Polarization-resolved microscopy reveals a muscle myosin motor-independent mechanism of molecular actin ordering during sarcomere maturation.

Sarcomeres are stereotyped force-producing mini-machines of striated muscles. Each sarcomere contains a pseudocrystalline order of bipolar actin and myosin filaments, which are linked by titin filaments. During muscle development, these three filament types need to assemble into long periodic chains of sarcomeres called myofibrils. Initially, myofibrils contain immature sarcomeres, which gradually mature into their pseudocrystalline order. Despite the general importance, our understanding of myofibril assembly and sarcomere maturation in vivo is limited, in large part because determining the molecular order of protein components during muscle development remains challenging. Here, we applied polarization-resolved microscopy to determine the molecular order of actin during myofibrillogenesis in vivo. This method revealed that, concomitantly with mechanical tension buildup in the myotube, molecular actin order increases, preceding the formation of immature sarcomeres. Mechanistically, both muscle and nonmuscle myosin contribute to this actin order gain during early stages of myofibril assembly. Actin order continues to increase while myofibrils and sarcomeres mature. Muscle myosin motor activity is required for the regular and coordinated assembly of long myofibrils but not for the high actin order buildup during sarcomere maturation. This suggests that, in muscle, other actin-binding proteins are sufficient to locally bundle or cross-link actin into highly regular arrays.

Effect of dry salt versus brine injection plus dry salt on the physicochemical characteristics of smoked salmon after filleting.

Smoked fish fillets are pre-salted as a food conservation and quality preservation measure. Here we investigated biochemical and sensory aspects of smoked salmon fillets. Left-side salmon fillets were dry-salted while the right-side fillets underwent a mixed salting method consisting of an injection of saturated brine followed by surface application of dry salt. After 6 h of salting, all the fillets were smoked. At each step of the process, quality was evaluated using instrumental measurements (pH, color, texture, water content, salt content, aw), and lipid distribution was visualized by MRI. Mixed-salted fillets had a higher salt content than dry-salted fillets and variability in salt distribution was dependent on the salting process. However, these variations had no effect on pH, color or texture, which showed similar values regardless of salting method. Fatty areas had a lower salt content due to slower diffusion of aqueous salt solutions through them. Mixed salting speeds up the salting of the muscle without significantly affecting the quality traits of the salmon fillet.

Structural changes in local Thai beef during sous-vide cooking.

Thai beef (Bos indicus) samples were sous-vide-cooked at temperatures of 60°C, 70°C or 80°C for 2 to 36 hrs and prepared for microstructure characterization by light and electron microscopy. Muscle fibers showed a first phase of lateral shrinkage during the first 6 hrs of cooking at 60-70°C and the first 2 hrs at 80°C followed by a second phase of significant alternations of shrinkage and swelling independently of water transfers. Swelling peaked at 12 hrs. Microstructural changes were more variable for samples cooked at 60-70°C than for samples cooked at 80°C that showed a larger cross-sectional myofibrillar mass area (CSA). Hypercontracted fibers were evidenced at all temperature-time combinations and were associated with adjacent wavy fibers and a characteristic structural evolution in the mitochondria. The role of thermal denaturation of proteins and the ultrastructural analogy of hypercontracted fibers with cold-shortened fibers are discussed.

Shockwave processing of beef brisket in conjunction with sous vide cooking: Effects on protein structural characteristics and muscle microstructure.

We studied the effect of shockwave processing and subsequent sous vide cooking on meat proteins (molecular size and thermal stability) and muscle structures (molecular, micro- and ultrastructure). Beef briskets were subjected to shockwave (11 kJ/pulse) and were sous vide-cooked at 60 °C for 12 h. Shockwave processing alone decreased the enthalpy and thermal denaturation temperature of the connective tissue proteins (second peak in the DSC thermogram, p < 0.05) compared to the control raw samples, while the protein gel electrophoresis profile remained unaffected. It led to disorganisation of the sarcomere structure and also modified the protein secondary structure. More severe muscle fibre coagulation and denaturation were observed in the shockwave-treated cooked meat compared to the cooked control. The results show that shockwave processing, with and without sous vide cooking, promotes structural changes in meat, and thus may have the potential to improve the organoleptic quality of the tough meat cuts.

Experimental validation of force inference in epithelia from cell to tissue scale.

