The impact of exercise on the gut microbiota in middle-aged amateur serious runners: a comparative study.

Introduction: Exercise, health, and the gut microbiota (GM) are strongly correlated. Research indicates that professional athletes, especially ultra-marathon runners, have unique GM characteristics. However, more research has focused on elite athletes, with little attention given to amateur sports enthusiasts, especially those in the middle-aged population. Therefore, this study focuses on the impact of long-term running on the composition and potential functions of the GM in middle-aged individuals. Methods: We compared the GM of 25 middle-aged serious runnerswith 22 sedentary healthy controls who had minimal exercise habitsusing 16S rRNA gene sequencing. Additionally, we assessed dietary habits using a food frequency questionnaire. Results and Discussion: Statistical analysis indicates that there is no significant difference in dietary patterns between the control group and serious runners. Diversity analysis results indicate that there is no significant difference in α diversity between the two groups of GM, but there is a significant difference in ß diversity. Analysis of the composition of GM reveals that Ruminococcus and Coprococcus are significantly enriched in serious runners, whereas Bacteroides, Lachnoclostridium, and Lachnospira are enriched in the control group. Differential analysis of functional pathway prediction results reveals significant differences in the functional metabolism levels of GM between serious runners and the control group. Further correlation analysis results indicate that this difference may be closely related to variations in GM. In conclusion, our results suggest that long-term exercise can lead to changes in the composition of the GM. These changes have the potential to impact the overall health of the individual by influencing metabolic regulation.

Structural characteristics of gut microbiota in longevity from Changshou town, Hubei, China.

The gut microbiota (GM) and its potential functions play a crucial role in maintaining host health and longevity. The aim of this study was to investigate the potential relationship between GM and longevity. We collected fecal samples from 92 healthy volunteers (middle-aged and elderly: 43-79 years old; longevity: ≥ 90 years old) from Changshou Town, Zhongxiang City, Hubei, China. In addition, we collected samples from 30 healthy middle-aged and elderly controls (aged 51-70 years) from Wuhan, Hubei. The 16S rDNA V3 + V4 region of the fecal samples was sequenced using high-throughput sequencing technology. Diversity analysis results showed that the elderly group with longevity and the elderly group with low body mass index (BMI) exhibited higher α diversity. However, no significant difference was observed in ß diversity. The results of the microbiome composition indicate that Firmicutes, Proteobacteria, and Bacteroidota are the core phyla in all groups. Compared to younger elderly individuals, Akkermansia and Lactobacillus are significantly enriched in the long-lived elderly group, while Megamonas is significantly reduced. In addition, a high abundance of Akkermansia is a significant characteristic of elderly populations with low BMI values. Furthermore, the functional prediction results showed that the elderly longevity group had higher abilities in short-chain fatty acid metabolism, amino acid metabolism, and xenobiotic biodegradation. Taken together, our study provides characteristic information on GM in the long-lived elderly population in Changshou Town. This study can serve as a valuable addition to the current research on age-related GM. KEY POINTS: • The gut microbiota of elderly individuals with longevity and low BMI exhibit higher alpha diversity • Gut microbiota diversity did not differ significantly between genders in the elderly population • Several potentially beneficial bacteria (e.g., Akkermansia and Lactobacillus) are enriched in long-lived individuals.

Analysis of differentially expressed lncRNAs and mRNAs associated with slow­transit constipation.

Slow transit constipation (STC) is a refractory gastrointestinal disease, accounting for approximately 13 âˆ¼ 37 % of chronic constipation. However, the molecular mechanism of STC remains poorly understood. Herein, this study aims to identify the key mRNAs and lncRNAs associated with STC. To this end, we performed high-throughput RNA sequencing to identify differentially expressed (DE) mRNAs and lncRNAs in the whole-layer sigmoid intestinal tissues from 4 STC patients and 4 non-STC patients. The identified DE lncRNAs and mRNAs were validated through quantitative real-time PCR. Weighted gene co-expression network analysis (WGCNA) and Pearson correlation analysis were conducted to determine the significantly correlated DE mRNA-lncRNA pairs. A total of 1420 DE lncRNAs and 1634 DE mRNAs were identified. Kyoto Encyclopedia of Genes and Genomes analysis of DE mRNAs indicated that these DE mRNAs might be associated with systemic lupus erythematosus, alcoholism, intestinal immune network for IgA production, inflammatory bowel disease, NF-kappa B signaling pathway. WGCNA and Pearson correlation analyses jointly identified 16,577 significantly correlated DE mRNA-lncRNA pairs. Furthermore, lncRNAs LINC00641, LINC02268, LINC03013 were identified as hub lncRNAs. The protein-protein interaction (PPI) network of proteins encoded by DE mRNAs was established, and PPI-based analysis revealed that Interleukin 2(IL2), CD80 molecule (CD80), interleukin-17A (IL-17A) might play significant roles in the development of STC. This study analyzes the expression profiles of lncRNAs and mRNAs associated with STC. Our findings will contribute to further understanding of the molecular mechanism of STC and provide potential diagnostic or therapeutic biomarkers for STC.

