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The processes of muscle growth and development, including myoblast proliferation, migration, differentiation, and fusion, are modified by a variety of regulatory factors. MYL4 plays an important role in atrial development, atrial cardiomyopathy, muscle-fiber size, and muscle development. The structural variation (SV) of MYL4 was found via the de novo sequencing of Ningxiang pigs, and the existence of SV was verified in the experiments. The genotype distribution of Ningxiang pigs and Large White pigs was detected, and it was found that Ningxiang pigs were mainly of the BB genotype and that Large White pigs were mainly of the AB genotype. However, the molecular mechanisms behind the MYL4-mediated regulation of skeletal muscle development need to be deeply explored. Therefore, RT-qPCR, 3′RACE, CCK8, EdU, Western blot, immunofluorescence, flow cytometry, and bioinformation analysis were used to explore the function of MYL4 in myoblast development. The cDNA of MYL4 was successfully cloned from Ningxiang pigs, and its physicochemical properties were predicted. The expression profiles in six tissues and four stages of Ningxiang pigs and Large White pigs were found to be the highest in the lungs and 30 days after birth. The expression of MYL4 increased gradually with the extension of the myogenic differentiation time. The myoblast function test showed that the overexpression of MYL4 inhibited proliferation and promoted apoptosis and differentiation. The knockdown of MYL4 showed the opposite result. These results enhance our understanding of the molecular mechanisms of muscle development and provide a solid theoretical foundation for further exploring the role of the MYL4 gene in muscle development.

Adipose tissue composition contributes greatly to the quality and nutritional value of meat. Transcriptomic and lipidomic techniques were used to investigate the molecular mechanism of fat deposition difference among Ningxiang, Berkshire and F1 pigs. Transcriptomic analysis identified 680, 592 and 380 differentially expressed genes (DEGs) by comparing the groups of Ningxiang pigs vs Berkshire pigs, Berkshire pigs vs F1 pigs, Ningxiang pigs vs F1 pigs. Lipidomic analysis screened 423, 252, and 50 significantly changed lipids (SCLs) by comparing the groups of Ningxiang pigs vs Berkshire pigs, Berkshire pigs vs F1 pigs, Ningxiang pigs vs F1 pigs. Lycine, serine and threonine metabolism pathway, fatty acid biosynthesis and metabolism related pathways were siginificant enriched in the groups of Berkshire pigs vs Ningxiang pigs and Berkshire pigs vs F1 pigs. The DEGs (PHGDH, LOC110256000) and the SCLs (Phosphatidylserines) may have a great impact on lycine, serine and threonine metabolism pathway. Moreover, the DEGs (FASN, ACACA, CBR4, SCD, ELOV6, HACD, CYP3A46, CYP2B22, GPX1, GPX3) and the SCLs (palmitoleic acid, linoleic acid, arachidonic acid, icosadienoic acid) play important role in fatty acid biosynthesis and metabolism. Thus, the fat deposition difference among Ningxiang, Berkshire and F1 pigs may be caused by the difference of the expression pattern of key genes in multiple enrichment KEGG pathways. The research presented multiple lipids that were potentially available biological indicator and screened key genes that were potentially gene targets for molecular design breeding. The research also explored the molecular mechanisms of fat deposition difference among Ningxiang, Berkshire and F1 pigs and provides insight into the selection of backfat thickness and fat composition of adipose tissue for future breeding strategies.

With the increasing popularity of genomic sequencing, breeders pay more attention to identifying the crucial molecular markers and quantitative trait loci for improving the body size and reproduction traits that could affect the production efficiency of pig-breeding enterprises. Nevertheless, for the Shaziling pig, a well-known indigenous breed in China, the relationship between phenotypes and their corresponding genetic architecture remains largely unknown. Herein, in the Shaziling population, a total of 190 samples were genotyped using the Geneseek Porcine 50K SNP Chip, obtaining 41857 SNPs for further analysis. For phenotypes, two body measurement traits and four reproduction traits in the first parity from the 190 Shaziling sows were measured and recorded, respectively. Subsequently, a genome-wide association study (GWAS) between the SNPs and the six phenotypes was performed. The correlation between body size and reproduction phenotypes was not statistically significant. A total of 31 SNPs were found to be associated with body length (BL), chest circumference (CC), number of healthy births (NHB), and number of stillborns (NSB). Gene annotation for those candidate SNPs identified 18 functional genes, such as GLP1R, NFYA, NANOG, COX7A2, BMPR1B, FOXP1, SLC29A1, CNTNAP4, and KIT, which exert important roles in skeletal morphogenesis, chondrogenesis, obesity, and embryonic and fetal development. These findings are helpful to better understand the genetic mechanism for body size and reproduction phenotypes, while the phenotype-associated SNPs could be used as the molecular markers for the pig breeding programs.

