Feed efficiency (FE) can be measured by feed conversion ratio (FCR) or residual feed intake (RFI). In this study, we measured the FE related phenotypes of 236 castrated purebred Yorkshire boars, and ...selected 10 extreme individuals with high and low RFI for transcriptome analysis. We used RNA-seq analyses to determine the differential expression of genes and miRNAs in skeletal muscle. There were 99 differentially expressed genes identified (q ≤ 0.05). The down-regulated genes were mainly involved in mitochondrial energy metabolism, including FABP3, RCAN, PPARGC1 (PGC-1A), HK2 and PRKAG2. The up-regulated genes were mainly involved in skeletal muscle differentiation and proliferation, including IGF2, PDE7A, CEBPD, PIK3R1 and MYH6. Moreover, 15 differentially expressed miRNAs (|log2FC| ≥ 1, total reads count ≥ 20, p ≤ 0.05) were identified. Among them, miR-136, miR-30e-5p, miR-1, miR-208b, miR-199a, miR-101 and miR-29c were up-regulated, while miR-215, miR-365-5p, miR-486, miR-1271, miR-145, miR-99b, miR-191 and miR-10b were down-regulated in low RFI pigs. We conclude that decreasing mitochondrial energy metabolism, possibly through AMPK - PGC-1A pathways, and increasing muscle growth, through IGF-1/2 and TGF-β signaling pathways, are potential strategies for the improvement of FE in pigs (and possibly other livestock). This study provides new insights into the molecular mechanisms that determine RFI and FE in pigs.
We report a general strategy to generate linear and circular gradients of active proteins or polymeric microparticles on planar surfaces by controlling the distribution of electrostatic field during ...electrohydrodynamic jet printing or electrospray process. Taking fibronectin as an example, we generated a circular gradient of fibronectin and investigated its effect on accelerating the migration of fibroblasts to suit for use in wound closure. In another demonstration, we created linear gradients of laminin in unidirectional and bidirectional patterns, respectively. We showed that such gradations significantly promoted the migration of human neuroblastoma cells with the increase of laminin content. When we changed fibronectin/laminin to electrosprayed poly(lactic-co-glycolic acid) (PLGA) microparticles, we found similar results in terms of guiding cell migration, except that the guidance cues varied from biological signal to topographic structure. Taken together, this method for generating linear/circular gradients of fibronectin/laminin and PLGA microparticles can be readily extended to different types of bioactive proteins and polymeric microparticles to suit wound closure, nerve repair, and related applications involving cell migration.
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•Various types of surface gradients were obtained by changing electrostatic field.•Circular gradient of fibronectin accelerated centripetal cell migration.•Linear gradient of laminin accelerated uniaxial cell migration.•Linear gradient of PLGA microparticles accelerated unidirectional cell migration.
The influence of metro tunnel excavation on the surrounding existing structures should not be ignored. According to the characteristics of the tunnel side passing through the Beiyuan viaduct pile of ...Jinan Metro Line R2, the construction process is simulated by using FLAC3D software. The influence of tunnel excavation on adjacent bridge piles is studied. The results show that during the construction of double track tunnel, the farther away from the tunnel axis, the smaller the disturbance caused by the construction. Due to the disturbance of the stratum caused by the construction of the large excavation section of the left line tunnel, the maximum settlement value of the surface is directly above the axis of the left line tunnel. The vertical deformation curve of the stratum presents an asymmetric double concave groove distribution. By adopting the advanced support scheme of WSS deep hole grouting + advanced small catheter, the pile deformation and the uneven settlement of the ground surface is effectively limited. The longer the length of the pile embedded in the rock mass, the smaller the horizontal displacement generated by the pile bottom. The research can provide reference for the construction of similar tunnel side crossing bridge piles in the future.
