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Cultured meat (CM) production has emerged as a breakthrough technology for the global food industry, with the potential to reduce challenges associated with environmental sustainability, global public health, animal welfare, and competition for food between humans and animals. The muscle stem cell lines currently used for CM production cannot be passaged in vitro for extended periods of time. Here, we successfully developed a directional differentiation system of porcine pregastrulation epiblast stem cells (pgEpiSCs) with stable cellular features and achieved serum-free myogenic differentiation of the pgEpiSCs. The pgEpiSCs-derived skeletal muscle progenitor cells and skeletal muscle fibers show typical muscle cell characteristics and display skeletal muscle transcriptional features during myogenic differentiation. Importantly, we further established a three-dimensional differentiation system for shaping cultured tissue by screening plant-based edible scaffolds of non-animal origin, followed by the generation of pgEpiSCs-derived CM. These advances provide a novel technical approach for the development of CM.

Pig epiblast-derived pluripotent stem cells are considered to have great potential and broad prospects for human therapeutic model development and livestock breeding. Despite ongoing attempts since the 1990s, no stably defined pig epiblast-derived stem cell line has been established. Here, guided by insights from a large-scale single-cell transcriptome analysis of pig embryos from embryonic day (E) 0 to E14, specifically, the tracing of pluripotency changes during epiblast development, we developed an in vitro culture medium for establishing and maintaining stable pluripotent stem cell lines from pig E10 pregastrulation epiblasts (pgEpiSCs). Enabled by chemical inhibition of WNT-related signaling in combination with growth factors in the FGF/ERK, JAK/STAT3, and Activin/Nodal pathways, pgEpiSCs maintain their pluripotency transcriptome features, similar to those of E10 epiblast cells, and normal karyotypes after more than 240 passages and have the potential to differentiate into three germ layers. Strikingly, ultradeep in situ Hi-C analysis revealed functional impacts of chromatin 3D-spatial associations on the transcriptional regulation of pluripotency marker genes in pgEpiSCs. In practice, we confirmed that pgEpiSCs readily tolerate at least three rounds of successive gene editing and generated cloned gene-edited live piglets. Our findings deliver on the long-anticipated promise of pig pluripotent stem cells and open new avenues for biological research, animal husbandry, and regenerative biomedicine.

Prefrontal cortex (PFC) is the cognitive center that integrates and regulates global brain activity. However, the whole-brain organization of PFC axon projections remains poorly understood. Using single-neuron reconstruction of 6,357 mouse PFC projection neurons, we identified 64 projectome-defined subtypes. Each of four previously known major cortico-cortical subnetworks was targeted by a distinct group of PFC subtypes defined by their first-order axon collaterals. Further analysis unraveled topographic rules of soma distribution within PFC, first-order collateral branch point-dependent target selection and terminal arbor distribution-dependent target subdivision. Furthermore, we obtained a high-precision hierarchical map within PFC and three distinct functionally related PFC modules, each enriched with internal recurrent connectivity. Finally, we showed that each transcriptome subtype corresponds to multiple projectome subtypes found in different PFC subregions. Thus, whole-brain single-neuron projectome analysis reveals organization principles of axon projections within and outside PFC and provides the essential basis for elucidating neuronal connectivity underlying diverse PFC functions.

Spatially ordered embryo-like structures self-assembled from blastocyst-derived stem cells can be generated to mimic embryogenesis in vitro. However, the assembly system and developmental potential of such structures needs to be further studied. Here, we devise a nonadherent-suspension-shaking system to generate self-assembled embryo-like structures (ETX-embryoids) using mouse embryonic, trophoblast and extra-embryonic endoderm stem cells. When cultured together, the three cell types aggregate and sort into lineage-specific compartments. Signaling among these compartments results in molecular and morphogenic events that closely mimic those observed in wild-type embryos. These ETX-embryoids exhibit lumenogenesis, asymmetric patterns of gene expression for markers of mesoderm and primordial germ cell precursors, and formation of anterior visceral endoderm-like tissues. After transplantation into the pseudopregnant mouse uterus, ETX-embryoids efficiently initiate implantation and trigger the formation of decidual tissues. The ability of the three cell types to self-assemble into an embryo-like structure in vitro provides a powerful model system for studying embryogenesis., The precise cellular patterning and decisions of early embryogenesis have been hard to mimic in vitro. Here, the authors culture murine embryonic and trophoblast stem cells together with extra-embryonic endoderm stem cells to form embryo-like structures (ETX-embryoids), which can initiate an implantation response.

