylation on glycolysis and myofibrillar proteins degradation in postmortem muscle.

The study investigated the different effects between protein phosphorylation and acetylation on glycolytic enzyme activity and myofibrillar protein degradation. Lamb longissimus thoracis lumborum muscles were homogenized and then inhibitors were added for incubation at 4 °C. Phosphatase inhibitor was added to produce a high phosphorylation level (PI group) and lysine deacetylase inhibitor was added to produce a high acetylation level (DI group). The lactate and ATP content in the PI group was inhibited compared with that in the DI group (P < 0.05). Phosphofructokinase (PFK) activity was negatively related with the phosphorylation level and was positively related with the acetylation level in the DI group (P < 0.05). The degradation of troponin T and desmin of the DI group were restrained when compared to that in the PI group (P < 0.05). Compared with initial PFK and desmin, the simulation of phosphorylation and acetylation of PFK and desmin showed different electrostatic potential at the surface and a more unstable structure. The phosphorylation level of the DI group was increased, suggesting that the changes of protein acetylation altered protein phosphorylation. In conclusion, compared with protein phosphorylation, protein acetylation had a greater effect on promoting glycolysis and inhibiting protein degradation.

Combined Effect of Cinnamon Bark Oil and Packaging Methods on Quality of Fresh Lamb Meat Patties during Storage at 4 °C.

The present study aimed to investigate the effects of adding cinnamon bark oil (CBO) on the quality of ground lamb meat, considering different packaging conditions, including modified atmospheric packaging (MAP) using Hengxian HX-300H and overwrapped packaging. The CBO was incorporated into lamb meat samples at three different levels: 0% (control), 0.025% and 0.05% (v/w). The samples were then subjected to three packaging methods: MAP1 (80% O2 + 20% CO2), MAP2 (40% O2 + 30% CO2 + 30% N2) and overwrapped packaging and stored at 4 °C for 0, 4, 8, 12 and 16 days. The findings of the present study revealed that the addition of 0.025% and 0.05% CBO under MAP1 condition significantly improved the color of the meat samples after 12 days of storage at 4 °C (p < 0.05). The overwrapped samples exhibited higher levels of thiobarbituric acid reactive substances (TBARS) compared to all other treatments, starting from day 4 of storage (p < 0.05). Furthermore, microbial counts were notably higher in the overwrapped samples than in all other samples after day 8 of storage (p < 0.05). In conclusion, the combination of 0.05% CBO with MAP proved to be an effective strategy for enhancing the color stability and oxidative stability of ground lamb meat. These results suggest that CBO can be utilized as a beneficial protective agent in meat packaging processes.

Phosphorylation and acetylation responses of glycolytic enzymes in meat to different chilling rates.

The aim of this study was to investigate the effects of chilling rate on phosphorylation and acetylation levels of the glycolytic enzymes in meat, including glycogen phosphorylase, phosphofructokinase, aldolase (ALDOA), triose-phosphate isomerase (TPI1), phosphoglycerate kinase, lactate dehydrogenase (LDH). The samples were assigned into three groups: Control, Chilling 1 and Chilling 2, corresponding to the chilling rates of 4.8 °C/h, 23.0 °C/h and 25.1 °C/h respectively. The contents of glycogen and ATP were significantly higher in samples from the chilling groups. The activity and phosphorylation level of the six enzymes were higher in samples at the chilling rate of 25.1 °C/h, while the acetylation level of ALDOA, TPI1 and LDH were inhibited. In brief, glycolysis was delayed and the activity of glycolytic enzymes were maintained at higher level by the changes of phosphorylation and acetylation levels at the chilling rates of 23.0 °C/h and 25.1 °C/h, which may partly explain why very fast chilling improves meat quality.

Phosphorylation of Calpastatin Negatively Regulates the Activity of Calpain.

Tenderness is an important characteristic of meat quality. Calpastatin and calpain play important roles in meat tenderization. However, it is not clear how phosphorylation affects the regulation of calpastatin on µ-calpain and, consequently, meat tenderness. Calpastatin with high and low phosphorylation levels were obtained in vitro corresponding to the treatments by protein kinase A (PKA) and alkaline phosphatase. Then, calpain was incubated with calpastatin with different phosphorylation levels, and the effect of calpastatin on calpain activity under different phosphorylation levels was analyzed. The results showed that PKA promoted the phosphorylation of calpastatin, and a high phosphorylation level was maintained during incubation. The degradation rate of µ-calpain in AP group was higher than that in the other groups, meaning there was lower inhibition of calpastatin on calpain activity. The degradation of calpastatin was lower and its structure was more stable after phosphorylation. One more serine 133 site of calpastatin was identified in PKA group compared with the other groups. Phosphorylation at serine 133 of calpastatin enhanced its inhibition on calpain activity by maintaining its structural stability, thus inhibiting the tenderization of meat.

