Machine learning and deep learning enabled age estimation on medial clavicle CT images.

The medial clavicle epiphysis is a crucial indicator for bone age estimation (BAE) after hand maturation. This study aimed to develop machine learning (ML) and deep learning (DL) models for BAE based on medial clavicle CT images and evaluate the performance on normal and variant clavicles. This study retrospectively collected 1049 patients (mean± SD: 22.50±4.34 years) and split them into normal training and test sets, and variant training and test sets. An additional 53 variant clavicles were incorporated into the variant test set. The development stages of normal MCE were used to build a linear model and support vector machine (SVM) for BAE. The CT slices of MCE were automatically segmented and used to train DL models for automated BAE. Comparisons were performed by linear versus ML versus DL, and normal versus variant clavicles. Mean absolute error (MAE) and classification accuracy was the primary parameter of comparison. For BAE, the SVM had the best MAE of 1.73 years, followed by the commonly-used CNNs (1.77-1.93 years), the linear model (1.94 years), and the hybrid neural network CoAt Net (2.01 years). In DL models, SE Net 18 was the best-performing DL model with similar results to SVM in the normal test set and achieved an MAE of 2.08 years in the external variant test. For age classification, all the models exhibit superior performance in the classification of 18-, 20-, 21-, and 22-year thresholds with limited value in the 16-year threshold. Both ML and DL models produce desirable performance in BAE based on medial clavicle CT.

Hepatic depletion of nucleolar protein mDEF causes excessive mitochondrial copper accumulation associated with p53 and NRF1 activation.

Copper is an essential component in the mitochondrial respiratory chain complex IV (cytochrome c oxidases). However, whether any nucleolar factor(s) is(are) involved in regulating the mitochondrial copper homeostasis remains unclear. The nucleolar localized Def-Capn3 protein degradation pathway cleaves target proteins, including p53, in both zebrafish and human nucleoli. Here, we report that hepatic depletion of mDEF in mice causes an excessive copper accumulation in the mitochondria. We find that mDEF-depleted hepatocytes show an exclusion of CAPN3 from the nucleoli and accumulate p53 and NRF1 proteins in the nucleoli. Furthermore, we find that NRF1 is a CAPN3 substrate. Elevated p53 and NRF1 enhances the expression of Sco2 and Cox genes, respectively, to allow more copper acquirement in the mDefloxp/loxp, Alb:Cre mitochondria. Our findings reveal that the mDEF-CAPN3 pathway serves as a novel mechanism for regulating the mitochondrial copper homeostasis through targeting its substrates p53 and NRF1.

Quality evaluation of cultured meat with plant protein scaffold.

Cultured meat technology is a promising new technology to solve the negative problems brought by traditional animal husbandry. Cultured meat should be further developed to appear on consumers' tables as alternative protein product. Therefore, this study used food grade peanut wire-drawing protein as scaffold to culture smooth muscle cells (SMCs) in vitro to obtain cultured meat productions containing both animal protein and plant protein. Multiple passages lead to the decline of the proliferation rate of SMCs in the proliferation stage and the differentiation ability in the differentiation stage, which means that the plasticity of cells decreased in the later stage of passage. SMCs can well adhere to the peanut wire-drawing protein scaffold and produce extracellular matrix protein and muscle protein, so as to form a cultured meat product with rich protein composition. This study provides a theoretical basis for the production of nutrient-rich cultured meat products.

Production of cultured fat with peanut wire-drawing protein scaffold and quality evaluation based on texture and volatile compounds analysis.

Cultured meat is an emergent technology that cultivates cells in three-dimensional scaffolds to generate tissue for consumption. Fat makes an important contribution to the flavor and texture of traditional meat, but there are few reports on cultured fat. Here, we demonstrated the construction of cultured fat by inoculating porcine adipose-derived mesenchymal stem cell (ADSC) on peanut wire-drawing protein (PWP) scaffolds. First, we demonstrated that basic fibroblast growth factor (bFGF) promoted cell proliferation and maintained adipogenic differentiation ability. Then, we generated cultured fat and found that cultured fat decreased the texture of PWP scaffolds. Moreover, 43 volatile compounds were detected by headspace gas chromatography-ion mobility spectrometry (GC-IMS), of which 17 volatile compounds showed no significant differences between cultured fat and porcine subcutaneous adipose tissue (pSAT), which indicated that cultured fat and pSAT had certain similarities. Collectively, this research has great promise for improving the quality of cultured meat.

Production of cultured meat by culturing porcine smooth muscle cells in vitro with food grade peanut wire-drawing protein scaffold.

