Tooth Enamel Proteins Enamelin and Amelogenin Cooperate To Regulate the Growth Morphology of Octacalcium Phosphate Crystals

To examine the hypothetical cooperative role of enamelin and amelogenin in controlling the growth morphology of enamel crystals in the postsecretory stage, we applied a cation selective membrane system for the growth of octacalcium phosphate (OCP) in the truncated recombinant porcine amelogenin (rP148) with and without the 32 kDa enamelin fragment. Enamelin alone inhibited the growth in the c-axis direction more than rP148, yielding OCP crystals with the smallest aspect ratio of all conditions tested. When enamelin was added to the amelogenin “gel-like matrix”, the inhibitory action of the protein mixture on the growth of OCP in the c-axis direction was diminished, while that in the b-axis direction was increased. As a result, the length to width ratio (aspect ratio) of OCP crystal was markedly increased. Addition of enamelin to amelogenin enhanced the potential of amelogenin to stabilize the amorphous calcium phosphate (ACP) transient phase. The ratio of enamelin and amelogenin was crucial for stabilization of ACP and the growth of OCP crystals with larger aspect ratio. The cooperative regulatory action of enamelin and amelogenin was attained, presumably, through coassembling of enamelin and amelogenin. These results have important implications in understanding the growth mechanism of enamel crystals with large aspect ratio

Effects of Stigmasterol on 3‑Chloropropane-1,2-diol Fatty Acid Esters and Aldehydes Formation in Heated Soybean Oil

In this study, we investigated the inhibitory effects of three soybean isoflavones and two soybean phytosterols on the formation of 3-chloropropane-1,2-diol fatty acid esters (3-MCPDE) and aldehydes in heated soybean oil model. 0.4 mM of genistin, genistein, daidzein, stigmasterol, and β-sitosterol significantly reduced 3-MCPDE formation by 25.7, 51.4, 21.4, 61.6, and 55.7%, and total aldehydes formation by 42.03, 43.94, 28.36, 54.74, and 39.23%, respectively. Further study showed that stigmasterol reduced the content of glycidyl esters (GEs) and glycidol, two key intermediates of 3-MCPDE, and prevented fatty acids degradation in the oils. Moreover, the effects of continuous frying time on the content of stigmasterol and the migration of stigmasterol were evaluated in the fried dough sticks model system. The content of stigmasterol in soybean oil was found to be significantly decreased with prolonged heating time. The concentrations of stigmasterol in fried dough sticks and the migration rates of stigmasterol from soybean oil to fried dough sticks decreased with repeated frying sessions. In addition, stigmasterol undergoes oxidative changes during heat treatment, and the oxidation products including 5,6α-epoxystigmasterol, 5,6β-epoxystigmasterol, 7α-hydroxystigmasterol, 7β-hydroxystigmasterol, stigmasterlol-3β,5α,6β-triol, and 7-ketostigmasterol were identified in the frying oils but not in the fried dough sticks. Overall, stigmasterol could be added to soybean oil to reduce 3-MCPDE and aldehydes formation, and reacting with GEs/glycidol and protection of lipid acids from oxidation may be the mechanism of action of stigmasterol

Insight into Ionic Strength-Induced Solubilization of Myofibrillar Proteins from Silver Carp (<i>Hypophthalmichthys molitrix</i>): Structural Changes and 4D Label-Free Proteomics Analysis

In this study, changes in the physical, structural, and assembly characteristics of silver carp myofibrillar proteins (MPs) at different ionic strength (I) values were investigated. Moreover, the differential proteomic profile of soluble MPs was analyzed using 4D proteomics based on timsTOF Pro mass spectrometry. Solubility of MPs significantly increased at high I (>0.3), and the increase in I enhanced the apparent viscosity, fluorescence intensity, surface hydrophobicity, and α-helix content of MPs solution. Particle size and sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns also supported the solubility profiles. Transmission electron microscopy and atomic force microscopy observations revealed the morphological assembly and disassembly of MPs under different I conditions. Finally, proteomic analysis revealed the evolution law of salt-induced solubilization of MPs and the critical molecular characteristics in different I environments. The number of differentially abundant proteins (DAPs) decreased with the increase of I, and most DAPs related to the muscle filament sliding, contraction and assembly, actinin binding, and actin filament binding. The soluble abundance of myosin and some structural proteins was dependent on I, and structural proteins in the Z-disk and M-band might contribute to the solubilization of myosin. Our findings provide insightful information about the impact of common I on the solubility pattern of MPs from freshwater fish

Postmortem Muscle Proteome Characteristics of Silver Carp (<i>Hypophthalmichthys molitrix</i>): Insights from Full-Length Transcriptome and Deep 4D Label-Free Proteomic