Paeonol promotes longevity and fitness in Caenorhabditis elegans through activating the DAF-16/FOXO and SKN-1/Nrf2 transcription factors.

Paeonol, as one of the most abundant plant-derived polyphenols, has multiple bioactivities including anti-inflammatory, anti-tumor, and anti-cardiovascular diseases. Nevertheless, the anti-aging effects and related mechanisms of paeonol are rarely reported. In this study, we found that paeonol significantly prolonged the mean lifespan of Caenorhabditis elegans (C. elegans) by 28.49% at a dose of 200 µM. Moreover, paeonol promoted the health of C. elegans by increasing the body bending and pharyngeal pumping rates and reducing the lipofuscin accumulation level. Meanwhile, paeonol induced the expression of stress-related genes or proteins by activating the transcription factors DAF-16/FOXO, SKN-1/Nrf2, and HSF-1, which in turn enhanced oxidative and thermal stress tolerance. The mechanism behind the anti-aging effect of paeonol occurred by down-regulating the insulin/IGF-1 signaling (IIS) pathway. Our findings shed new light on the application of paeonol for longevity promotion and human health.

Integrated analysis of genome-wide association studies and 3D epigenomic characteristics reveal the BMP2 gene regulating loin muscle depth in Yorkshire pigs.

BACKGROUND: The lack of integrated analysis of genome-wide association studies (GWAS) and 3D epigenomics restricts a deep understanding of the genetic mechanisms of meat-related traits. With the application of techniques as ChIP-seq and Hi-C, the annotations of cis-regulatory elements in the pig genome have been established, which offers a new opportunity to elucidate the genetic mechanisms and identify major genetic variants and candidate genes that are significantly associated with important economic traits. Among these traits, loin muscle depth (LMD) is an important one as it impacts the lean meat content. In this study, we integrated cis-regulatory elements and genome-wide association studies (GWAS) to identify candidate genes and genetic variants regulating LMD. RESULTS: Five single nucleotide polymorphisms (SNPs) located on porcine chromosome 17 were significantly associated with LMD in Yorkshire pigs. A 10 kb quantitative trait locus (QTL) was identified as a candidate functional genomic region through the integration of linkage disequilibrium and linkage analysis (LDLA) and high-throughput chromosome conformation capture (Hi-C) analysis. The BMP2 gene was identified as a candidate gene for LMD based on the integrated results of GWAS, Hi-C meta-analysis, and cis-regulatory element data. The identified QTL region was further verified through target region sequencing. Furthermore, through using dual-luciferase assays and electrophoretic mobility shift assays (EMSA), two SNPs, including SNP rs321846600, located in the enhancer region, and SNP rs1111440035, located in the promoter region, were identified as candidate SNPs that may be functionally related to the LMD. CONCLUSIONS: Based on the results of GWAS, Hi-C, and cis-regulatory elements, the BMP2 gene was identified as an important candidate gene regulating variation in LMD. The SNPs rs321846600 and rs1111440035 were identified as candidate SNPs that are functionally related to the LMD of Yorkshire pigs. Our results shed light on the advantages of integrating GWAS with 3D epigenomics in identifying candidate genes for quantitative traits. This study is a pioneering work for the identification of candidate genes and related genetic variants regulating one key production trait (LMD) in pigs by integrating genome-wide association studies and 3D epigenomics.

Role of macrophage polarisation in skin wound healing.

