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Long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) are important classes of small noncoding RNAs that can regulate numerous biological processes. To understand the role of message RNA (mRNAs, lncRNAs and circRNAs) in the regulation of intramuscular fat (IMF) deposition, in this study the expression profiles of longissimus dorsi (LD) muscle from six Laiwu pigs (three with extremely high and three with extremely low IMF content) were sequenced based on rRNA-depleted library construction. In total, 323 differentially expressed protein-coding genes (DEGs), 180 lncRNAs (DELs) and 105 circRNAs (DECs) were detected between the high IMF and low IMF groups. Functional analysis indicated that most DEGs, and some target genes of DELs, were enriched into GO terms and pathways related to adipogenesis, suggesting their important roles in regulating IMF deposition. In addition, 12 DELs were observed to exhibit a positive relationship with stearoyl-CoA desaturase (SCD), phosphoenolpyruvate carboxykinase 1 (PCK1), and adiponectin (ADIPOQ), suggesting they are highly likely to be the target genes of DELs. Finally, we constructed a source gene-circRNA-miRNA connective network, and some of miRNA of the network have been reported to affect lipid metabolism or adipogenesis. Overall, this work provides a valuable resource for further research and helps to understand the potential functions of lncRNAs and circRNAs in IMF deposition.

Transient receptor potential channel M2 (TRPM2) is a Ca2+-permeable channel that is activated by reactive oxygen species (ROS). In many cell types, ROS activate TRPM2 to induce excessive Ca2+ influx, resulting in Ca2+ overload and consequent cell death. Recent studies suggest that TRPM2 may also regulate autophagy in pericytes and cancer cells by acting on the early step of autophagy, i.e. autophagic induction. However, there is no report on the role of TRPM2 in autophagic degradation, which is the late stage of autophagy. In the present study, we found abundant TRPM2 expression in lysosomes/autolysosomes in the primary cultured mouse aortic smooth muscle cells (mASMCs). Nutrient starvation stimulated autophagic flux in mASMCs mainly by promoting autophagic degradation. This starvation-induced autophagic degradation was reduced by TRPM2 knockout. Importantly, starvation-induced lysosomal/autolysosomal acidification and cell death were also substantially reduced by TRPM2 knockout. Taken together, the present study uncovered a novel mechanism that lysosomal TRPM2 facilitates lysosomal acidification to stimulate excessive autolysosome degradation and consequent cell death.

Atherosclerosis is a chronic inflammatory arterial disease characterized by build-up of atheromatous plaque, which narrows the lumen of arteries. Hypercholesterolemia and excessive oxidative stress in arterial walls are among the main causative factors of atherosclerosis. Transient receptor potential channel M2 (TRPM2) is a Ca2+-permeable cation channel activated by oxidative stress. However, the role of TRPM2 in atherosclerosis in animal models is not well studied. In the present study, with the use of adeno-associated virus (AAV)-PCSK9 and TRPM2 knockout (TRPM2−/−) mice, we determined the role of TRPM2 in hypercholesterolemia-induced atherosclerosis. Our results demonstrated that TRPM2 knockout reduced atherosclerotic plaque area in analysis of En face Oil Red O staining of both whole aortas and aortic-root thin sections. Furthermore, TRPM2 knockout reduced the expression of CD68, α-SMA, and PCNA in the plaque region, suggesting a role of TRPM2 in promoting macrophage infiltration and smooth-muscle cell migration into the lesion area. Moreover, TRPM2 knockout reduced the expression of ICAM-1, MCP-1, and TNFα and decreased the ROS level in the plaque region, suggesting a role of TRPM2 in enhancing monocyte adhesion and promoting vascular inflammation. In bone-marrow-derived macrophages and primary cultured arterial endothelial cells, TRPM2 knockout reduced the production of inflammatory cytokines/factors and decreased ROS production. In addition, a TRPM2 antagonist N-(p-amylcinnamoyl) anthranilic acid (ACA) was able to inhibit atherosclerotic development in an ApoE−/− mouse model of atherosclerosis. Taken together, the findings of our study demonstrated that TRPM2 contributes to the progression of hypercholesterolemia-induced atherosclerosis. Mechanistically, TRPM2 channels may provide an essential link that can connect ROS to Ca2+ and inflammation, consequently promoting atherosclerotic progression.