Phosphorylation and acetylation of glycolytic enzymes cooperatively regulate their activity and lamb meat quality.

This study examined cooperative regulation of phosphorylation and acetylation of glycolytic enzymes on their activity and lamb meat quality. Muscle samples were divided into two groups (fast and slow) according to their glycolysis rate as defined by pH decline rate from 1 h to 1 d postmortem. In slow glycolysis rate group, the activity of hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK) was lower and meat sample showed lower a*, higher shear force and cooking loss. The acetylation and phosphorylation of HK were positively correlated with HK activity. The acetylation and phosphorylation of PFK were correlated with shear force and negatively associated with PFK activity. The acetylation and phosphorylation of PK were significantly correlated with each other but showed insignificant correlations with PK activity. Briefly, the phosphorylation and acetylation of HK, PFK and PK coregulate glycolysis through different crosstalk patterns on their activity and this might affect meat quality.

Potentials of circSOBP in the diagnosis and prognosis of gastric cancer.

OBJECTIVES: Gastric cancer (GC) causes no symptoms at early stages. However, with the progression, GC causes symptoms mimicking normal gastrointestinal issues, such as irritable bowel syndrome (IBS), gastroesophageal reflux disease (GERD), gastritis, or peptic ulcers (PU). CircRNA circSOBP has been characterized as a critical regulator in prostate cancer. The present study aimed to study its involvement in GC. MATERIALS AND METHODS: Plasma samples were collected from GC patients (n = 64), IBS patients (n = 64), GERD patients (n = 64), gastritis patients (n = 64), PU patients (n = 64), and healthy controls (HCs, n = 64). Paired GC and non-tumor samples were from all GC patients (n = 64). Tissue and plasma samples were subjected to RT-qPCR to determine circSOBP expression. The role of circSOBP in distinguishing GC patients from other patients was analyzed by ROC curve. The 64 patients were followed up for 5 years to study the role of circSOBP in predicting the survival of GC patients. RESULTS: Decreased circSOBP RNA accumulation was observed in GC tissues compared to normal tissue samples. Decreased plasma circSOBP accumulation was only observed in GC patients, but not other patients, compared to HCs. With plasma circSOBP as a biomarker, GC patients were separated from other patients and HCs. Patients with high plasma or tissue levels of circSOBP showed better survival conditions. In addition, plasma and tissue circSOBP levels were only closely correlated with GC patients' tumor metastasis, but not other clinical factors. CONCLUSIONS: Decreased circSOBP accumulation may be applied in clinical practice to improve the diagnosis and prognosis of GC.

Characterization of the thickness of the Tear Film Lipid Layer in Meibomian Gland Dysfunction using high resolution optical microscopy.

PURPOSE: To evaluate the thickness of the tear film lipid layer (TFLL) in meibomian gland dysfunction (MGD) using a high-resolution optical microscope. METHODS: The Ocular Surface Disease Index (OSDI) and meibum grade score (MGS) were used to classify 190 subjects into four groups: normal (OSDI<13 and MGS<10), mixed (OSDI≥13 and MGS<10), asymptomatic MGD (OSDI<13 and MGS≥10), and MGD (OSDI≥13 and MGS≥10). The high-resolution optical microscope was used to capture TFLL images in vivo. The histograms of TFLL thickness were analyzed and curve-fitted using probability density functions (PDFs). RESULTS: There were three obvious peaks in the distributions of TFLL across the groups. From the curve-fitting process, the main outcomes are displayed according to each Gaussian function with the position of peak (µ) and the summed percentage within the range of standard deviation (σ). The normal group had distribution as follows: 33.3 ± 0.005 nm, 26%; 53.9 ± 0.019 nm, 40%; 79.4 ± 0.064 nm, 12%. The mixed group had a distribution as follows: 33.8 ± 0.004 nm, 32%; 53.1 ± 0.115 nm, 21%; 71.7 ± 0.232 nm, 27%. The asymptomatic MGD group had a distribution as follows: 33.5 ± 0.004 nm, 20%; 49.2 ± 0.041 nm, 25%; 62.9 ± 0.063 nm, 47%. The MGD group had a distribution as follows: 34.3 ± 0.004 nm, 34%; 53.7 ± 0.022 nm, 28%; 74.9 ± 0.060 nm, 16%. CONCLUSIONS: The MGD and mixed groups had the largest percentages of TFLL thicknesses fall within the thinnest modes (peak 34.3 and 33.8 nm, respectively). These data show that measures of central tendency (e.g., averages, medians) do not fully appreciate the variable distributions of TFLL across disease spectra.