Skin wound healing is a complex pathophysiological change that is driven by macrophages and their secreted related factors. Depending on the stimuli, macrophages can be polarised into two subtypes of macrophages with completely different phenotypes and functions, namely M1 and M2. The aim of this study was to explore the role of M1 and M2 macrophages in skin healing in order to develop new drugs for the treatment of refractory wounds. Primary bone marrow-derived macrophages (BMDMs) were isolated from rats and expanded in vitro using macrophage colony stimulating factor. In addition, the BMDMs were polarised into the M1 and M2 subtypes using lipopolysaccharides (LPS) and interleukin-4 (IL-4), respectively. Cytokine levels in the culture supernatants were measured by an enzyme linked immunosorbent assay. Epidermal wounds were made on the dorsal surface of rats, and treated with M1 or M2 cell suspensions or phosphate buffered saline. Wound healing was recorded on days 1, 3, 7, 10, and 14 after stamping, and the wound healing rate was measured by haematoxylin-eosin and Masson staining. A total of 3 to 4 × 107 bone marrow cells were extracted from each rat femur. The BMDM culture had 87.1% CD45+ cells, 89.2% CD68+ cells, and 86.5% CD45+ CD68+ cells. Furthermore, IL-12 (P < .05) and IL-10 (P ≥ .05) levels, respectively, increased and decreased in the culture supernatants of the M1 cells after LPS stimulation compared with those in the M0 (unstimulated) group. Likewise, IL-4 stimulation led to a significant increase in IL-10 levels (P < .01) in the conditioned media of M2 cells, while that of IL-12 decreased slightly (P ≥ .05). In the rat model, the infusion of M2 cells accelerated wound healing and tissue regeneration, whereas the M1 cells delayed the recruitment of inflammatory cells, granulation growth, and collagen deposition, which impaired wound healing. Macrophage polarisation and activation are critical for skin wound healing. While exogenous M1 cell infusion delayed wound healing, the M2 cells promoted wound healing in a rat model.

Identification of Differentially Expressed miRNAs in Porcine Adipose Tissues and Evaluation of Their Effects on Feed Efficiency.

Feed efficiency (FE) is a very important trait affecting the economic benefits of pig breeding enterprises. Adipose tissue can modulate a variety of processes such as feed intake, energy metabolism and systemic physiological processes. However, the mechanism by which microRNAs (miRNAs) in adipose tissues regulate FE remains largely unknown. Therefore, this study aimed to screen potential miRNAs related to FE through miRNA sequencing. The miRNA profiles in porcine adipose tissues were obtained and 14 miRNAs were identified differentially expressed in adipose tissues of pigs with extreme differences in FE, of which 9 were down-regulated and 5 were up-regulated. GO and KEGG analyses indicated that these miRNAs were significantly related to lipid metabolism and these miRNAs modulated FE by regulating lipid metabolism. Subsequently, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) of five randomly selected DEMs was used to verify the reliability of miRNA-seq data. Furthermore, 39 differentially expressed target genes of these DEMs were obtained, and DEMs-target mRNA interaction networks were constructed. In addition, the most significantly down-regulated miRNAs, ssc-miR-122-5p and ssc-miR-192, might be the key miRNAs for FE. Our results reveal the mechanism by which adipose miRNAs regulate feed efficiency in pigs. This study provides a theoretical basis for the further study of swine feed efficiency improvement.

A histone deacetylase inhibitor enhances rice immunity by derepressing the expression of defense-related genes.

Histone deacetylase (HDAC) inhibitors (HDACis) have been widely used in plants to investigate the role of histone acetylation, particularly the function of HDACs, in the regulation of development and stress response. However, how histone acetylation is involved in rice (Oryza sativa L.) disease resistance has hardly been studied. In this paper, four HDACis including Sodium butyrate (NaBT), Suberoylanilide Hydroxamic Acid (SAHA), LBH-589 and Trichostatin A (TSA) were used to treat rice seedlings at different concentrations before inoculation of Magnaporthe oryzae. We found that only 10mM NaBT treatment can significantly enhanced rice blast resistance. However, treatment of the four HDACis all increased global histone acetylation but at different sites, suggesting that the inhibition selectivity of these HDACis is different. Notably, the global H3K9ac level was dramatically elevated after both NaBT and LBH589 treatment although LBH589 could not enhance rice blast resistance. This indicates that the HDACs they inhibit target different genes. In accordance with the phenotype, transcriptomic analysis showed that many defense-related genes were up-regulated by NaBT treatment. Up-regulation of the four genes bsr-d1, PR10B, OsNAC4, OsKS4 were confirmed by RT-qPCR. ChIP-qPCR results revealed that H3K9ac level on these genes was increased after NaBT treatment, suggesting that these defense-related genes were repressed by HDACs. In addition, by promoter motif analysis of the genes that induced by both NaBT treatment and rice blast infection, we found that the motifs bound by ERF and AHL transcription factors (TFs) were the most abundant, which demonstrates that ERF and AHL proteins may act as the candidate TFs that recruit HDACs to defense-related genes to repress their expression when plants are not infected by rice blast.

Differences in liver microRNA profiling in pigs with low and high feed efficiency.