In the present work, a new kind of porous polyvinyl alcohol/sodium alginate/hydroxyapatite (PVA/SA/HA) composite hydrogels with tunable structure and mechanical properties have been fabricated by dual-crosslinking method. The morphologies, moisture content, porosity and mechanical properties of the composite hydrogels have been investigated in detail. The PVA/SA/HA hydrogels present uniform, interpenetrating porous structure. The FTIR and XRD results indicate that PVA, SA and HA could be uniformly compounded. The mechanical properties, moisture content and porosity of the samples could be controlled by changing the mass ratio of PVA/SA/HA. The optimized compression modulus (41.74 ± 7.86 kPa), moisture content (86.99 ± 0.72%) and porosity (79.98 ± 1.61%) of the composite hydrogels could be achieved

Muscle growth and fat deposition are the two important biological processes in the development of pigs which are closely related to the pig production performance. Long intergenic noncoding RNAs (lincRNAs), with lack of coding potential and the length of at least 200nt, have been extensively studied to play important roles in many biological processes. However, the importance and molecular regulation mechanism of lincRNAs in the process of muscle growth and fat deposition in pigs are still to be further studied comprehensively. In our study, we used the data, including liver, abdominal fat, and longissimus dorsi muscle of 240 days’ age of two F2 full-sib female individuals from the white Duroc and Erhualian crossbreed, to identify 581 putative lincRNAs associated with pig muscle growth and fat deposition. The 581 putative lincRNAs shared many common features with other mammalian lincRNAs, such as fewer exons, lower expression levels, and shorter transcript lengths. Cross-tissue comparisons showed that many transcripts were tissue-specific and were involved in the important biological processes in their corresponding tissues. Gene ontology and pathway analysis revealed that many potential target genes (PTGs) of putative lincRNAs were involved in pig muscle growth and fat deposition-related processes, including muscle cell proliferation, lipid metabolism, and fatty acid degradation. In Quantitative Trait Locus (QTLs) analysis, some PTGs were screened from putative lincRNAs, MRPL12 is associated with muscle growth, GCGR and SLC25A10 were associated with fat deposition, and PPP3CA, DPYD, and FGGY were related not only to muscle growth but also to fat deposition. Therefore, it implied that these lincRNAs might participate in the biological processes related to muscle growth or fat deposition through homeostatic regulation of PTGs, but the detailed molecular regulatory mechanisms still needed to be further explored. This study lays the molecular foundation for the in-depth study of the role of lincRNAs in the pig muscle growth and fat deposition and further provides the new molecular markers for understanding the complex biological mechanisms of pig muscle growth and fat deposition.