Sarcoplasmic and myofibrillar phosphoproteins profile of beef M. longissimus thoracis with different pHu at different days postmortem.

BACKGROUND: The abnormal ultimate pH (pHu ) in postmortem muscles affect the meat quality and results in substantial economic losses. Dark, firm, and dry (DFD) meat linked with the higher postmortem pHu values and exhibited many quality issues such as dark color, tough texture and shorter shelf life. This research aimed to investigate the effect of protein phosphorylation on variations in beef pHu in order to explore the possible mechanisms underlying DFD meat formation. RESULTS: Glycogen and lactate contents were higher, while L* and a* were lower in high pHu beef. Shear force was higher in intermediate pHu group. Global phosphorylation of sarcoplasmic proteins was higher in low pHu samples on day 1 and of myofibrillar proteins was higher in intermediate pHu meat on days 1 and 2 postmortem. Sarcoplasmic protein bands with different phosphorylation levels were identified as containing some glycometabolism and stress response proteins and phosphorylated myofibrillar protein bands were identified sarcomeric and metabolic proteins. CONCLUSIONS: Phosphorylation of multiple proteins of glycolytic pathway and contractile machinery may play critical roles in development of DFD beef. © 2021 Society of Chemical Industry.

Human meibum and tear film derived cholesteryl and wax esters in meibomian gland dysfunction and tear film structure.

PURPOSE: This study evaluated the presence and roles of cholesteryl esters (CEs) and wax esters (WEs) from human tear film and meibum in meibomian gland dysfunction (MGD). METHODS: Out of 195 enrolled subjects, 164 and 179 subjects provided tear and meibum samples, respectively. Subjects were classified into normal, asymptomatic MGD, MGD, and mixed (MGD & aqueous deficient). The precorneal tear film (PCTF) thinning rate (evaporation) was measured using optical coherence tomography. Lipids extracted from tear and meibum samples were infused into a SCIEX 5600 TripleTOF mass spectrometer. CE and WE intensities quantified with Analyst 1.7 TF and LipidView 1.3 were compared across disease groups in MetaboAnalyst 5.0 and correlated with PCTF thinning rates. RESULTS: The numbers of unique CEs and WEs identified in the samples were 125 and 86, respectively. Unsupervised Principal Component (PC) analysis and supervised Partial Least Square Discriminant analysis exhibited little separation among groups for both CEs and WEs in tears and meibum. Spearman's correlation analyses showed no association between either the first or second PC scores with PCTF thinning rates. CONCLUSION: The abundances of human PCTF and meibum-derived CEs and WEs were independent of MGD disease status and PCTF thinning (evaporation). CEs and WEs alterations do not contribute to alterations in tear film dynamics in MGD, such as has been demonstrated by the (O-acyl) ω-hydroxy fatty acids (OAHFAs).

Acetylation of Sarcoplasmic and Myofibrillar Proteins were Associated with Ovine Meat Quality Attributes at Early Postmortem.

The objective of this study was to examine the relationship between meat quality attributes and the changes of sarcoplasmic protein acetylation and myofibrillar protein acetylation in lamb longissimus thoracis et lumborum muscles at different postmortem phases. Protein acetylation, color, pH, shear force, myofibril fragmentation index and cooking loss were measured. The total level of acetylated sarcoplasmic proteins showed a negative relation with pH, a positive relation with a*, b* and cooking loss at the pre-rigor phase. Sarcoplasmic proteins acetylation affected postmortem pH by regulating glycolysis, which in turn affects color and cooking loss. The total level of acetylated myofibrillar proteins showed a positive relation with shear force at the pre-rigor phase. Myofibrillar proteins acetylation affected meat tenderness by regulating muscle contraction. This study indicated that acetylation played a regulatory role of meat color, water-holding capacity, and tenderization process at early postmortem.

Human Meibum and Tear Film Derived (O-Acyl)-Omega-Hydroxy Fatty Acids as Biomarkers of Tear Film Dynamics in Meibomian Gland Dysfunction and Dry Eye Disease.