Feed cost is the main factor affecting the economic benefits of pig industry. Improving the feed efficiency (FE) can reduce the feed cost and improve the economic benefits of pig breeding enterprises. Liver is a complex metabolic organ which affects the distribution of nutrients and regulates the efficiency of energy conversion from nutrients to muscle or fat, thereby affecting feed efficiency. MicroRNAs (miRNAs) are small non-coding RNAs that can regulate feed efficiency through the modulation of gene expression at the post-transcriptional level. In this study, we analyzed miRNA profiling of liver tissues in High-FE and Low-FE pigs for the purpose of identifying key miRNAs related to feed efficiency. A total 212~221 annotated porcine miRNAs and 136~281 novel miRNAs were identified in the pig liver. Among them, 188 annotated miRNAs were co-expressed in High-FE and Low-FE pigs. The 14 miRNAs were significantly differentially expressed (DE) in the livers of high-FE pigs and low-FE pigs, of which 5 were downregulated and 9 were upregulated. Kyoto Encyclopedia of Genes and Genomes analysis of liver DE miRNAs in high-FE pigs and low-FE pigs indicated that the target genes of DE miRNAs were significantly enriched in insulin signaling pathway, Gonadotropin-releasing hormone signaling pathway, and mammalian target of rapamycin signaling pathway. To verify the reliability of sequencing results, 5 DE miRNAs were randomly selected for quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The qRT-PCR results of miRNAs were confirmed to be consistent with sequencing data. DE miRNA data indicated that liver-specific miRNAs synergistically acted with mRNAs to improve feed efficiency. The liver miRNAs expression analysis revealed the metabolic pathways by which the liver miRNAs regulate pig feed efficiency.

Silencing of Nesprin-2 inhibits the differentiation of myofibroblasts from fibroblasts induced by mechanical stretch.

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.

Manipulating electrostatic field to control the distribution of bioactive proteins or polymeric microparticles on planar surfaces for guiding cell migration.

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.

Transcriptomic and Epigenomic Assessment Reveals Epigenetic Regulation of WRKY Genes in Response to Magnaporthe oryzae Infection in Rice.

Background: Histone acetylations acting as active hallmarks for gene transcription is involved in regulating numerous developmental and stress-responsive gene expression. Methods: The data from chromatin immunoprecipitation sequencing (ChIP-seq) was performed by using histone H3 lysine 9 acetylation (H3K9ac) antibody, and RNA sequencing (RNA-seq) utilizing rice seedlings inoculated by Magnaporthe oryzae (M. oryzae) were integrated. Results: RNA-seq data revealed that 422, 460 and 466 genes were up-regulated at 12h, 24h and 48h after inoculation. ChIP-seq data showed that 60%-80% of blast up-regulated genes at different time points were marked with H3K9ac, which was prone to be enriched in both TSS and gene body region. However, the H3K9ac level at a rather small proportion of the up-regulated genes was elevated after M. oryzae inoculation. We found that seven WRKY genes induced by rice blast fungus harbor H3K9ac. For different WRKY genes, blast fungus induction led to the increase of H3K9ac in distinct regions, including promoter, TSS or gene body, indicating that histone acetylation may play diverse roles in the activation of defense-related genes. By searching DNA-binding motifs of transcription factors in the promoter of genes with increased H3K9ac after M. oryzae infection, we found that ERF family protein-binding motifs were enriched with high -log P-value (>20), including ERF1, DEAR3, DREB2C, RAP2.6, RRTF1_3ARY, all of which contain GCC-box (GCCGCC). Conclusion: In this study, we revealed that the vast majority of genes induced by fungus M. oryzae were marked with H3K9ac preferring both TSS and gene body regions. However, H3K9ac enrichment was increased, responding to M. oryzae inoculation only at a low proportion of these genes, including several WRKY genes. Besides, for different genes, the increment of H3K9ac occurred in different regions. Finally, ERF proteins that have been proved to bind GCC-box might be one of the potential transcription factors for recruiting histone acetyltransferases to deposit histone acetylation at defense-related genes in rice.

Harnessing biocompatible nanofibers and silver nanoparticles for wound healing: Sandwich wound dressing versus commercial silver sulfadiazine dressing.

Owing to the structural replication of native extracellular matrix, nonwoven mats of electrospun nanofibers have great potential for use in wound healing. Herein, we report the design and fabrication of a sandwich wound dressing to balance its antimicrobial activity and biocompatibility. This success mainly relies on the incorporation of silver nanoparticles (AgNPs) into electrospun nanofibers, together with the rational design of a sandwich structure for the dressing. The bottom layer was composed of hydrophilic nanofibers made from a blend of polycaprolactone (PCL) and gelatin (Gel). The top layer consisted of hydrophobic PCL nanofibers. AgNP-loaded PCL/Gel nanofibers were sandwiched between the two layers. When compared with a commercial silver sulfadiazine dressing, the designed wound dressing showed competitive antimicrobial properties, lower cell toxicity, and accelerated wound closure for mouse skin injury. By balancing the biocompatibility of electrospun nanofibers and the broad-spectrum antibacterial activity of AgNPs within a sandwich structure, the novel multifunctional wound dressing could be valuable for effective wound healing and related applications.