TRAPPC9 deficiency is an autosomal recessive disorder mainly associated with intellectual disability (ID), microcephaly, and obesity. Previously, TRAPPC9 deficiency has not been associated with biochemical abnormalities.Exome sequencing was performed in 3 individuals with ID and dysmorphic features. N-Glycosylation analyses were performed in the patients' blood samples to test for possible congenital disorder of glycosylation (CDG). TRAPPC9 gene, TRAPPC9 protein expression, and N-glycosylation markers were assessed in patient fibroblasts. Complementation with wild-type TRAPPC9 and immunofluorescence studies to assess TRAPPC9 expression and localization were performed. The metabolic consequences of TRAPPC9 deficiency were evaluated using tracer metabolomics.All 3 patients carried biallelic missense variants in TRAPPC9 and presented with an N-glycosylation defect in blood, consistent with CDG type I. Extensive investigations in patient fibroblasts corroborated TRAPPC9 deficiency and an N-glycosylation defect. Tracer metabolomics revealed global metabolic changes with several affected glycosylation-related metabolites.We identified 3 TRAPPC9 deficient patients presenting with ID, dysmorphic features, and abnormal glycosylation. On the basis of our findings, we propose that TRAPPC9 deficiency could lead to a CDG (TRAPPC9-CDG). The finding of abnormal glycosylation in these patients is highly relevant for diagnosis, further elucidation of the pathophysiology, and management of the disease.

RNA editing is an essential post-transcriptional regulatory mechanism. However, few studies have investigated the functional RNA edits in the economic traits of livestock on a genome-wide scale. Pigs are one of the most important livestock species and their fat is the principal organ involved in the regulation of adipose deposition. Here, we used three full-sibling pairs, with each pair comprising a pig with higher backfat (BF) thickness and lower backfat thickness, to identify RNA editing events based on whole-genome and transcriptome sequencing data. A total of 60,903 non-redundant RNA editing sites with 59,472 (97.7%) A-to-G edits were detected using a revised bioinformatics pipeline. A specific sequence context with G preference was found one base downstream of the edited site, and the editing level was associated with the distribution of nucleotides across nearly sites. Moreover, the A-to-G editing sites mostly occurred in the pig-special short interspersed nuclear elements, Pre0_SS. Comparing the difference between pigs with higher BF and lower BF, we found 211 differentially edited sites (DESites). Functional enrichment analyses revealed a significant enrichment of genes containing DESites in terms of adipose deposition. The DESites located in the six adipose-related genes (SKP1, GSK3B, COL5A3, MDM4, NT5C2, and DENND2A) were selected as candidate RNA editing sites associated with adipose deposition, and thus require further evaluation. This study mined the potentially functional RNA editing sites in pig adipose tissue and indicated that RNA editing may play an important role in adipose deposition, which provides a new insight into the post-transcriptionally mediated regulation mechanism of fat development.

The genomic breed composition (GBC) reflects the genetic relationship between individual animal and ancestor breeds in composite or hybrid breeds. Also, it can estimate the genomic contribution of each breed (ancestor) to the genome of each individual animal. Using genomic SNP information to estimate Ningxiang pig GBC is of great significance. First of all, GBC was widely used in cattle and had significant effects, but there is almost no using experience in Chinese endemic pig breeds. Importantly, High-density SNPs are expensive but can be economized by deploying a relatively small number of highly informative SNP scattered evenly across the genome. Moreover, the impact of low-density SNPs selection strategy on estimating the GBC of individual animals has not been fully explained. Using SNP data from different databases and organizations, we established reference (N = 2015) and verification (N = 302) data sets. Twelve successively smaller SNP panels (500, 1K, 5K, 10K) were built from those SNP in the reference data by three selection methods (uniform, maximized the Euclidean distance (MED) and random distribution method). For each panel, the GBC of Ningxiang pigs in the reference dataset was estimated. Then combining Shannon entropy and the GBC results, the optimal panel (the 10K SNP panel constructed by MED method) was picked out to estimate the GBC of verification Ningxiang pig, which detected that 230 individuals were purebred Ningxiang pigs and the remaining 72 impure individuals contained 6.44% blood related with Rongchang pigs and 4.09% with Bamaxiang pigs in the verification Ningxiang population. Finally, the genetic structure analysis of verification population was performed combining with the results of GBC, multi-dimensional scaling (MDS) analysis and hierarchical cluster analysis. These results showed: (a) GBC could accurately identify purebred Ningxiang pigs and, scientifically, calculate the genomic contribution of each breed of each hybrid animal. (b) GBC could carry out population genetic structure and understand the genetic background of Ningxiang pigs. Such findings highlight a variety of opportunities to better protect and identify other endangered local breeds in China facing the same situation as Ningxiang pig and provide more accurate, economical and efficient new technical support in GBC estimation breeding work.

Background. Danlou tablet (DLT), the traditional Chinese medicine has been commonly used for dyslipidemia, atherosclerosis, and coronary heart disease. Whether it was effective against vascular injury caused by CIH has remained unknown. The aim of the current study was to observe the effects of DLT on chronic intermittent hypoxia (CIH)-induced vascular injury via regulation of blood lipids and to explore potential mechanisms. Methods. Sixteen 12-week-old male ApoE−/− mice were randomly divided into four groups. The sham group was exposed to normal room air, whereas the other three groups were exposed to CIH. Mice in the CIH + normal saline (NS) group were gavaged with NS. Mice in the CIH + Angptl4-ab group were intraperitoneally injected with Angptl4-antibody. Mice in the CIH + DLT group were gavaged with DLT. After four weeks of intervention, serum lipid concentrations, and serum lipoprotein lipase (LPL) activity were detected. The changes in atherosclerosis in vascular tissue were detected by hematoxylin and eosin (H&E) staining. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analysis were applied to detect the expression levels of hypoxia-induciblefactor-1 (HIF-1), factor-inhibiting HIF-1 (FIH-1), angiopoietin-like 4 (Angptl4), and LPL in different tissues. Results. CIH exposure increases serum lipid levels, decreases serum LPL activity, and exacerbates atherosclerosis. Both Angptl4-ab and DLT treatment reversed the changes in lipid concentration, LPL activity, and atherosclerosis caused by CIH. In the epididymal fat pad, CIH exposure decreased the expression of FIH-1 and increased the expression of HIF-1, whereas DLT treatment increased the expression of FIH-1 and LPL and inhibited the expression of HIF-1 and Angptl4. In heart tissue, the expression levels of LPL and Angptl4 were not affected by modeling or treatment. Conclusions. DLT improved vascular damage by improving the increase in blood lipids induced by CIH, potentially by upregulating FIH-1 and downregulating HIF-1 and Angptl4 in adipose tissue. Therefore, DLT may be a promising agent for the prevention and treatment of CIH-induced vascular injury.