Highlights • Higher step count is inversely associated with the risk of premature death and cardiovascular events. • As measured by accelerometers, 8959 steps/day (Q3) had a 40.36% lower risk of all-cause mortality than 4183 steps/day (Q1). • As measured by accelerometers, 9500 steps/day (Q3) had a 35.05% lower risk of cardiovascular events than 3500 steps/day(Q1). • These associations were in nonlinear dose–response patterns., Background A goal of 10,000 steps per day is widely advocated, but there is little evidence to support that goal. Our purpose was to examine the dose–response relationships between step count and all-cause mortality and cardiovascular disease risk. Methods Cochrane Central Register of Controlled Trials, EMBASE, OVID, PubMed, Scopus, and Web of Science databases were systematically searched for studies published before July 9, 2021, that evaluated the association between daily steps and at least 1 outcome. Results Sixteen publications (12 related to all-cause mortality, 5 related to cardiovascular disease; and 1 article contained 2 outcomes: both all-cause death and cardiovascular events) were eligible for inclusion in the meta-analysis. There was evidence of a nonlinear dose–response relationship between step count and risk of all-cause mortality or cardiovascular disease (p = 0.002 and p = 0.014 for nonlinearity, respectively). When we restricted the analyses to accelerometer-based studies, the third quartile had a 40.36% lower risk of all-cause mortality and a 35.05% lower risk of cardiovascular event than the first quartile (all-cause mortality: Q1 = 4183 steps/day, Q3 = 8959 steps/day; cardiovascular event: Q1 = 3500 steps/day, Q3 = 9500 steps/day; respectively). Conclusion Our meta-analysis suggests inverse associations between higher step count and risk of premature death and cardiovascular events in middle-aged and older adults, with nonlinear dose–response patterns., Graphical abstract Image, graphical abstract

Introduction. To investigate whether the membrane-associated transporter protein SLC45A2 is differentially expressed in the skin of sheep with different coat colors and to determine its correlation with coat color establishment in sheep. Material and methods. The expression of SLC45A2 in sheep skin samples with different coat colors was qualitatively and quantitatively analyzed by PCR amplification, RT-PCR, immunohistochemical staining and Western blotting. Results. A 193-bp SLC45A2 CDS sequence was successfully amplified from sheep skin samples with diverse coat colors. RT-PCR analysis revealed that SLC45A2 mRNA was expressed in all sheep skin samples tested, with relative expression levels of 512.74 ± 121.51 in black skin, 143.38 ± 119.31 and 1.36 ± 0.09 in black dots and white dots of piebald skin, respectively, and 1.02 ± 0.23 in white skin (p < 0.01**). Positive SLC45A2 protein bands were also detected in all skin samples by Western blot analysis, with relative expression levels of 0.85 ± ± 0.17** in black skin, 0.60 ± 0.05** and 0.34 ± 0.07 in black dots and white dots of piebald skin, respectively, and 0.20 ± 0.05 in white skin (p < 0.01**). Immunohistochemical assays revealed that SLC45A2 was expressed in the hair follicle matrix, the inner and outer root sheath, and the dermal papilla in the skin tissues with different coat colors. These patterns were quantified by optical density (OD) analysis, which yielded relative expression levels of 0.23 ± 0.11 in black skin, 0.19 ± 0.09 and 0.10 ± 0.03 in black dots and white dots of piebald skin, respectively, and 0.08 ± 0.01 in white skin (p < 0.05*). Conclusion. SLC45A2 is detectably expressed in sheep skin of all coat colors, though at significantly different levels. SLC45A2 may participate in the establishment of coat color by regulating the synthesis and trafficking of melanin.