Purpose: To investigate the association between precorneal tear film (PCTF)- and meibum-derived (O-Acyl)-omega-hydroxy fatty acids (OAHFAs) and PCTF thinning in meibomian gland health and dysfunction. Methods: Of 195 eligible subjects (18-84 years, 62.6% female), 178 and 170 subjects provided both PCTF optical coherence tomography (OCT) imaging and mass spectrometry data for tears (n = 178) and meibum (n = 170). The PCTF thinning rate was measured in the right eye using an ultra-high-resolution, custom-built OCT. Tear and meibum samples from the right eye were infused into the SCIEX 5600 TripleTOF mass spectrometer in the negative ion mode. Intensities (m/z) of preidentified OAHFAs were measured with Analyst 1.7TF and LipidView 1.3 (SCIEX). Principal component (PC) analyses and Spearman's correlations (ρ) were performed to evaluate the association between OAHFAs and PCTF thinning rates. Results: In meibum and tear samples, 76 and 78 unique OAHFAs were detected, respectively. The first PC scores of the meibum-derived OAHFAs had statistically significant correlations with PCTF thinning rates (ρ = 0.18, P = 0.016). Among 10 OAHFAs with the highest first PC loadings, six OAHFAs had negative correlations with PCTF thinning rate (18:2/16:2, ρ = -0.19, P = 0.01; 18:2/30:1, ρ = -0.21, P = 0.008; 18:1/28:1, ρ = -0.22, P = 0.004; 18:1/30:1, ρ = -0.22, P = 0.005; 18:1/25:0, ρ = 0.22, P = 0 .006; and 18:1/26:1, ρ = -0.22, P = 0.006), while one OAHFA had a positive correlation with PCTF thinning rate (18:2/18:1, ρ = 0.48, P = 0.006). Tear film-derived OAHFAs had no association with the PCTF thinning rate. Conclusions: Several human meibum-derived OAHFAs showed significant associations with PCTF thinning, suggesting that these OAHFAs could be implicated in the mechanism underlying the stabilization and thinning of the PCTF. The tear-film derived OAHFAs were, however, independent of the rate of PCTF thinning.

Effects of acetylation on dissociation and phosphorylation of actomyosin in postmortem ovine muscle during incubation at 4 °C in vitro.

This study aimed to assess the effects of acetylation levels on actomyosin disassociation and phosphorylation of lamb during incubation at 4 °C. Samples of whole proteins from lamb longissimus thoracis muscles were prepared and assigned into three treatments (high, middle and low acetylation groups). The results showed that deacetylation of myosin heavy chain and actin was inhibited by lysine deacetylase inhibitor trichostatin A and nicotinamide in this study. Phosphorylation levels of myosin heavy chain and actin were inhibited by their acetylation during incubation in vitro. Actomyosin disassociation degree in high acetylation group was significantly lower than that in middle and low acetylation groups (P < 0.05). The ATPase activity in high acetylation group was significantly higher than that in middle and low acetylation groups (P < 0.05). In conclusion, acetylation of myosin heavy chain and actin inhibited actomyosin dissociation by inhibiting their phosphorylation at 4 °C in vitro.

Human precorneal tear film and lipid layer dynamics in meibomian gland dysfunction.

PURPOSE: To evaluate the precorneal tear film (PCTF) and lipid layer (TFLL) thicknesses and thinning rates in meibomian gland dysfunction (MGD) using a combined ultra-high-resolution optical coherence tomography (OCT) and thickness dependent fringe (TDF) interferometry system. METHODS: Based on the Tear Film and Ocular Surface Society (TFOS) International Workshop on Meibomian Gland Dysfunction diagnostic algorithm, the Ocular Surface Disease Index (OSDI) and meibum grade score (MGS) were used to classify subjects into four groups: Normal (OSDI<13 and MGS<10), MGD (OSDI≥13 and MGS≥10), Asymptomatic MGD (OSDI<13 and MGS≥10), and Mixed (OSDI≥13 and MGS<10). The OCT/TDF system was used to capture PCTF and TFLL thicknesses and thinning rates. Kruskal-Wallis was used to compare median PCTF and TFLL thicknesses and thinning rates. RESULTS: There were 190 subjects categorized into four groups: Normal (n = 63), MGD (n = 51), Asymptomatic MGD (n = 29), and Mixed (n = 47). The PCTF was significantly thinner in the Mixed group (3.3 [1.2]) than in the Normal (p < 0.001), MGD (p < 0.001) and Asymptomatic MGD (p = 0.009) groups. Relative to the Normal (4.5 [4.5] µm/min) and Mixed (5.0 [2.0] µm/min) groups, the rate of PCTF thinning was faster in the MGD (8.1 [3.0] µm/min, both p < 0.001) and Asymptomatic MGD (6.9 [3.1] µm/min, p = 0.009 and p = 0.04, respectively) groups. The correlation between PCTF thinning rate and TFLL thickness was ρ = -0.46, p < 0.001. CONCLUSIONS: Symptomatic and asymptomatic MGD shows rapid PCTF thinning rates (evaporation), while the PCTF thickness was reduced in mixed disease. Thicker lipid layers were associated with slower PCTF thinning.