Pancreatic ductal adenocarcinoma (PDAC) is notorious for high mortality due to limited options of appropriate chemotherapy drugs. Here we report that Aurora kinase-A expression is elevated in both human and mouse PDAC samples. MLN8237, an inhibitor of Aurora kinase-A, efficiently reduced the proliferation and motility of PDAC cells in vitro as well as tumor growth in orthotropic xenograft model and genetic pancreatic cancer animal models (p53/LSL/Pdx-Cre mice) in vivo. MLN8237 exhibited tumor inhibitory effect through inhibiting proliferation and migration, and inducing apoptosis and senescence. These results provide the molecular basis for a novel chemotherapy strategy for PDAC patients.

To detect whether Danlou Tablet (丹蒌片, DLT) regulates the hypoxia-induced factor (HIF)-1 α-angiopoietin-like 4 (Angptl4) mRNA signaling pathway and explore the role of DLT in treating chronic intermittent hypoxia (CIH)-induced dyslipidemia and arteriosclerosis. The mature adipocytes were obtained from 3T3-L1 cell culturation and allocated into 8 groups including control groups (Groups 1 and 5, 0.1 mL of cell culture grade water); DLT groups (Groups 2 and 6, 0.1 mL of 1,000 µg/mL DLT submicron powder solution); dimethyloxalylglycine (DMOG) groups (Groups 3 and 7, DMOG and 0.1 mL of cell culture grade water); DMOG plus DLT groups (Groups 4 and 8, DMOG and 0.1 mL of 1,000 µg/mL DLT submicron powder solution). Groups 1–4 used mature adipocytes and groups 5–8 used HIF-1 α-siRNA lentivirus-transfected mature adipocytes. After 24-h treatment, real-time polymerase chain reaction and Western blot were employed to determine the mRNA and protein expression levels of HIF-1 α and Angptl4. In animal experiments, the CIH model in ApoE−/− mice was established. Sixteen mice were complete randomly divided into 4 groups including sham group, CIH model group [intermittent hypoxia and normal saline (2 mL/time) gavage once a day]; Angptl4 Ab group [intermittent hypoxia and Angptl4 antibody (30 mg/kg) intraperitoneally injected every week]; DLT group [intermittent hypoxia and DLT (250 mg/kg) once a day], 4 mice in each group. After 4-week treatment, enzyme linked immunosorbent assay was used to detect the expression levels of serum total cholesterol (TC) and triglyceride (TG). Hematoxylin-eosin and CD68 staining were used to observe the morphological properties of arterial plaques. Angptl4 expression was dependent on HIF-1 α, with a reduction in mRNA expression and no response in protein level to DMOG or DLT treatment in relation to siHIF-1 α -transfected cells. DLT inhibited HIF-1 α and Angptl4 mRNA expression (P

Residual feed intake (RFI) is defined as the difference between the observed and expected feed intake for maintenance and growth requirements. In this study, the expression profiles of mRNAs and long noncoding RNAs (lncRNAs) from skeletal muscle in Duroc pigs with divergent RFI phenotypes were investigated by Illumina sequencing. Finally, a total of 2195 annotated lncRNAs and 1976 novel lncRNAs were obtained. About 210 mRNAs and 43 lncRNAs were differentially expressed among high and low RFI pigs. The differentially expressed mRNAs were potentially involved in the biological processes of lipid metabolism, extracellular matrix organization, cell proliferation, and cell adhesion. The lipolysis in skeletal muscle was increased in high RFI pigs, suggesting that high RFI pigs might need more energy than low RFI pigs. However, skeletal muscle development was increased in low RFI pigs. These results suggested that low RFI pigs might be more efficient in energy utilization during skeletal muscle growth. The function of lncRNA was also analyzed by target prediction. Nine lncRNAs might be candidate lncRNAs for the determination of RFI phenotype, by the regulation of the biological processes of lipid metabolism, cell proliferation, and cell adhesion. This study should facilitate a further understanding of the molecular mechanism for the determination of RFI phenotype in pigs.