Mechanical force plays a pivotal role in the pathogenesis of hypertrophic scar (HTS). Dermal fibroblasts and myofibroblasts are the key cells involved in HTS. Myofibroblasts in HTS possess different ...biochemical and biophysical characteristics by which myofibroblasts are often distinguished from fibroblasts. The role of mechanotransducers outside the nucleus in the pathogenesis of HTS has been reported in many studies. However, the role of Nesprin‐2 in HTS is not clear. Hence, we aim to construct a cell model of HTS and explore the role of Nesprin‐2 in this process. Myofibroblasts and fibroblasts were isolated from HTS and healthy skin tissues of the same patient. Fibroblasts were exposed to cyclic stretch with 10% magnitude and a frequency of 0.1 Hz for 3 days, 5 days, and 7 days, respectively. After the cell model was confirmed, fibroblasts transfected with siRNA targeting human Nesprin‐2 were exposed to cyclic stretch. The mechanical behaviour and biochemical reaction of the dermal fibroblasts were analysed. The stretched fibroblasts at day 5 showed the same mechanotransductive and biochemical features as unstretched myofibroblasts. Mechanical strain could induce the myofibroblasts differentiation and a cell model of HTS was established successfully at day 5. The expressions of lamin A/C, alpha‐smooth muscle actin, transforming growth factor beta 1, and collagen type I in fibroblasts were reduced by the silencing of Nesprin‐2. Mechanical strain could induce the myofibroblasts differentiation and silencing of Nesprin‐2 could block the mechanical stimulation of terminal myofibroblasts differentiation. Nesprin‐2 might be a potential target to treat the HTS.
Homeobox C10 (HOXC10) has been reported to participate in various cancers. However, the involvement of HOXC10 in melanoma is still unknown. Here, we attempted to determine whether HOXC10 can affect ...the development of melanoma. We separated melanoma tissues and the matched tumor-adjacent normal tissues from melanoma patients, and examined HOXC10 expression in the melanoma cells and tissues. Comparing with the tumor-adjacent normal tissues, HOXC10 was up-regulated in melanoma tissues. Melanoma cells also displayed an up-regulation of HOXC10. Moreover, HOXC10 inhibition suppressed cell proliferation, clone formation and promoted apoptosis of melanoma cells. Knockdown of HOXC10 also retarded migration, invasion and epithelial–mesenchymal transition (EMT) in melanoma cells. Additionally, HOXC10 accelerated Slug expression by interacting with Slug, and activating the promoter of Slug. Slug activated the YAP/TAZ signaling pathway, which was reversed by HOXC10 silencing. The in vitro assays demonstrated that inhibition of HOXC10 significantly repressed tumor growth and lung metastasis of melanoma in mice by inhibiting Slug and YAP/TAZ signaling pathway. In conclusion, this work demonstrated that HOXC10 promoted growth and migration of melanoma by regulating Slug to activate the YAP/TAZ signaling pathway. Therefore, this study suggests that inhibition of HOXC10 has therapeutic potential in melanoma.
Microphthalmia-associated transcription factor (MITF) is a key regulator for the development and function of melanocytes in skin, eye, and plumage pigmentations. Thus, the MITF was selected as a ...candidate gene associated with plumage coloration in ducks. This study analyzed the mRNA expression, promoter methylation, and polymorphisms in the MITF gene in ducks with different plumage colors (Putian Black, Putian White, Liancheng White, and Longsheng Jade-green). No expression of the MITF melanin-specific isoform (MITF-M) was detected in white feather bulbs. By contrast, the mRNA expression levels of MITF-M were high in black feather bulbs. Bioinformatics analysis showed that two CpG islands were present in the promoter region of the MITF gene. The methylation level of the second CpG island was significantly lower in black feather bulbs than in white feather bulbs. However, the methylation level of the first CpG island was not different among the feather bulbs with various colors except Liancheng White feather bulbs. The methylation status of the whole CpG island significantly and negatively correlated with the mRNA expression of MITF-M (P<0.05). Furthermore, four novel SNPs (single nucleotide polymorphisms) were identified in the 5′UTR, exon 4, intron 7, and intron 8 of the MITF gene. Allele T in g.39807T>G and allele G in g.40862G>A were the predominant alleles only found in Putian White, whereas the variant A allele in g.32813G>A exhibited a high allele frequency in Liancheng White. Collectively, these results contributed to the understanding of the function of the MITF gene in duck plumage coloration.