Identification of new semen trait-related candidate genes in Duroc boars through genome-wide association and weighted gene co-expression network analyses.

Semen traits are crucial in commercial pig production since semen from boars is widely used in artificial insemination for both purebred and crossbred pig production. Revealing the genetic architecture of semen traits potentially promotes the efficiencies of improving semen traits through artificial selection. This study is aimed to identify candidate genes related to the semen traits in Duroc boars. First, we identified the genes that were significantly associated with three semen traits, including sperm motility (MO), sperm concentration (CON), and semen volume (VOL) in a Duroc boar population through a genome-wide association study (GWAS). Second, we performed a weighted gene co-expression network analysis (WGCNA). A total of 2, 3, and 20 single-nucleotide polymorphisms were found to be significantly associated with MO, CON, and VOL, respectively. Based on the haplotype block analysis, we identified one genetic region associated with MO, which explained 6.15% of the genetic trait variance. ENSSSCG00000018823 located within this region was considered as the candidate gene for regulating MO. Another genetic region explaining 1.95% of CON genetic variance was identified, and, in this region, B9D2, PAFAH1B3, TMEM145, and CIC were detected as the CON-related candidate genes. Two genetic regions that accounted for 2.23% and 2.48% of VOL genetic variance were identified, and, in these two regions, WWC2, CDKN2AIP, ING2, TRAPPC11, STOX2, and PELO were identified as VOL-related candidate genes. WGCNA analysis showed that, among these candidate genes, B9D2, TMEM145, WWC2, CDKN2AIP, TRAPPC11, and PELO were located within the most significant module eigengenes, confirming these candidate genes' role in regulating semen traits in Duroc boars. The identification of these candidate genes can help to better understand the genetic architecture of semen traits in boars. Our findings can be applied for semen traits improvement in Duroc boars.

Genome-wide association and transcriptome studies identify candidate genes and pathways for feed conversion ratio in pigs.

BACKGROUND: The feed conversion ratio (FCR) is an important productive trait that greatly affects profits in the pig industry. Elucidating the genetic mechanisms underpinning FCR may promote more efficient improvement of FCR through artificial selection. In this study, we integrated a genome-wide association study (GWAS) with transcriptome analyses of different tissues in Yorkshire pigs (YY) with the aim of identifying key genes and signalling pathways associated with FCR. RESULTS: A total of 61 significant single nucleotide polymorphisms (SNPs) were detected by GWAS in YY. All of these SNPs were located on porcine chromosome (SSC) 5, and the covered region was considered a quantitative trait locus (QTL) region for FCR. Some genes distributed around these significant SNPs were considered as candidates for regulating FCR, including TPH2, FAR2, IRAK3, YARS2, GRIP1, FRS2, CNOT2 and TRHDE. According to transcriptome analyses in the hypothalamus, TPH2 exhibits the potential to regulate intestinal motility through serotonergic synapse and oxytocin signalling pathways. In addition, GRIP1 may be involved in glutamatergic and GABAergic signalling pathways, which regulate FCR by affecting appetite in pigs. Moreover, GRIP1, FRS2, CNOT2, and TRHDE may regulate metabolism in various tissues through a thyroid hormone signalling pathway. CONCLUSIONS: Based on the results from GWAS and transcriptome analyses, the TPH2, GRIP1, FRS2, TRHDE, and CNOT2 genes were considered candidate genes for regulating FCR in Yorkshire pigs. These findings improve the understanding of the genetic mechanisms of FCR and may help optimize the design of breeding schemes.

HOXC10 promotes growth and migration of melanoma by regulating Slug to activate the YAP/TAZ signaling pathway.

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.

Comparison of gene disruption induced by cytosine base editing-mediated iSTOP with CRISPR/Cas9-mediated frameshift.

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.

Integrated Analysis of mRNA Expression, CpG Island Methylation, and Polymorphisms in the MITF Gene in Ducks (Anas platyrhynchos).

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.

Candidate Gene Identification of Feed Efficiency and Coat Color Traits in a C57BL/6J × Kunming F2 Mice Population Using Genome-Wide Association Study.

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.