Aims Acute aortic dissection (AAD) is a life-threatening disease with high morbidity and mortality. Previous studies have showed that vascular smooth muscle cell (VSMC) phenotype switching modulates vascular function and AAD progression. However, whether an endogenous signaling system that protects AAD progression exists, remains unknown. Our aim is to investigate the role of Anxa1 in VSMC phenotype switching and the pathogenesis of AAD. Methods and results We first assessed Anxa1 expression levels by immunohistochemical staining in control aorta and AAD tissue from mice. A strong increase of Anxa1 expression was seen in the mouse AAD tissues. In line with these findings, micro-CT scan results indicated that Anxa1 plays a role in the development of AAD in our murine model, with systemic deficiency of Anxa1 markedly progressing AAD. Conversely, administration of Anxa1 mimetic peptide, Ac2-26, rescued the AAD phenotype in Anxa1-/- mice. Transcriptomic studies revealed a novel role for Anxa1 in VSMC phenotype switching, with Anxa1 deficiency triggering the synthetic phenotype of VSMCs via down-regulation of the JunB/MYL9 pathway. The resultant VSMC synthetic phenotype rendered elevated inflammation and enhanced matrix metalloproteinases (MMPs) production, leading to augmented elastin degradation. VSMC-restricted deficiency of Anxa1 in mice phenocopied VSMC phenotype switching and the consequent exacerbation of AAD. Finally, our studies in human AAD aortic specimens recapitulated key findings in murine AAD, specifically that the decrease of Anxa1 is associated with VSMC phenotype switch, heightened inflammation, and enhanced MMP production in human aortas. Conclusions Our findings demonstrated that Anxa1 is a novel endogenous defender that prevents acute aortic dissection by inhibiting vascular smooth muscle cell phenotype switching, suggesting that Anxa1 signaling may be a potential target for AAD pharmacological therapy. Translational perspective Our studies herein may lead to a paradigm shift for pharmacologic therapy towards acute aortic dissection. Through careful examination of the pathological changes that occur during AAD onset in experimental animal models, we demonstrated that VSMC phenotype switching plays a critical role in the development of AAD. Inhibition of VSMC phenotype switching and its attendant impacts on aortic function may be a viable approach for future treatment. Toward that end, our studies highlighted the protective benefit of Anxa1 and its mimetic peptide Ac2-26 in AAD through prevention of the switching of VSMC to a synthetic phenotype.

Aim: This study aims to couple DNA methylation changes and evolution of retrogenes. Materials & methods: A new two-step strategy was developed to screen retrogenes. Further, reduced representation bisulfite sequencing and RNA-seq data of eight tissues were used to analyze retrogenes. Results: A total of 964 retrocopies were identified and new retrocopies were available for the synthesis of glycans and lipids corresponding to pig phenotypic traits. Retrogenes were consistently hypermethylated. Hypomethylation of parental genes presented more susceptibility to retroposition. Promoter DNA methylation of retrogenes was negatively correlated with evolutionary time and played important roles in regulating retrogene tissue-specific expression pattern. Conclusion: A two-step procedure is effective and necessary for identifying retrogenes. DNA methylation drives origination, survival, evolution and expression of retrogenes.

Long non-coding RNAs (lncRNAs) have been implicated in fundamental and diverse biological processes, including myogenesis. However, the molecular mechanisms involved in this process remain largely unexplored. This study found that H19 affected the differentiation of porcine satellite cells (PSCs) by directly binding to the DNA/RNA-binding protein TDP43. Functional analyses showed that TDP43 knockdown decreased PSC differentiation, whereas TDP43 overexpression exerted opposite effects in vitro. Furthermore, rescue experiments demonstrated that TDP43 can rescue the decrease in PSC differentiation caused by H19 knockdown. Mechanistically, H19 may act as a scaffold to recruit TDP43 to the promoters of MYOD and thereby activate the transcription of MYOD, leading to PSC differentiation. In summary, we elucidate the molecular mechanism by which H19 and TDP43 regulate myogenesis.

Aims Eva-1 homologue 1 (Eva1a) is a novel protein involved in the regulation of cardiac remodelling and plaque stability, but little is known about its role in re-endothelialization and the development of atherosclerosis (AS). Thus, in the present study, we aimed to elucidate the function of Eva1a in re-endothelialization and AS. Methods and results Wire injuries of carotid and femoral arteries were established in Eva1a−/− mice. Eva1a-deficient mice were crossed with apolipoprotein E−/− (ApoE−/−) mice to evaluate AS development and re-endothelialization of carotid artery injuries. Denudation of the carotid artery at 3, 5, and 7 days was significantly aggravated in Eva1a−/− mice. The neointima of the femoral artery at 14 and 28 days was consequently exacerbated in Eva1a−/− mice. The area of atherosclerotic lesions was increased in Eva1a−/−ApoE−/− mice. To explore the underlying mechanisms, we performed transwell, scratch migration, cell counting kit-8, and bromodeoxyuridine assays using cultured human aorta endothelial cells (HAECs), which demonstrated that EVA1A promoted HAEC migration and proliferation. Proteomics revealed that the level of actin-related protein 2/3 complex subunit 1B (Arpc1b) was decreased, while Eva1a expression was absent. Arpc1b was found to be a downstream molecule of EVA1A by small interfering RNA transfection assay. Activation of Rac1 and Cdc42 GTPases was also regulated by EVA1A. Conclusion This study provides insights into anti-atherogenesis effects of Eva1a by promoting endothelium repair. Thus, Eva1a is a promising therapeutic target for AS.