Ningxiang pigs, a fat-type pig, are native to Ningxiang County in Hunan Province, with thousands of years of breeding history. This study aims to explore the expression profiles and functional networks on messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) in the liver. Liver tissue of Ningxiang piglets was collected at 30, 90, 150, and 210 days after birth (four development stages), and the mRNA and lncRNA expression was profiled. Compared to mRNA and lncRNA expression profiles, most differentially expressed mRNAs (DEmRNAs) were upregulated at 30 days, however, most DElncRNAs were downregulated at 210 days. Via Short Time-series Expression Miner (STEM) analysis and weighted gene co-expression network analysis (WGCNA), a complex interaction between mRNAs and lncRNAs was identified, indicating that lncRNAs may be a critical regulatory element for mRNAs. One module of genes in particular (module profile 4) was related to fibril organization, vasculogenesis, GTPase activator activity, and regulation of kinase activity. The mRNAs and lncRNAs in module profile 4 had a similar pattern of expression, indicating that they have functional and regulatory relationships. Only CAV1, PACSIN2, and CDC42 in the particular mRNA profile 4 were the target genes of lncRNAs in that profile, which shows the possible regulatory relationship between lncRNAs and mRNAs. The expression of these genes and lncRNAs in profile 4 was the highest at 30 days, and it is believed that these RNAs may play a critical role during the suckling period in order to meet the dietary requirements of piglets. In the lncRNA–mRNA co-expression network, the identified gene hubs and associated lncRNAs were shown to be involved in saccharide, lipid, and glucose metabolism, which may play an important role in the development and health of the liver. This result will lead to further investigation of liver lncRNA functions at various stages of development in Ningxiang pigs.

Pathogenic alterations in the DPM2 gene have been previously described in patients with hypotonia, progressive muscle weakness, absent psychomotor development, intractable seizures, and early death. We identified biallelic DPM2 variants in a 23-year-old male with truncal hypotonia, hypertonicity, congenital heart defects, intellectual disability, and generalized muscle wasting. His clinical presentation was much less severe than that of the three previously described patients. This is the second report on this ultra-rare disorder. Here we review the characteristics of previously reported individuals with a defect in the DPM complex while expanding the clinical phenotype of DPM2-Congenital Disorders of Glycosylation. In addition, we offer further insights into the pathomechanism of DPM2-CDG disorder by introducing glycomics and lipidomics analysis. ispartof: MOLECULAR GENETICS AND METABOLISM vol:132 issue:1 pages:27-37 ispartof: location:United States status: published

Background In recent years, thousands of different circular RNAs (circRNAs) have been identified through comparative analysis of different pig breeds. However, very few studies have investigated the spatiotemporal expression patterns of circRNA during organ development, which is crucial for functional and evolutionary analysis. Results In this study, we systematically analyzed circRNAs associated with fatty acid metabolism in the three main organs (muscle, fat and liver) a tfour growth time points (30 d, 90 d, 150 d and 210 d after birth) for Ningxiang pigs, awell-known native pig breed in China known for its excellent meat quality. We identified 61,683 circRNAs and analyzed their molecular characteristics, potential functions, and interactions with miRNAs. The circRNAs exhibited notable spatiotemporal specificity in the form of dynamic expression. At 90 d, the number and complexity of differentially expressed circRNAs in the liver was the highest.We used STEM (Short Time-series Expression Miner) to perform time series analysis on differentially expressed circRNAs and observed that there were multiple model spectra that were significantly enriched with time changes in all three organs. All the significantly changed circRNAs, miRNAs and mRNAs obtained from STEM analysis of each tissue were utilized to confirm that the circRNA and mRNA have a targeting relationship with miRNA. Notably, we screened 2384 (fat), 2672 (muscle) and 9187 (liver) circRNA-miRNA-mRNA network pairs related to developmental time changes. Finally, we used fluorescence in situ hybridization (FISH) to demonstrate the dynamic subcellular localization of circRNA during development. Conclusion These data suggest that circRNA is highly abundant in muscle, fat and liver and is dynamically expressed in a spatiotemporal manner. These results suggest that circRNAs play important roles in fat metabolism in Ningxiang pigs. Our findings also provide new ideas and perspectives for the study of Ningxiang pigs' lipid metabolism regulation pathway and meat quality traits.

Background: In recent years, thousands of different circular RNA (circRNAs) have been discovered by comparative analysis of different pig breeds. However, very few studies have been conducted on the spatiotemporal expression pattern of circRNA during organ development which is crucial for functional and evolutionary analysis.Results: Here, we systematically analyzed circRNAs associated with fatty acid metabolism in the three main organs (muscle, fat and liver) for four growth time points (30d, 90d, 150d and 210d after birth) of Ningxiang pigs, a famous native pig breeds in China, known for its excellent meat quality. We identified 61,683 circRNAs and analyzed their molecular characteristics, potential functions, and interaction with miRNA. The circRNAs showed striking spatiotemporal specificity in the form of dynamic expression. At 90d, the number and complexity of differentially expressed circRNAs in the liver is the most obvious. We used STEM (Short Time Series Expression Miner) to perform time series analysis on differentially expressed circRNAs, and found that there are multiple model spectra that are significantly enriched with time changes in all the three organs. All the significantly changed circRNAs, miRNAs and mRNAs obtained from STEM analysis of each tissue were used to confirm that the circRNA and mRNA have a targeting relationship with miRNA. It is worth noting that we screened out 2384 (fat), 2672 (muscle) and 9187 (liver) circRNAs-miRNA-mRNA network pairs related to developmental time changes. Finally, we used fluorescence in situ hybridization（FISH）to show the dynamic subcellular localization of circRNA during development.Conclusion: To a certain extent, these data indicate that circRNA is highly abundant in muscle, fat and liver, and is dynamically expressed in a spatiotemporal manner. These results suggest that circRNAs play an important role in fat metabolism of Ningxiang pigs. Our findings also provide new ideas and perspectives for the study of Ningxiang pigs' lipid metabolism regulation pathway and meat quality traits.