Objectives
Recently developed CRISPR‐dependent cytosine base editor (CBE), converting four codons (CAA, CAG, CGA and TGG) into stop codons without DNA double‐strand breaks (DSB), serves as an ...efficient gene disruption strategy besides uncontrollable CRISPR‐mediated frameshift. However, the detailed difference of gene knockout between the two systems has not been clarified.
Materials and methods
Here, we selected some sgRNAs with different position background, then HEK293T cells were transfected with CBE/Cas9 plasmids together with sgRNAs. GFP‐positive cells were harvested by fluorescence‐activated cell sorting (FACS) 48 hours after transfection. Genomic DNA was collected for deep sequencing to analyse editing efficiency and genotype. RNA and protein were extracted to analyse gene mRNA level using qPCR analysis and Western blot.
Results
Here, we compared the gene disruption by CBE‐mediated iSTOP with CRISPR/Cas9‐mediated frameshift. We found BE‐mediated gene knockout yielded fewer genotypes. BE‐mediated gene editing precisely achieved silencing of two neighbouring genes, while CRISPR/Cas9 may delete the large fragment between two target sites. All of three stop codons could efficiently disrupt the target genes. It is worth notifying, Cas9‐mediated gene knockout showed a more impact on neighbouring genes mRNA level than the BE editor.
Conclusions
Our results reveal the differences between the two gene knockout strategies and provide useful information for choosing the appropriate gene disruption strategy.
Background: Melanoma, a type of skin cancer, is a leading cause of death worldwide. Currently available therapy shows numerous side effects; therefore, there is an urgent need to develop safe drugs ...to treat this malignancy. Recently, 2-hydroxy-4-methoxy benzoic acid (HMBA), a bioactive plant component, has been reported to demonstrate various biological functions. Objectives: In this study, the anticancer effect of HMBA was assessed against SK-MEL-28 cells. Materials and Methods: DNA damage was assessed using DNA strand break assay (comet assay), apoptosis-mediated cell death by Annexin-V, and Terminal deoxynucleotide transferase dUTP Nick End Labeling experiments. Western blot analysis was performed to assess the phosphorylation of ERK, p38, and JNK. Results: Increasing the time of exposure decreases the IC25, IC50, and IC75 value of HMBA. Activity of lactate dehydrogenase in the culture medium of control and HMBA-exposed cells directly correlate its cytotoxic property. HMBA caused dose-dependent DNA damage and induced apoptosis in SK-MEL-28 cells. AO-staining showed autophagy in HMBA-treated cells, whereas Western blot analysis revealed increase in the phosphorylation of ERK, p38, and JNK. Furthermore, our results show that ERK phosphorylation is responsible for the activation of autophagy protein such as LC3 and p62. These observations reveal that the activation of caspase-3 and commencement of autophagy is mediated through activation of ERK phosphorylation. Conclusion: Therefore, HMBA inhibits propagation of SK-MEL-28 cells via stimulation of apoptosis and autophagy. In addition, HMBA promotes apoptosis and autophagy by phosphorylation of vital signaling protein such as ERK, however other vital signaling such as p38 and JNK also phosphorylate. Thus, HMBA may be of therapeutic importance for the treatment of melanoma.
Feed efficiency (FE) is a very important trait in livestock industry. Identification of the candidate genes could be of benefit for the improvement of FE trait. Mouse is used as the model for many ...studies in mammals. In this study, the candidate genes related to FE and coat color were identified using C57BL/6J (C57) × Kunming (KM) F2 mouse population. GWAS results showed that 61 and 2 SNPs were genome-wise suggestive significantly associated with feed conversion ratio (FCR) and feed intake (FI) traits, respectively. Moreover, the Erbin, Msrb2, Ptf1a, and Fgf10 were considered as the candidate genes of FE. The Lpl was considered as the candidate gene of FI. Further, the coat color trait was studied. KM mice are white and C57 ones are black. The GWAS results showed that the most significant SNP was located at chromosome 7, and the closely linked gene was Tyr. Therefore, our study offered useful target genes related to FE in mice; these genes may play similar roles in FE of livestock. Also, we identified the major gene of coat color in mice, which would be useful for better understanding of natural mutation of the coat color in mice.
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