Long intergenic non-coding RNAs (lincRNAs) play essential roles in numerous biological processes and are widely studied. The skeletal muscle is an important tissue that plays an essential role in individual movement ability. However, lincRNAs in pig skeletal muscles are largely undiscovered and their biological functions remain elusive. In this study, we assembled transcriptomes using RNA-seq data published in previous studies of our laboratory group and identified 323 lincRNAs in porcine leg muscle. We found that these lincRNAs have shorter transcript length, fewer exons and lower expression level than protein-coding genes. Gene ontology and pathway analyses indicated that many potential target genes (PTGs) of lincRNAs were involved in skeletal-muscle-related processes, such as muscle contraction and muscle system process. Combined our previous studies, we found a potential regulatory mechanism in which the promoter methylation of lincRNAs can negatively regulate lincRNA expression and then positively regulate PTG expression, which can finally result in abnormal phenotypes of cloned piglets through a certain unknown pathway. This work detailed a number of lincRNAs and their target genes involved in skeletal muscle growth and development and can facilitate future studies on their roles in skeletal muscle growth and development.

>Emerging evidence indicates that microRNA (miRNA) plays an important role in adipogenesis and disease pathogenesis. To investigate the miRNA involved in regulating different periods of adipogenesis, we performed a comprehensive study on microRNAome during the stimulation of adipogenesis by an adipogenic differentiation cocktail in C2C12 myoblasts at 0, 2, 4, and 7 days using the Solexa sequencing technology. In this study, we identified 52 differentially expressed (DE) miRNA. Functional annotation indicated that the target genes of DE miRNA were mostly enriched in adipogenic transdifferentiation and fat metabolism-related pathways, including Wnt, mitogen-activated protein kinase (MAPK), and insulin signaling. The insulin signaling pathway was further analyzed for its close connections to Wnt and MAPK signaling pathways as well as for the containing of thymoma viral proto-oncogene-3 (Akt3) and glycogen synthase kinase-3 beta (Gsk3b), which were both target genes predicted by most DE miRNA. CLIP-seq (crosslinking-immunprecipitation and high-throughput sequencing) data showed that Akt3 was targeted by seven DE miRNA, including five upregulated (miR-203-3p, miR-181c-5p, miR-322-5p, miR-351-5p, and miR-181a-5p) and two downregulated (miR-15b-5p and miR-17-5p) ones. Likewise, Gsk3b was targeted by six DE miRNA, including four upregulated (miR-199a-3p, miR-126-5p, miR-26a-5p, and miR-101a-3p) and two downregulated (miR-150-5p and miR-140-3p) ones. Moreover, Akt3 could regulate the key transcription factor (TF) Foxo1, targeted by two downregulated miRNA (miR-96-5p and miR-183-5p). The expression levels of Akt3 and Gsk3b were downregulated, and TF Foxo1, which worked on the transcription axis of Pgc1a-Pparα-Rxrg-Pparγ to regulate adipogenesis, was upregulated. In conclusion, DE miRNA stimulated the adipogenesis of C2C12 myoblasts through the targeted insulin signaling pathway involving the genes Akt3, Gsk3b, and TF Foxo1.