Vascular dysfunction and insulin resistance (IR) are associated with obstructive sleep apnea (OSA), which is characterized by frequent episodes of nocturnal intermittent hypoxia (IH). While it is recognized that the balance between vasoconstrictive (endothelin-1) and vasodilatory molecules (nitric oxide, NO) determine vascular profile, molecular mechanisms contributing to vascular dysfunction and IR in OSA are not completely understood. Caveolin-1 is a membrane protein which regulates endothelial nitric oxide synthase (eNOS) activity which is responsible for NO generation and cellular insulin-signaling. Hence, we examined the effects of IH on caveolin-1, eNOS, and endothelin-1 in human coronary artery endothelial cells in the context of IR. Chronic 3-day IH exposure up-regulated caveolin-1 and endothelin-1 expression while reducing NO. Also, IH altered insulin-mediated activation of AKT but not ERK resulting in increased endothelin-1 transcription. Similarly, caveolin-1 overexpression attenuated basal and insulin-stimulated NO synthesis along with impaired insulin-dependent activation of AKT and eNOS, with no effect on insulin-stimulated ERK1/2 phosphorylation and endothelin-1 transcription. Our data suggest that IH contributes to a vasoconstrictive profile and to pathway-selective vascular IR, whereby insulin potentiates ET-1 expression. Moreover, IH may partly mediate its effects on NO and insulin-signaling via upregulating caveolin-1 expression.

Currently, an effective targeted therapy for pancreatitis is lacking. Hereditary pancreatitis (HP) is a heritable, autosomal-dominant disorder with recurrent acute pancreatitis (AP) progressing to chronic pancreatitis (CP) and a markedly increased risk of pancreatic cancer. In 1996, mutations in PRSS1 were linked to the development of HP. Here, we developed a mouse model by inserting a full-length human PRSS1(R122H) gene, the most commonly mutated gene in human HP, into mice. Expression of PRSS1(R122H) protein in the pancreas markedly increased stress signaling pathways and exacerbated AP. After the attack of AP, all PRSS1(R122H) mice had disease progression to CP, with similar histologic features as those observed in human HP. By comparing PRSS1(R122H) mice with PRSS1(WT) mice, as well as enzymatically inactivated Dead-PRSS1(R122H) mice, we unraveled that increased trypsin activity is the mechanism for R122H mutation to sensitize mice to the development of pancreatitis. We further discovered that trypsin inhibition, in combination with anticoagulation therapy, synergistically prevented progression to CP in PRSS1(R122H) mice. These animal models help us better understand the complex nature of this disease and provide powerful tools for developing and testing novel therapeutics for human pancreatitis.

Intra-acinar trypsinogen activation occurs in the earliest stages of pancreatitis and is believed to play important roles in pancreatitis pathogenesis. However, the exact role of intra-acinar trypsin activity in pancreatitis remains elusive. Here, we aimed to examine the specific effects of intra-acinar trypsin activity on the development of pancreatitis using a transgenic mouse model. This transgenic mouse model allowed for the conditional expression of a mutant trypsinogen that can be activated specifically inside pancreatic acinar cells. We found that expression of this active mutated trypsin had no significant effect on triggering spontaneous pancreatitis. Instead, several protective compensatory mechanisms, including SPINK1 and heat shock proteins, were upregulated. Notably, these transgenic mice developed much more severe acute pancreatitis, compared with control mice, when challenged with caerulein. Elevated tissue edema, serum amylase, inflammatory cell infiltration and acinar cell apoptosis were dramatically associated with increased trypsin activity. Furthermore, chronic pathological changes were observed in the pancreas of all transgenic mice, including inflammatory cell infiltration, parenchymal atrophy and cell loss, fibrosis, and fatty replacement. These changes were not observed in control mice treated with caerulein. The alterations in pancreata from transgenic mice mimicked the histological changes common to human chronic pancreatitis. Taken together, we provided in vivo evidence that increased intra-acinar activation of trypsinogen plays an important role in the initiation and progression of both acute and chronic pancreatitis. NEW & NOTEWORTHY Trypsinogen is activated early in pancreatitis. However, the roles of trypsin in the development of pancreatitis have not been fully addressed. Using a genetic approach, we showed trypsin activity is critical for the severity of both acute and chronic pancreatitis.

Obstructive sleep apnea (OSA) is a common sleep disorder associated with obesity. Emerging evidence suggest that OSA increases the risk of cardiovascular morbidity and mortality partly via accelerating the process of cellular aging. Thus, we sought to examine the effects of intermittent hypoxia (IH), a hallmark of OSA, on senescence in human white preadipocytes. We demonstrate that chronic IH is associated with an increased generation of mitochondrial reactive oxygen species along with increased prevalence of cells with nuclear localization of γH2AX & p16. A higher prevalence of cells positive for senescence-associated β-galactosidase activity was also evident with chronic IH exposure. Intervention with aspirin, atorvastatin or renin-angiotensin system (RAS) inhibitors effectively attenuated IH-mediated senescence-like phenotype. Importantly, the validity of in vitro findings was confirmed by examination of the subcutaneous abdominal adipose tissue which showed that OSA patients had a significantly higher percentage of cells with nuclear localization of γH2AX & p16 than non-OSA individuals (20.1 ± 10.8% vs. 10.3 ± 2.7%, Padjusted