Morphogenesis relies on the active generation of forces, and the transmission of these forces to surrounding cells and tissues. Hence measuring forces directly in developing embryos is an essential task to study the mechanics of development. Among the experimental techniques that have emerged to measure forces in epithelial tissues, force inference is particularly appealing. Indeed it only requires a snapshot of the tissue, as it relies on the topology and geometry of cell contacts, assuming that forces are balanced at each vertex. However, establishing force inference as a reliable technique requires thorough validation in multiple conditions. Here we performed systematic comparisons of force inference with laser ablation experiments in four epithelial tissues from two animals, the fruit fly and the quail. We show that force inference accurately predicts single junction tension, tension patterns in stereotyped groups of cells, and tissue-scale stress patterns, in wild type and mutant conditions. We emphasize its ability to capture the distribution of forces at different scales from a single image, which gives it a critical advantage over perturbative techniques such as laser ablation. Overall, our results demonstrate that force inference is a reliable and efficient method to quantify the mechanical state of epithelia during morphogenesis, especially at larger scales when inferred tensions and pressures are binned into a coarse-grained stress tensor.

In situ characterization of acidic and thermal protein denaturation by infrared microspectroscopy.

Foods meet acid pH during gastric digestion after cooking. An in situ infrared microspectroscopy approach was developed to detect the effects of heat and acid treatments on protein structure separately. Infrared spectra were obtained from meat samples treated with heat and/or acid, and wavenumbers accounting independently for the treatments were extracted by principal component regression. Extreme-acid treatment (pHinitial 2.0) was well predicted (0.5% error) by a simple ratio of as-observed spectral intensities at 1211 and 1396 cm-1, reflecting a perturbation in the vibration of amino acid residues (phenylalanine, tyrosine and aspartic acid) by protein unfolding and protonation. Using the imaging mode of an IR microscope, meat protein acidification was evidenced with high spatial resolution. The heat effect was well discriminated from the acid effect by the ratio of as-observed intensities at 1666 and 1697 cm-1 (0.9% error), indicating content of aggregated ß-sheets relative to α-helix structure.

High pressure processing of meat: effects on ultrastructure and protein digestibility.

The effects of high pressure processing (HPP, at 175 and 600 MPa) on the ultrastructure and in vitro protein digestion of bovine longissimus dorsi muscle meat were studied. HPP caused a significant change in the visual appearance and texture of the meat subjected to HPP at 600 MPa so that it appeared similar to cooked meat, unlike the meat subjected to HPP at 175 MPa that showed no significant visible change in the colour and texture compared to the raw meat. The muscles were subjected to digestion under simulated gastric conditions for 1 h and then under simulated small-intestinal conditions for a further 2 h. The digests were analysed using gel electrophoresis (SDS-PAGE) and ninhydrin assay for amino N. The effect of the acid conditions of the stomach alone was also investigated. Reduced SDS-PAGE results showed that pepsin-digested (60 min) HPP meats showed fewer proteins or peptides of high molecular weight than the pepsin-digested untreated meat, suggesting more breakdown of the parent proteins in HPP-treated meats. This effect was more pronounced in the muscles treated at 600 MPa. These results are in accordance with microscopy results, which showed greater changes in the myofibrillar structure after simulated gastric digestion of the sample processed at 600 MPa than at 175 MPa. Transmission electron microscopy also showed the presence of protein aggregates in the former sample, resulting probably from protein denaturation of sarcoplasmic proteins, in the subcellular space and between myofibrils; along with cell contraction (similar to that caused by heating) in the former.

Quantitative study of the relationships among proteolysis, lipid oxidation, structure and texture throughout the dry-cured ham process.

Temperature, salt and water contents are key processing factors in dry-cured ham production. They affect how proteolysis, lipid oxidation, structure and texture evolve, and thus determine the sensory properties and final quality of dry-cured ham. The aim of this study was to quantify the interrelationships and the time course of (i) proteolysis, (ii) lipid oxidation, (iii) five textural parameters: hardness, fragility, cohesiveness, springiness and adhesiveness and (iv) four structural parameters: fibre numbers, extracellular spaces, cross section area, and connective tissue area, during the dry-cured ham process. Applying multiple polynomial regression enabled us to build phenomenological models relating proteolysis, salt and water contents to certain textural and structural parameters investigated. A linear relationship between lipid oxidation and proteolysis was also established. All of these models and relationships, once combined with salt penetration, water migration and heat transfer models, can be used to dynamically simulate all of these phenomena throughout dry-cured ham manufacturing.