Multi-omics analysis of TLCD1 as a promising biomarker in pan-cancer.

Background: The TLC Domain Containing 1 (TLCD1) protein, a key regulator of phosphatidylethanolamine (PE) composition, is distributed across several cellular membranes, including mitochondrial plasma membranes. Existing research has revealed the impact of TLCD1 on the development of non-alcoholic fatty liver disease. However, there remains a gap in comprehensive pan-cancer analyses of TLCD1, and the precise role of TLCD1 in cancer patient prognosis and immunological responses remains elusive. This study aims to provide a comprehensive visualization of the prognostic landscape associated with TLCD1 across a spectrum of cancers, while shedding light on the potential links between TLCD1 expression within the tumor microenvironment and immune infiltration characteristics. Methods: TLCD1 expression data were obtained from GTEx, TCGA, and HPA data repositories. Multiple databases including TIMER, HPA, TISIDB, cBioPortal, GEPIA2, STRING, KEGG, GO, and CancerSEA were used to investigate the expression pattern, diagnostic and prognostic significance, mutation status, functional analysis, and functional status of TLCD1. In addition, we evaluated the relationship between TLCD1 expression and immune infiltration, tumor mutational burden (TMB), microsatellite instability (MSI), and immune-related genes in pan-cancer. Furthermore, the association of TLCD1 with drug sensitivity was analyzed using the CellMiner database. Results: We found that TLCD1 is generally highly expressed in pan-cancers and is significantly associated with the staging and prognosis of various cancers. Furthermore, our results also showed that TLCD1 was significantly associated with immune cell infiltration and immune regulatory factor expression. Using CellMiner database analysis, we then found a strong correlation between TLCD1 expression and sensitivity to anticancer drugs, indicating its potential as a therapeutic target. The most exciting finding is that high TLCD1 expression is associated with worse survival and prognosis in GBM and SKCM patients receiving anti-PD1 therapy. These findings highlight the potential of TLCD1 as a predictive biomarker for response to immunotherapy. Conclusion: TLCD1 plays a role in the regulation of immune infiltration and affects the prognosis of patients with various cancers. It serves as both a prognostic and immunologic biomarker in human cancer.

RNA Sequencing Reveals the Regulation of Betaine on Chicken Myogenesis.

Betaine is trimethylglycine and a universal methyl donor which could provide methyl and glycine for cells and animals. As a new star in epigenetics, N6-Methyladenosine has been reported to regulate multiple biological activities, but the regulatory mechanism of betaine on N6-Methyladenosine as well as myogenesis was little studied. In this study, we treated chicken primary myoblast cells with different concentrations of betaine (0, 10, 25, and 50 mmol/L) and found that myoblast cell proliferation was inhibited, although the cell cycle was promoted in the S phase by betaine, where the myotube area was increased as well as the differentiation marker genes MyoD, MyoG, MyHC, Myomarker, and Ckm. RNA sequencing obtained a total of 61 differentially expressed genes (DEGs); DEGs caused by 50 mmol/L betaine were mainly enriched in the regulation of skeletal muscle tissue regeneration and some amino acid metabolic processes. The gene expression pattern trends of all DEGs were mainly clustered into 2 profiles, with the increase in betaine concentration, the gene expression pattern either increased or decreased continuously. Overall, a low concentration betaine can increase the N6-Methyladenosine modification level and myotube area but depresses myoblast cell proliferation in vitro.

miR-1283 Contributes to Endoplasmic Reticulum Stress in the Development of Hypertension Through the Activating Transcription Factor-4 (ATF4)/C/EBP-Homologous Protein (CHOP) Signaling Pathway.

BACKGROUND Hypertension-related microRNA(miR)-1283 and its target gene, activating transcription factor-4 (ATF4), can regulate vascular endothelial dysfunction. This study aimed to explore whether miR-1283 prevents hypertension through targeting ATF4. MATERIAL AND METHODS Transcriptome sequencing was performed after overexpression or inhibition of miR-1283 in human amniotic epithelial cells (HAECs). After miR-1283 was overexpressed or inhibited in HAECs, ATF4+/- and wild-type mice were induced with a high-salt diet. We detected the expression of ATF4, C/EBP-homologous protein (CHOP), BH3-interacting domain death agonist (BID), Bcl-2, Bcl-2-like protein 11 (BIM), Bcl-2-like protein 1 (BCL-X), and caspase-3 by PCR and western blotting. We detected the changes of vasoactive substances including nitric oxide (NO), endothelin 1 (ET-1), endothelial protein C receptor (EPCR), thrombin (TM), and von Willebrand factor (vWF) by ELISA. RESULTS Compared with that of the miR-1283- inhibited group, NO was higher in the miR-1283 overexpression group, while the expression of ET-1, EPCR, TM, and vWF were lower. Similarly, compared with that of the miR-1283 inhibited group, the expression of ATF4, CHOP, BID, BIM, and caspase-3 in the miR-1283 overexpression group was downregulated, while the expression of BCL-2 and BCL-X was upregulated (P<0.05). In vivo experiments showed the lack of ATF4 gene could prevent hypertension in mice induced by high-salt diet and protect endothelial function. CONCLUSIONS The mechanism of regulating blood pressure and endothelial function of the miR-1283/ATF4 axis was related to inhibiting endoplasmic reticulum stress and cell apoptosis through the ATF4/CHOP signaling pathway. Therefore, the miR-1283/ATF4 axis may be a target for the prevention and treatment of hypertension.