α1-Antitrypsin (AAT) is widely used to treat patients with congenital AAT deficiency. Cohn Fraction IV (Cohn F IV) is normally discarded during the manufacturing process of albumin but contains approximately 33% of plasma AAT. We established a new process for large-scale purification of AAT from it. liquid chromatography-electrospray ionization-tandem mass spectrometry and high-performance liquid chromatography were applied for qualitative identification and composition analysis, respectively. Stabilizers were optimized for AAT activity protection during lyophilization and dry-heat. Virus inactivation by dry-heat and solvent/detergent (S/D) was validated on a range of viruses. AAT with purity of 95.54%, specific activity of 3,938.5 IU/mg, and yield of 26.79%, was achieved. More than 95% activity was reserved after S/D. More than 96% activity was obtained after lyophilization or dry-heat. After S/D, pseudorabies virus (PRV) and vesicular stomatitis virus (VSV) were inactivated below detectable level within 1 H. Virus titer reductions of more than 5.50 log10 and 5.38 log10 were achieved for PRV and VSV, respectively. Porcine parvovirus and encephalomyocarditis virus were inactivated by 3.17 log10 and 5.88 log10 reduction after dry-heat. The advantages of this process, including suitability for large-scale production, high purity, better utilization of human plasma, viral safety, commercial and inexpensive chromatography medium, may facilitate its further application.

As a widely used nanomaterial in daily life, silver nanomaterials may cause great concern to female reproductive system as they are found to penetrate the blood-placental barrier and gain access to the ovary. However, it is largely unknown about how silver nanomaterials influence ovarian physiology and functions such as hormone production. This study performs in vitro toxicology study of silver nanomaterials, focusing especially on cytotoxicity and steroidogenesis and explores their underlying mechanisms. This study exposes primary rat granulosa cells to gold nanorod core/silver shell nanostructures (Au@Ag NRs), and compares outcomes with cells exposed to gold nanorods. The Au@Ag NRs generate more reactive oxygen species and reduce mitochondrial membrane potential and less production of adenosine triphosphate. Au@Ag NRs promote steroidogenesis, including progesterone and estradiol, in a time- and dose-dependent manner. Chemical reactivity and transformation of Au@Ag NRs are then studied by electron spin resonance spectroscopy and X-ray absorption near edge structure, which analyze the generation of free radical and intracellular silver species. Results suggest that both particle-specific activity and intracellular silver ion release of Au@Ag NR contribute to the toxic response of granulosa cells.

A water-soluble polysaccharide, FI, extracted from the mycelium of Granoderma tsugae, was fractionated and purified by ion-exchange chromatography, gel filtration, and affinity chromatography. Sixteen polysaccharides obtained were examined for antitumor effects on Sarcoma 180 in mice. The three active polysaccharides obtained were as follows: FIo-a: A glycan-protein complex containing 9.3% protein, and having a hetero-glyco-chain of mannose and xylose. FIo-b-alpha: Molecular weight 10,000, glucan-protein complex containing 25.8% protein. The inhibition ratio was 61.8% against the solid cancer Sarcoma 180/mice; the survival ratio was more than 194% of the control group (100). FA-1-b-alpha: Molecular weight 16,000, a complex of glycan:protein = 42:58 w/w, consisting of glucose as a main component, and associated with arabinose, mannose, xylose, and galactose. This had a tumor inhibition ratio of 56% and a survival ratio of more than 182%. Comparison of active glycan with the fruiting body and mycelium: Among water-soluble polysaccharides of fruiting body, FIo-a and FA-1, with antitumor activity, were both glucogalactan-protein complexes of molecular weight 10,000, but that of mycelium was a homoglucan protein complex in FIo-b-alpha and heteroglucan protein in both FA-1-a and FA-1-b-alpha. The heteroglucan had a low tumor inhibition ratio, but caused a high survival ratio in mice.