The coverage of the protein database directly determines the results of shotgun proteomics. In this study, PacBio single-molecule real-time sequencing technology was performed on postmortem silver carp muscle transcripts. A total of 42.43 Gb clean data, 35,834 nonredundant transcripts, and 15,413 unigenes were obtained. In total, 99.32% of the unigenes were successfully annotated and assigned specific functions. PacBio long-read isoform sequencing (Iso-Seq) analysis can provide more accurate protein information with a higher proportion of complete coding sequences and longer lengths. Subsequently, 2671 proteins were identified in deep 4D proteomics informed by a full-length transcriptomics technique, which has been shown to improve the identification of low-abundance muscle proteins and potential protein isoforms. The feature of the sarcomeric protein profile and information on more than 30 major proteins in the white dorsal muscle of silver carp were reported here for the first time. Overall, this study provides valuable transcriptome data resources and the comprehensive muscle protein information detected to date for further study into the processing characteristic of early postmortem fish muscle, as well as a spectral library for data-independent acquisition and data processing. This batch of muscle-specific dependent acquisition data is available via PRIDE with identifier PXD043702

Postmortem Muscle Proteome Characteristics of Silver Carp (<i>Hypophthalmichthys molitrix</i>): Insights from Full-Length Transcriptome and Deep 4D Label-Free Proteomic

The coverage of the protein database directly determines the results of shotgun proteomics. In this study, PacBio single-molecule real-time sequencing technology was performed on postmortem silver carp muscle transcripts. A total of 42.43 Gb clean data, 35,834 nonredundant transcripts, and 15,413 unigenes were obtained. In total, 99.32% of the unigenes were successfully annotated and assigned specific functions. PacBio long-read isoform sequencing (Iso-Seq) analysis can provide more accurate protein information with a higher proportion of complete coding sequences and longer lengths. Subsequently, 2671 proteins were identified in deep 4D proteomics informed by a full-length transcriptomics technique, which has been shown to improve the identification of low-abundance muscle proteins and potential protein isoforms. The feature of the sarcomeric protein profile and information on more than 30 major proteins in the white dorsal muscle of silver carp were reported here for the first time. Overall, this study provides valuable transcriptome data resources and the comprehensive muscle protein information detected to date for further study into the processing characteristic of early postmortem fish muscle, as well as a spectral library for data-independent acquisition and data processing. This batch of muscle-specific dependent acquisition data is available via PRIDE with identifier PXD043702

Insight into Ionic Strength-Induced Solubilization of Myofibrillar Proteins from Silver Carp (<i>Hypophthalmichthys molitrix</i>): Structural Changes and 4D Label-Free Proteomics Analysis

In this study, changes in the physical, structural, and assembly characteristics of silver carp myofibrillar proteins (MPs) at different ionic strength (I) values were investigated. Moreover, the differential proteomic profile of soluble MPs was analyzed using 4D proteomics based on timsTOF Pro mass spectrometry. Solubility of MPs significantly increased at high I (>0.3), and the increase in I enhanced the apparent viscosity, fluorescence intensity, surface hydrophobicity, and α-helix content of MPs solution. Particle size and sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns also supported the solubility profiles. Transmission electron microscopy and atomic force microscopy observations revealed the morphological assembly and disassembly of MPs under different I conditions. Finally, proteomic analysis revealed the evolution law of salt-induced solubilization of MPs and the critical molecular characteristics in different I environments. The number of differentially abundant proteins (DAPs) decreased with the increase of I, and most DAPs related to the muscle filament sliding, contraction and assembly, actinin binding, and actin filament binding. The soluble abundance of myosin and some structural proteins was dependent on I, and structural proteins in the Z-disk and M-band might contribute to the solubilization of myosin. Our findings provide insightful information about the impact of common I on the solubility pattern of MPs from freshwater fish

Serratia marcescens Causes the Brown Discoloration of Frozen Steamed Stuffed Buns during Resteaming

Brown discoloration was observed in the crust of commercial frozen steamed stuffed buns (FSSBs) during resteaming. Culture-dependent and culture-independent analyses demonstrated that Serratia marcescens, a prodigiosin-producing species, was more abundant in spoiled samples than in unspoiled samples. Inoculation of experimental FSSBs with S. marcescens isolated from spoiled FSSBs confirmed that this species causes brown discoloration of FSSBs during resteaming. S. marcescens formed prodigiosin only between 15 and 28 °C but brown discoloration appeared only upon resteaming after storage at 4 °C. High-performance liquid chromatography analyses revealed that prodigiosin was absent from yellow–brown FSSBs. The pigmentation observed during resteaming is thus likely attributable to the intermediate 2-methyl-3-amylpyrrole. These findings provide valuable insights into the microbial contamination of FSSBs and will facilitate the prevention of spoilage of FSSBs