Effects of protein posttranslational modifications on meat quality: A review.

Meat quality plays an important role in the purchase decision of consumers, affecting producers and retailers. The formation mechanisms determining meat quality are intricate, as several endogenous and exogenous factors contribute during antemortem and postmortem periods. Abundant research has been performed on meat quality; however, unexpected variation in meat quality remains an issue in the meat industry. Protein posttranslational modifications (PTMs) regulate structures and functions of proteins in living tissues, and recent reports confirmed their importance in meat quality. The objective of this review was to provide a summary of the research on the effects of PTMs on meat quality. The effects of four common PTMs, namely, protein phosphorylation, acetylation, S-nitrosylation, and ubiquitination, on meat quality were discussed, with emphasis on the effects of protein phosphorylation on meat tenderness, color, and water holding capacity. The mechanisms and factors that may affect the function of protein phosphorylation are also discussed. The current research confirms that meat quality traits are regulated by multiple PTMs. Cross talk between different PTMs and interactions of PTMs with postmortem biochemical processes need to be explored to improve our understanding on factors affecting meat quality.

Influence of adding cinnamon bark oil on meat quality of ground lamb during storage at 4 °C.

The study explored the preservative effects of adding different levels of cinnamon bark oil on meat quality of ground lamb. The longissimus thoracis et lumborum (LTL) meat samples were treated with 0 (control), 0.01%, 0.025%, 0.05%, and 0.5% of cinnamon bark oil and stored at 4 °C for 16 days. Microbial populations of TVC, lactic acid bacteria, and Enterobacteriaceae were reduced up to 0.6 to 1.9 log CFU/g than control from day 4 to 16 during storage. The samples adding 0.025% and 0.05% cinnamon bark oil showed lower thiobarbituric acid reactive substance (TBARS) and pH values but higher L⁎, a⁎, R630/580 and Chroma values than all the other samples (P < 0.05). The relative content of oxymyoglobin was higher for 0.01%, 0.025%, and 0.05% cinnamon bark oil samples after 12 days of storage than other treatments (P < 0.05). In conclusion, cinnamon bark oil of 0.025% and 0.05% had a better preservative effect on the lamb meat quality during storage.

Role of phosphorylation on characteristics of glycogen phosphorylase in lamb with different glycolytic rates post-mortem.

The relationship between glycogen phosphorylase activity and phosphorylation levels in the longissimus thoracis muscle post-mortem was studied. Sixty lamb samples were collected at 0.5 h, 2 h, 6 h, 12 h, 24 h, 48 h, and 72 h post-mortem and divided into three groups (n = 6) with different glycolytic rates (fast, intermediate, and slow) according to the pH at 6 h post-mortem. The phosphorylation level and activity and expression of glycogen phosphorylase were determined. The results showed that the phosphorylation level and activity of glycogen phosphorylase in the slow pH decline group was lower than that in the fast pH decline group during 24 h post-mortem (P < .05). There was a significant positive correlation between the glycogen phosphorylase activity and the phosphorylation level. In conclusion, these data demonstrated that the glycogen phosphorylase activity in lambs was affected by phosphorylation levels and postmortem duration.

Effects of phosphorylation on the activity of glycogen phosphorylase in mutton during incubation at 4 °C in vitro.

The aim of this study was to assess the effects of the phosphorylation levels of glycogen phosphorylase on its activity in mutton sarcoplasmic protein samples during incubation at 4 °C. Samples of sarcoplasmic proteins from mutton longissimus thoracis muscles were prepared and separated into three treatment groups to obtain glycogen phosphorylase with different phosphorylation levels, which were (1) treated with protein kinase A, (2) treated with alkaline phosphatase, and (3) left untreated (control). Glycogen phosphorylase phosphorylation levels and activity as well as the levels of related endogenous substances were assessed. The results showed that phosphorylation of glycogen phosphorylase in mutton promoted its activity during incubation at 4 °C. The activity of glycogen phosphorylase was also influenced by other factors (glycogen, glucose, glucose 6-phosphate, ATP, etc.) in vitro. The combined effects of phosphorylation and endogenous substances on glycogen phosphorylase activity varied at different incubation times.