Genome diversity of Chinese indigenous chicken and the selective signatures in Chinese gamecock chicken.

Gamecock chickens are one of the earliest recorded birds in China, and have accumulated some unique morphological and behavioral signatures such as large body size, muscularity and aggressive behavior, whereby being excellent breeding materials and a good model for studying bird muscular development and behavior. In this study, we sequenced 126 chicken genomes from 19 populations, including four commercial chicken breeds that are commonly farmed in China, 13 nationwide Chinese typical indigenous chicken breeds (including two Chinese gamecock breeds), one red jungle fowl from Guangxi Province of China and three gamecock chickens from Laos. Combined with 31 published chicken genomes from three populations, a comparative genomics analysis was performed across 157 chickens. We found a severe confounding effect on potential cold adaptation exerted by introgression from commercial chickens into Chinese indigenous chickens, and argued that the genetic introgression from commercial chickens into indigenous chickens should be seriously considered for identifying selection footprint in indigenous chickens. LX gamecock chickens might have played a core role in recent breeding and conservation of other Chinese gamecock chickens. Importantly, AGMO (Alkylglycerol monooxygenase) and CPZ (Carboxypeptidase Z) might be crucial for determining the behavioral pattern of gamecock chickens, while ISPD (Isoprenoid synthase domain containing) might be essential for the muscularity of gamecock chickens. Our results can further the understanding of the evolution of Chinese gamecock chickens, especially the genetic basis of gamecock chickens revealed here was valuable for us to better understand the mechanisms underlying the behavioral pattern and the muscular development in chicken.

Vinculin presents unfavorable prediction in ovarian cancer and prevents proliferation and migration of ovarian cancer cells.

The influences of Vinculin on many cancers were blurry, including ovarian cancer. Thus, we concentrated on the efficient role of Vinculin in ovarian cancer and explored the potential mechanism(s). Expression of Vinculin in ovarian cancer tissues and cell lines was investigated by real-time polymerase chain reaction, immunohistochemistry, and Western blot. The Kaplan-Meier manner with the logrank was performed to assess overall survival. We further evaluated the relations between Vinculin expression and clinicopathological features of ovarian cancer. Moreover, Vinculin was overexpressed or silenced by respectively transfection with pcDNA-Vinculin or small interfering (si-Vinculin) into human ovarian cancer cell line Caov3 or human ovarian epithelial cell line (HOEpiC). Thereafter, cell viability, cell apoptosis, and migration were checked by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, flow cytometer, and scratch assay, respectively. Likewise, the apoptosis- and migration-related proteins were distinguished by Western blot. Compared to the nontumor tissues or HOEpiC cells, Vinculin was significantly lower expressed in the ovarian cancer tissues and cells. Furthermore, we found out that Vinculin was primarily distributed at the cell membrane and cytoplasm. Moreover, Vinculin was negatively associated with International Federation of Gynecology and Obstetrics stage, grade, and distant metastasis. Overexpression of Vinculin dramatically weakened cell viability and migration and stimulated apoptosis. Conversely, suppression of Vinculin showed opposite results. Vinculin presents unfavorable prediction in ovarian cancer and inhibits ovarian cancer proliferation and migration.

LncRNA EMX2OS Induces Proliferation, Invasion and Sphere Formation of Ovarian Cancer Cells via Regulating the miR-654-3p/AKT3/PD-L1 Axis.