Figure S1. Overview of the experimental design. Figure S2. Results of the Sanger sequencing validation of all 64 selected editing sites. For each candidate editing site (indicated by genome coordinates and red arrow), raw chromatograms of sequences derived from cDNA and matched DNA samples are shown. Unverified sites are marked with an asterisk. Figure S3. Distribution of RNA editing types in repetitive sequences. Figure S4. Distribution of A-to-G sites across major repeat families. Figure S5. Overlap of RNA editing sites among the three samples of each tissue. Figure S6. Dot plot of the top five enriched GO biological process terms in each tissue. Dot color indicates statistical significance of the enrichment (q-value); dot size represents the fraction of genes annotated to each term. Figure S7. Dot plot of the enriched KEGG pathways after removing the tissue-specific expressed genes. Dot color indicates the statistical significance of the enrichment (q-value); dot size represents the fraction of genes annotated to each term. Figure S8. RNA editing vs expression of ADARs in all samples. a) We correlated the number of A-to-G sites and ADAR1 expression levels in all samples (n = 21). b) We correlated the number of A-to-G sites and ADAR2 expression levels in all samples (n = 21). c) and d) We calculated the correlations between the overall RNA editing levels and the expression values of ADAR1 and ADAR2 in all samples (n = 21). Correlation coefficients (r) and P-values are shown in each graph. Figure S9. RNA editing vs the expression of ADARs in tissue groups. a) We correlated the number of A-to-G sites and ADAR1 expression levels in all tissue groups (n = 7). b) We correlated the number of A-to-G sites and ADAR2 expression levels in all tissue groups (n = 7). c) and d) We calculated the correlations between the overall RNA editing levels and the expression values of ADAR1 and ADAR2 in tissue groups (n = 7). Correlation coefficients (r) and P-values are displayed in each graph. (PDF 87057 kb)

Ear size exhibits remarkable diversity in pig breeds. LEM domain-containing 3 ( LEMD3 ) on chromosome 5 is considered as an important candidate for porcine ear size. This is the first study on cloning and characterization of LEMD3 cDNA. The complete cDNA contains 4 843 bp, including a 2 736-bp open reading frame (ORF), a 37-bp 5′-untranslated region (UTR) and a 2 070-bp 3′-UTR. The complete LEMD3 gene is 126 241-bp and contains 13 exons and 12 introns. The ORF encodes a deduced LEMD3 protein of 911 amino acids, which shares 82–94% nucleic acid and 51–96% amino acid identity with other species. A phylogenetic tree constructed based on the amino acid sequences revealed that the porcine LEMD3 protein was closely related with cattle LEMD3. Resequencing of the ORF and promoter of LEMD3 from Minzhu pig and Large White revealed three single nucleotide polymorphisms (SNPs): L964C>A in the complete coding region, L4625A>G in the 3’ UTR, and L-394T>C in the promoter region. Genome-wide association study (GWAS) revealed that all of SNPs were shown significant association with ear size in Large White×Minzhu pig intercross population. With conditional GWAS, –log 10 (P-value) decreased by more than 80% when each of three SNPs was included as a fixed effect. These results suggested direct involvement of LEMD3 or close linkage to the causative mutation for ear size. The findings of this study might form the basis for understanding the genetic mechanism of ear size variation in pigs and provide potential molecular markers for screening ear size diversity in pig breeds.

Litter uniformity, which is usually represented by within-litter weight coefficient of variation at birth (CVB), could influence litter performance of sows and the profitability of pig enterprises. The objective of this study was to characterize CVB and its effect on other reproductive traits in Large White sows. Genetic parameters and genetic correlation of the reproductive traits, including CVB, within-litter weight coefficient of variation at three weeks (CVT), total number born (TNB), number born alive (NBA), number born dead (NBD), gestation length (GL), piglet mortality at birth (M0), piglet mortality at three weeks (M3), total litter weight at birth (TLW0), and total litter weight at three weeks (TLW3) were estimated for 2 032 Large White litters. The effects of parity and classified litter size on CVB, CVT, TNB, NBA, NBD, GL, M0, M3, TLW0, and TLW3 were also estimated. The heritabilities of these reproductive traits ranged from 0.06 to 0.17, with the lowest heritability for CVB and the highest heritability for TLW0. Phenotypic and genetic correlations between these reproductive traits were low to highly positive and negative (ranging from −0.03 to 0.93, and −0.53 to 0.93, respectively). The genetic correlations between TNB and CVB, and between M0 and CVB were 0.32 and 0.29, respectively. In addition, CVB was significantly influenced by parity and litter size class (P