Cultivating meat is a promising solution to the negative problems brought by traditional animal husbandry. To make cultured meat have the sensory and nutritional characteristics of conventional meat as much as possible, many studies have been conducted on various cell types and scaffold characteristics. Therefore, this study aims to produce a low-cost cultured meat with a quality closer to that of conventional meat. Tissue generation requires three-dimensional (3D) scaffolds to support cells and simulate extracellular matrix (ECM). Here, we used peanut wire-drawing protein (a biomaterial based on edible porous protein) as a new culture meat scaffold to culture cells. The scaffold can support cell attachment and proliferation to create 3D engineered porcine muscle tissue. The differentiation of smooth muscle cells (SMCs) was induced by a low serum medium to produce more extracellular matrix proteins. After differentiation, it was found that peanut wire-drawing protein scaffolds could be used for porcine smooth muscle cell adhesion and growth. The ECM protein and muscle protein produced by SMCs can endow cultured meat with better quality. This technology provides an innovative pathway for the industrialized production of cultured meat.

Production of cultured meat from pig muscle stem cells.

Cultured meat is meat for consumption produced in a more sustainable way. It involves cell harvesting and expansion, differentiation into myotubes, construction into muscle fibres and meat structuring. We isolated 5.3 × 104 porcine muscle stem cells from 1 g of neonatal pig muscle tissue. According to calculations, we need to expand muscle stem cells 106-107 times to produce 100 g or 1 kg of cultured meat. However, the cells gradually lost the ability to express stemness and mature muscle cell markers (PAX7, MyHC). To tackle this critical issue and maintain cell function during cell expansion, we found that long-term culture with (100 µM) l-Ascorbic acid 2-phosphate (Asc-2P) accelerated cell proliferation while preserving the muscle cell differentiation. We further optimized a scalable PDMS mold. Porcine muscle stem cells formed structurally-organized myotubes similar to muscle fibres in the mold. Asc-2P enhanced porcine muscle cells grown as 3D tissue networks that can produce a relatively large 3D tissue networks as cultured meat building blocks, which showed improved texture and amino acid content. These results established a realistic workflow for the production of cultured meat that mimics the pork meat structurally and is potentially scalable for industry.

Evaluation of the effect of smooth muscle cells on the quality of cultured meat in a model for cultured meat.

While the research on improving the meat quality of cultured meat is in full swing, few studies have focused on the effect of smooth muscle cells (SMCs) on the meat quality of cultured meat. Therefore, this study aimed at building a cultured meat model containing smooth muscle cells, and further evaluating the effect of smooth muscle cells on the quality of cultured meat, so as to reveal the contribution of smooth muscle cells in the production of cultured meat. In this study, we isolated high purity of smooth muscle cells from vascular tissues. The addition of basic fibroblast growth factor (bFGF) to the medium significantly increased the growth rate of smooth muscle cells and the expression of extracellular matrix related genes, especially collagen and elastin. Smooth muscle cells were seeded in a collagen gel to construct a culture meat model. It was found that the pressure loss of the model meat significantly decreased from 98.5 % in control group to 54 % with the extension of culture time for 9 days, while the total collagen content of model meat increased significantly (P < 0.05). In addition, the hydrogel tissue with smooth muscle cells compacted more dramatically and were more tightly, accompanied by significantly increased hardness, springiness and chewiness compared to the control one (P < 0.05). These results indicate that smooth muscle cells can secrete extracellular matrix proteins such as collagen, which can significantly enhance the texture of cultured meat models prepared by hydrogel.

YAP Promotes Cell Proliferation and Stemness Maintenance of Porcine Muscle Stem Cells under High-Density Condition.

Muscle stem cells (MuSCs) isolated ex vivo are essential original cells to produce cultured meat. Currently, one of the main obstacles for cultured meat production derives from the limited capacity of large-scale amplification of MuSCs, especially under high-density culture condition. Here, we show that at higher cell densities, proliferation and differentiation capacities of porcine MuSCs are impaired. We investigate the roles of Hippo-YAP signaling, which is important regulators in response to cell contact inhibition. Interestingly, abundant but not functional YAP proteins are accumulated in MuSCs seeded at high density. When treated with lysophosphatidic acid (LPA), the activator of YAP, porcine MuSCs exhibit increased proliferation and elevated differentiation potential compared with control cells. Moreover, constitutively active YAP with deactivated phosphorylation sites, but not intact YAP, promotes cell proliferation and stemness maintenance of MuSCs. Together, we reveal a potential molecular target that enables massive MuSCs expansion for large-scale cultured meat production under high-density condition.