MicroRNAs (miRNAs) play an essential role in the regulation of almost all the biological processes, including melanogenesis. MiR-27a-3p is nearly six times higher in white alpaca skin compared to brown skin, which indicates that miR-27a-3p may be a candidate regulator for melanogenesis. Wnt3a plays an important role in promoting melanoblasts to differentiate into melanocytes and melanogenesis. To confirm the function of miR-27a-3p to melanogenesis in mammals, miR-27a-3p mimic, inhibitor and their negative control were transfected into mouse melanocytes. As a result, miR-27a-3p inhibits melanogenesis by repressing Wnt3a at post-transcriptional level. A significant decrease in Wnt3a luciferase activity was observed in 293T cells co-transfected with the matched luciferase reporter vector and pre-miR-27a. Furthermore, the presence of exogenous miR-27a-3p significantly decreased Wnt3a protein expression rather than mRNA and reduced β-catenin mRNA levels in melanocytes. The over-expression of miR-27a-3p significantly increased the melanin content of melanocytes. However, miR-27a-3p inhibitor performs an opposite effect on melanogenesis. Wnt3a is one target of miR-27a-3p. MiR-27a-3p could inhibit Wnt3a protein amount by post-transcriptional regulation and melanogenesis in mouse melanocytes. Previous studies reported that Wnt3a promoted melanogenensis in mouse melanocytes. Thus, miR-27-3p inhibits melanogenesis by repressing Wnt3a protein expression.

Tyrosinase‑related protein 2 (TRP‑2) is one of the most important members of the tyrosinase family, and is a key enzyme involved in melanin biosynthesis. In the present study, a skin transcriptome profile, immunohistochemistry, western blotting and reverse transcription‑quantitative polymerase chain reaction were used to investigate TRP‑2 expression in sheep with different coat colors, namely, black, white and black‑white. TRP‑2 was overexpressed in melanocytes in order to study the effect of TRP‑2 on melanin production. Results revealed differing TRP‑2 levels in sheep of different coat colors and in various parts of the coat with different colors in the same sheep. TRP‑2 expression levels in dark‑colored areas were significantly increased compared with light‑colored areas in piebald sheep. TRP‑2 overexpression may regulate melanogenesis and significantly increase melanogenesis associated transcription factor expression in vitro. Therefore, TRP‑2 may affect melanin production in sheep, and different expression levels determine coat color. The results may provide novel approaches for developing therapeutic strategies for skin diseases associated with pigmentation disorders.

Fibroblast growth factor 21 (FGF21) is known as a metabolic regulator to regulate the metabolism of glucose and lipids. However, the underlying mechanism of FGF21 on melanin synthesis remains unknown. Therefore, the current study investigates the effect of FGF21 on melanogenesis in alpaca melanocytes. We transfected the FGF21 into alpaca melanocytes, then detected the melanin contents, protein and mRNA levels of pigmentation-related genes in order to determine the melanogenesis-regulating pathway of FGF21. The results showed that FGF21 overexpression suppressed melanogenesis and decreased the expression of the major target genes termedmicrophthalmia-associated transcription factor(MITF) and its downstream genes, including tyrosinase (TYR) and tyrosinase-related protein 2 (TRP2). However FGF21 increased the expression of phospho-extracellular signal-regulated kinase (p-Erk1/2). In contrast, FGF21-siRNA, a small interference RNA mediating FGF21 silencing, abolished the inhibition of melanogenesis. Altogether, FGF21 may decrease melanogenesis in alpaca melanocytes via ERK activation and subsequent MITF downregulation, which is then followed by the suppression of melanogenic enzymes and melanin production.

Research literature suggests that there are age and gender effects on numinous constructs, but little is known about how spirituality and religiousness evolve over time and differ between genders. The purpose of this study was to determine whether observed gender and age effects would be evidenced on the Assessment of Spirituality and Religious Sentiments (ASPIRES) and whether the underlying factor structure of the ASPIRES was consistent across age and gender groups. These sample data (N 1,534 women and 697 men) demonstrated that (a) there are significant age and gender effects on the ASPIRES scales, and (b) the underlying factor structure of the scales remains unchanged in all groups, suggesting that the groups appear to understand and experience spirituality and religiousness in a similar manner. The results of this study demonstrate that although the expression of spirituality and religious sentiments may vary across age and between genders, the fundamental meaning of these constructs remains the same.