Body fat distribution contributes to obesity-related metabolic and cardiovascular disorders. Visceral fat is more detrimental than subcutaneous fat. However, the mechanisms underlying visceral fat-mediated cardiometabolic dysregulation are not completely understood. Localized increases in expression of the renin angiotensin system (RAS) in adipose tissue (AT) may be implicated. We therefore investigated mRNA and protein expression of RAS components in visceral versus subcutaneous AT using paired samples from individuals undergoing surgery (N = 20, body mass index: 45.6 ± 6.2 kg/m2, and age: 44.6 ± 9.1 years). We also examined RAS-related proteins in AT obtained from individuals on renin angiotensin aldosterone system (RAAS) targeted drugs (N = 10, body mass index: 47.2 ± 9.3 kg/m2, and age: 53.3 ± 10.1 years). Comparison of protein expression between subcutaneous and visceral AT samples showed an increase in renin (p = 0.004) and no change in angiotensinogen (p = 0.987) expression in visceral AT. Among proteins involved in angiotensin peptide generation, angiotensin converting enzyme (p = 0.02) was increased in subcutaneous AT while chymase (p = 0.001) and angiotensin converting enzyme-2 (p = 0.001) were elevated in visceral fat. Furthermore, visceral fat expression of angiotensin II type-2 receptor (p = 0.007) and angiotensin II type-1 receptor (p = 0.031) was higher, and MAS receptor (p < 0.001) was lower. Phosphorylated-p53 (p = 0.147), AT fibrosis (p = 0.138) and average adipocyte size (p = 0.846) were similar in the two depots. Nonetheless, visceral AT showed increased mRNA expression of inflammatory (TNFα, p < 0.001; IL-6, p = 0.001) and oxidative stress markers (NOX2, p = 0.038; NOX4, p < 0.001). Of note, mRNA and protein expression of RAS components did not differ between subjects taking or not taking RAAS related drugs. In summary, several RAS related proteins are differentially expressed in subcutaneous versus visceral AT. This differential expression may not alter AngII but likely increases Ang1-7 generation in visceral fat. These potential differences in active angiotensin peptides and receptor expression in the two depots suggest that localized RAS may not be involved in differences in visceral vs subcutaneous AT function in obese individuals. Our findings do not support a role for localized RAS differences in visceral fat-mediated development of cardiovascular and metabolic pathology.

Dimethylarginine dimethylaminohydrolases (DDAHs) are known to degrade asymmetric dimethylarginine, an endogenous inhibitor of NOS, and maintain vascular homeostasis; however, the regulatory pathways of DDAHs remain unclear. In this study, we aimed to define the role of transmembrane glycoprotein neuropilin-1 (NRP1) in the expression of DDAHs and investigate the potential roles of NRP1 in regulation of blood pressure. Short hairpin RNA-mediated knockdown of NRP1 reduced the level and mRNA stability of DDAH1 but not DDAH2 in HUVECs, whereas overexpression of NRP1 increased the mRNA stability of DDAH1. Meanwhile, mesenteric arteries and lung vascular endothelial cells of tamoxifen-inducible endothelial cell-specific NRP1 knockout mice exhibited decreased expression of DDAH1 and slightly increased expression of DDAH2. Mechanistically, the regulation of NRP1 on DDAH1 expression is mediated by a posttranscriptional mechanism involving miR-219-5p in HUVECs. Although the endothelial cell-specific NRP1 knockout mice did not exhibit any significant change in blood pressure at the basal level, they were more sensitive to low-dose angiotensin II infusion-induced increases in blood pressure. Our results show that NRP1 is required for full expression of DDAH1 in endothelial cells and that NRP1 contributes to protection from low-dose angiotensin II-induced increases in blood pressure.-Wang, Y., Wang, E., Zhang, Y., Madamsetty, V. S., Ji, B., Radisky, D. C., Grande, J. P., Misra, S., Mukhopadhyay, D. Neuropilin-1 maintains dimethylarginine dimethylaminohydrolase 1 expression in endothelial cells, and contributes to protection from angiotensin II-induced hypertension.

Background: Leptin contributes to obesity-related cardiometabolic pathology. While the role of adipose tissue dysfunction in altering metabolic profile in obesity is recognized, the direct effects of leptin signaling in adipose tissue are not completely understood. Among factors impacting adipose tissue function in obesity, adipocyte death is associated with increased inflammation with consequent adipose tissue insulin-resistance. Furthermore, apoptosis of adipocyte progenitor cells (preadipocytes) is important as it lowers adipose tissue capacity to maintain and expand fat storage. Objective: To determine the effects of leptin on regulation of proteins important to apoptotic pathways in cultured human white preadipocytes (HWP) and differentiated HWP (dHWP). Methods: Cells were exposed to increasing concentrations of leptin to evaluate the role of leptin on regulation of anti-apoptotic Bcl2, and pro-apoptotic Bax and CD95 expression. Next, we examined if pre-exposure to leptin attenuated TNF-α and FasL induced apoptosis. Results: Leptin upregulated transcription of Bcl2 (p=0.02) and down-regulated transcription of Bax (p=0.02) and CD95 (p=0.04) in a leptin-concentration dependent manner in dHWP. Leptin dependent increases in Bcl2 (p=0.004) and decreases in Bax (p=0.003) and CD95 (P=0.03) protein expression were also observed. Furthermore, 24 hour pre-exposure to leptin protected cells from TNF-α (p=0.01; p=0.006) and FasL (p=0.03; p=0.007) induced apoptosis in dHWP and HWP respectively. Additionally, leptin attenuated TNF-mediated activation of the p53 pathway, suggesting another mechanism by which leptin mediates its inhibitory effects on TNF-induced apoptosis. Conclusion: Leptin regulates the expression of proteins involved in the apoptotic pathway such that it decreases the apoptotic potential of adipose tissue by increasing anti-apoptotic Bcl2 and decreasing pro-apoptotic Bax and CD95 protein expression. In contrast to our in-vitro findings, prior studies in adipose tissue from obese humans report decreased Bcl2 and increased Bax protein expression, along with high leptin expression. This suggests that increased adipose tissue apoptosis in obesity may at least in part be secondary to altered leptin signaling.