A systematic method of extraction, fractionation, and purification of polysaccharides from Songshan Lingzhi (Ganoderma tsugae) with antitumor activity was established. Seven glycans with strong antitumor activities were obtained from 14 water-soluble, and 15 water-insoluble fractions: FIo-a, FA-1, FII-1, FIII-2, and FIII-2-a, -b, and -c. FIo-a and FA-1 were protein-containing glucogalactans associated with mannose and fucose. FII-1 was a (1--3)-beta-D-glucan having a lower protein content. The water-insoluble fractions FIII-2-a, -b, and -c were extracted with alkali, and were found to be protein-containing (1--3)-beta-D-glucans showing the strongest activity. Chemical properties and structure of each antitumor polysaccharide were compared with three fungi of the Ganoderma family, Kofukitake (G. applanatum), Mannentake (G. lucidum), and Songshan Lingzhi (G. tsugae).

Protein-containing polysaccharides extracted from fruiting bodies of a Chinese fungus named Feng Wei Gu, were fractionated and purified, and their antitumor activities were tested, out of which the following active fractions were obtained. FIo-a: A protein-containing xyloglucan, MW 280,000, polysaccharide: protein = 76:24 (w/w), polysaccharide consisting of Man:Gal:Xyl:Glc = 2:12:42:42 (molar ratio). [alpha]D23 + 25.3 degrees. FA-2: A protein-containing mannogalactan, MW 120,000, polysaccharide: protein = 76:16 (w/w), consisting of Xyl:Man:Gal = 9:35:56 (molar ratio), [alpha]D23 + 98.5 degrees. FII-1: A Protein-containing xylan (62:21 w/w). MW 200,000, [alpha]D23 + 8.7 degrees. FIII-1a: A protein-containing glucoxylan (15:71 w/w), [alpha]D23 + 30.7 degrees, MW 90,000, consisting of Glc:Xyl = 40:44 (molar ratio). FIII-2a: A protein-containing xyloglucan, MW 70,000, polysaccharide:protein = 69:3 (w/w), polysaccharide consisting of Xyl:Glc = 36:62 (molar ratio). [alpha]D23 + 38.6 degrees.

Novel diterpenoids, erinacines H (1) and I (3), were isolated from the cultured mycelia of Hericium erinaceum. The structures of the compounds were determined by interpretation of the spectral data. Erinacine H showed stimulating activity of nerve growth factor (NGF)-synthesis.

Two novel eudesmane-type sesquiterpenes, dictyophorines A and B, and a known compound, teucrenone, were isolated from the mushroom Dictyophora indusiata. Dictyophorines A and B promoted nerve growth factor (NGF)-synthesis by astroglial cells.

A water-soluble polysaccharide, FI, and water-insoluble polysaccharides, FII and FIII were extracted from Pleurotus citrinopileatus mushrooms. After fractionation, the antitumor activity of the fractions against Sarcoma 180 implanted in mice were examined, and the following active polysaccharides were obtained: Water-soluble polysaccharides FIo: A heteropolysaccharide containing 9.8% protein, and composed of glucose, mannose, arabinose, and galactose. FA-2-b-beta: A glycoprotein consisting of glycan:protein = 40:60 w/w, with the glyco-chain composed of glucose, xylose, mannose, galactose, and fucose. FA-3: A glycoprotein consisting of glycan:protein = 50:50 w/w, and glycan moiety consisting of glucose, galactose, xylose, mannose, and fucose. Water-insoluble polysaccharides. FIII-1: Protein-containing beta-D-glucans, FIII-1-a, and -b were obtained from FIII-1 by gel filtration, composed of glucan:protein = 80:20, and 68:32 w/w, respectively. The glucan moieties of both were almost all (1--3)-beta-D-glucan, and their molecular weights were 68 x 10(4) and 40 x 10(4). FIII-2: Protein-containing beta-D-glucans, FIII-2-a, and -b, were obtained from FIII-2 by gel filtration, with molecular weights 190 x 10(4) and 120 x 10(4), respectively. Both were almost all composed of glucan:protein = 87:13 w/w. Both glucan moieties were mainly (1--3)-beta-D-glucan.