MicroRNAs (miRNAs) play an important role in regulating almost all biological processes. miRNAs bind to the 3′ untranslated region (UTR) of mRNAs by sequence matching. In a previous study, we demonstrated that miR-21 was differently expressed in alpaca skin with different hair color. However, the molecular and cellular mechanisms for miR-21 to regulate the coat color are not yet completely understood. In this study, we transfected miR-21a-5p into mouse melanocytes and demonstrated its function on melanogenesis of miR-21a-5p by targeting Sox5, which inhibits melanogenesis in mouse melanocytes. The results suggested that miR-21a-5p targeted Sox5 gene based on the binding site in 3′ UTR of Sox5 and overexpression of miR-21a-5p significantly down-regulated Sox5 mRNA and protein expression. Meanwhile, mRNA and protein expression of microphthalmia transcription factor (MITF) and Tyrosinase (TYR) were up-regulated, which subsequently make the melanin production in melanocytes increased. The results suggest that miR-21a-5p regulates melanogenesis via MITF by targeting Sox5.

In the present paper, a fully symmetric Galerkin multi-zone boundary element method (GMZBEM) is proposed for the cohesive crack growth. For interface problems, the system matrix of each subdomain formulated by GMZBEM is not completely symmetric. Based on regarding the coefficient matrix as the sum of a symmetric part and a non-symmetric one, this problem is solved by adopting a proper assembly. According to this approach, the cohesive crack growth is simulated by marching one of craze-tips step-by-step and by using a special iterative algorithm. During the fracture process, the crack is simulated by means of the cohesive crack model, and the materials in this paper follow linear-elastic and isotropic models. The iterative enforcement of the cohesive crack involves the solution from boundary element equations at each iteration, but these equations in each subdomain need be factored only once for the entire problem unless the interfaces are modified. Numerical results obtained by using the present method are compared with the results from previous methods, which shows that GMZBEM is accurate and effective.

Conventional plant transformation typically includes preparation of competent plant cells or tissues, delivery of foreign genes into cells, transformed cell selection with stable incorporated foreign genes, and regeneration of transformed cells into intact plants. This process traditionally relies on tissue culture, and cotton has not been an exception to this paradigm. Though the commercialization of transgenic cotton is a resounding success, cotton transformation, which is the first step in producing transgenic cotton, is a burdensome process since there is a very long tissue culture process and a limited number of cultivars that can be regenerated. An improved process which is easier to handle and more genotype independent could efficiently generate more transgenic plants and allow meaningful analyses of gene function and transgenic plants. Cotton pistil drip by inoculating Agrobacterium tumefaciens onto the pistil after pollination gave rise to stable transformants. Since this transformation process in cotton occurs following pollination and during fertilization (postanthesis) but not during preanthesis as in Arabidopsis, the mechanism by which Agrobacterium enters plant cells and integrates into the cotton genome may differ from that in Arabidopsis. This chapter provides the detailed protocol for pistil drip, a simple in planta transformation method without the plant tissue culture process.

Conventional plant transformation typically includes preparation of competent plant cells or tissues, delivery of foreign genes into cells, transformed cell selection with stable incorporated foreign genes, and regeneration of transformed cells into intact plants. This process traditionally relies on tissue culture, and cotton has not been an exception to this paradigm. Though the commercialization of transgenic cotton is a resounding success, cotton transformation, which is the first step in producing transgenic cotton, is a burdensome process since there is a very long tissue culture process and a limited number of cultivars that can be regenerated. An improved process which is easier to handle and more genotype independent could efficiently generate more transgenic plants and allow meaningful analyses of gene function and transgenic plants. Cotton pistil drip by inoculating Agrobacterium tumefaciens onto the pistil after pollination gave rise to stable transformants. Since this transformation process in cotton occurs following pollination and during fertilization (postanthesis) but not during preanthesis as in Arabidopsis, the mechanism by which Agrobacterium enters plant cells and integrates into the cotton genome may differ from that in Arabidopsis. This chapter provides the detailed protocol for pistil drip, a simple in planta transformation method without the plant tissue culture process.

One of the major tasks in the design and optimization process of a new landing gear system for a lunar lander is to accurately determine the loads and energy absorption capability during the landing event. The works of this paper describes a new approach of landing impact dynamic analysis using nonlinear finite element method. Abaqus/Explicit, which is part of Abaqus suite of finite element analysis software, is selected to simulate the landing event for its excellent nonlinear, transient dynamics capabilities. The aluminum honeycomb shock absorber is modeled by plastic crushable foam material model, while the lunar soil is modeled by Drucker-Prager/Cap material model. Simulation results, including the load at connector between structure and landing gear, acceleration response of structure and dissipated energy by the shock absorber and lunar soil, are given and discussed. It shows that the performance of landing gear meets the design requirements.