Statin use is associated with increased calorie intake and consequent weight gain. It is speculated that statin‐dependent improvements in lipid profile may undermine the perceived need to follow lipid‐lowering and other dietary recommendations leading consequently to increased calorie intake. However, increases in calorie intake in statin users may also be related to statin‐dependent decreases in satiety factors such as leptin, an adipocyte‐derived adipokine. The objective of our study was to examine the direct effects of statins on leptin expression. Adipocytes are the main source of circulating leptin. Therefore, we examined the effects of atorvastatin and simvastatin on leptin expression in cultured human white adipocytes. We show that treatment of white adipocytes with simvastatin and atorvastatin decreases leptin mRNA expression (simvastatin: P = 0.008, atorvastatin: P = 0.03) and leptin secretion (simvastatin: P = 0.0001, atorvastatin: P = 0.0001). Both simvastatin and atorvastatin mediate decreases in leptin expression via extracellular‐signal‐regulated kinases 1/2 and peroxisome proliferator‐activated receptor gamma pathways (simvastatin: P = 0.01, atorvastatin: P = 0.026). Additionally, statin treatment also induced expected increases in adiponectin, while decreasing monocyte chemoattractant protein 1 (MCP1) mRNA. Furthermore, statins increased secretion of both total as well as high molecular weight adiponectin while decreasing MCP1 secretion. To conclude, statins act directly on human white adipocytes to regulate adipokine secretion and decrease leptin expression. Leptin is an important satiety factor. Hence, statin‐dependent decreases in leptin may contribute, at least in part, to increases in food intake in statin users.

Although quantitative trait loci (QTLs) for number of thoracic-lumbar vertebrae have been identified on Sus scrofa chromosomes (SSCs) 1 and 7, the influence of these QTLs on the thoracic and lumbar vertebrae is not clear. The aim of this study was to identify single nucleotide polymorphisms (SNPs) associated with total number of thoracic-lumbar vertebrae and for each trait (number of thoracic and lumbar vertebrae) separately. A total of 581 individuals from an F 2 Large White×Minzhu population were genotyped using an SNP60K chip. Performing a genome-wide association study (GWAS) for total number of thoracic-lumbar vertebrae, 38 significant SNPs were identified in two QTL regions located on SSC1 and SSC7. Performing a GWAS for number of thoracic vertebrae only, 72 significant SNPs were located on SSC7. While performing a GWAS for number of lumbar vertebrae only, 17 significant SNPs were identified on SSC1. Gene mining suggested that the gene encoding orphan nuclear receptor, germ cell nuclear factor ( NR6A1 ) on SSC1 was a strong candidate affecting the number of lumbar vertebrae in pigs. Additionally, genes encoding vertnin ( VRTN ), prospero homeobox 2 ( PROX2 ), Finkel-Biskis-Jinkins murine osteosarcoma viral oncogene homolog (FOS) , and transforming growth factor beta 3 ( TGFB3 ) may be important candidates affecting the number of thoracic vertebrae in pigs. QTLs on SSC1 and SSC7 independently influenced the numbers of thoracic and lumbar vertebrae. These results shed light on the complex genetic background of vertebrae development in pigs.

We investigated improvements to the mechanical properties of iron matrix composites for the case where the surfaces of the reinforcing particles are coated with copper and a hybrid mixture of different types of reinforcing ceramic particles (hybrid particles mixture) is used. Copper coating on the surfaces of SiC, TiC, and TiN particles can eliminate interfacial defects to significantly improve the composites’ mechanical properties. The addition of uncoated hybrid particles mixture has little effect on the tensile strength improvement of composites compared with composites reinforced by monolithic particles, whereas copper-coated hybrid reinforcement has a significant effect. Any composite reinforced with copper-coated hybrid particles mixture will always have higher strength than that reinforced with monolithic particles. Our findings suggest that the load transfer between the matrix and reinforcing particles improved because of different elastic moduli, coefficients of thermal expansion and reaction heats of different types of reinforcing particles.

The pulmonary surfactant-associated protein (SFTPA1, SP-A) gene has been studied as a candidate gene for lung disease resistance in humans and livestock. The objective of the present study was to identify polymorphisms of the porcine SFTPA1 gene coding region and its association with acute lung injury (ALI). Through DNA sequencing and the PCR-single-strand conformation polymorphism method, a novel 9-bp nucleotide insertion (+) or deletion (-) was detected on exon 2 of SFTPA1, which causes a change in three amino acids, namely, alanine (Ala), glycine (Gly) and proline (Pro). Individuals of three genotypes (-/-, +/- and +/+) were divided into equal groups from 60 Rongchang pigs that were genotyped. These pigs were selected for participation in the oleic acid (OA)-ALI model by 1-h and 3-h injections of OA, and there were equal numbers of pigs in the control and injection groups. The lung water content, a marker for acute lung injury, was measured in this study; there is a significant correlation between high lung water content and the presence of the 9-bp indel polymorphism (P < 0.01). The lung water content of the OA injection group was markedly higher than that of the control group and lung water content for the +/+ genotype was significantly higher than that of the others in the 1-h group (P < 0.01). No differences in the expression of the SFTPA1 gene were found among individuals with different SFTPA1 genotypes, indicating that the trait is not caused by a linked polymorphism causing altered expression of the gene. The individuals with the -/- genotype showed lower lung water content than the +/+ genotype pigs, which suggests that polymorphism could be a potential marker for lung disease-resistant pig breeding and that pig can be a potential animal model for human lung disease resistance in future studies.