PURPOSE: Long noncoding RNA (lncRNA) deregulation is frequent in human ovarian cancers (OCs), but the role of specific miRNAs involved in this disease remains elusive. LncRNA EMX2OS was previously reported to act as an oncogene in human cancers. However, their accurate expression, function and underlying mechanisms in OC are largely unclear. MATERIALS AND METHODS: The levels of EMX2OS in OC tissues and cell lines were determined by quantitative real-time PCR, and the function of EMX2OS was then analyzed both in vitro and in vivo. Luciferase assays and immunoprecipitation assays were performed to analyze the association between EMX2OS and miR-654 expression in OC cells. RESULTS: EMX2OS is overexpressed in human ovarian cancer tissues. Knockdown of EMX2OS reduced, while overexpression of EMX2OS enhanced the proliferation, invasion and sphere formation of OC cells. In addition, EMX2OS enhanced tumor growth in an in vivo xenograft model of human OC. We discovered that EMX2OS directly binds to miR-654 and suppresses its expression, thus leading to the upregulation of AKT3, which served as a direct target of miR-654. Moreover, miR-654 inhibited cell proliferation, invasion and sphere formation, and restoration of AKT3 reversed the effects of EMX2OS silencing or miR-654 overexpression. Furthermore, PD-L1 was identified as the key oncogenic component acting downstream of AKT3 in OC cells. Ectopic expression of PD-L1 reversed the anti-cancer functions by EMX2OS knockdown, AKT3 silencing or miR-654 upregulation in OC cells. CONCLUSION: These results demonstrated that the EMX2OS/miR-654/AKT3/PD-L1 axis confers aggressiveness in ovarian cancer and may represent a therapeutic target for OC metastasis.

Chinese tree shrews as a primate experimental animal eligible for the study of alcoholic liver disease: characterization and confirmation by MRI.

There has been a lack of suitable fatty liver models and characterization techniques for histopathological evaluation of alcoholic fatty liver (AFL). This work aimed to exploit an magnetic resonance imaging (MRI) technique for characterizing an alcohol-induced fatty liver model established in tree shrews (Tupaia belangeri chinese). The animals were treated with 15% alcohol for two weeks instead of drinking water to induce AFL. Blood alanine aminotransferase (ALT), aspartate aminotransferase (AST), alcohol, and liver malondialdehyde (MDA) concentrations were determined, and the histopathology of the liver was checked by hematoxylin & eosin (HE) and Oil red O staining on day 0 and on the 4th, 7th and 14th days after alcohol feeding. MRI was used to trace the histopathological changes in the liver of tree shrews in real time. Compared with the control group, the levels of ALT, AST, and MDA significantly increased in the alcohol-induced group and were positively correlated with the induction time. HE and Oil red O staining revealed that a moderate fatty lesion occurred in the liver on the 4th day and that a serious AFL was successfully induced on the 14th day. MRI further confirmed the formation of AFL. MRI, as noninvasive examination technique, provides an alternative tool for accurate characterization of AFL in live subjects. It is comparable to HE or Oil red O staining for histopathological examination, but is more suitable by virtue of its high flexibility and compliance. The AFL model of tree shrews combined with MRI characterization can work as a platform for studying fatty liver diseases and medications for their treatment.

Bioinformatics analysis of microRNAs related to blood stasis syndrome in diabetes mellitus patients.

In traditional Chinese medicine (TCM), blood stasis syndrome (BSS) is mainly manifested by the increase of blood viscosity, platelet adhesion rate and aggregation, and the change of microcirculation, resulting in vascular endothelial injury. It is an important factor in the development of diabetes mellitus (DM). The aim of the present study was to screen out the potential candidate microRNAs (miRNAs) in DM patients with BSS by high-throughput sequencing (HTS) and bioinformatics analysis. Human umbilical vein endothelial cells (HUVECs) were incubated with 10% human serum to establish models of DM with BSS, DM without BSS (NBS), and normal control (NC). Total RNA of each sample was extracted and sequenced by the Hiseq2000 platform. Differentially expressed miRNAs (DE-miRNAs) were screened between samples and compared with known changes in mRNA abundance. Target genes of miRNAs were predicted by softwares. Gene Ontology (GO) and pathway enrichment analysis of the target genes were conducted. According to the significantly enriched GO annotations and pathways (P-value ≤ 0.001), we selected the key miRNAs of DM with BSS. It showed that the number of DE-miRNAs in BSS was 32 compared with non-blood stasis syndrome (NBS) and NC. The potential candidate miRNAs were chosen from GO annotations in which target genes were significantly enriched (-log10 (P-value) > 5), which included miR-140-5p, miR-210, miR-362-5p, miR-590-3p, and miR-671-3p. The present study screened out the potential candidate miRNAs in DM patients with BSS by HTS and bioinformatics analysis. The miRNAs will be helpful to provide valuable suggestions on clinical studies of DM with BSS at the gene level.

Effect of the Diabetic Environment On the Expression of MiRNAs in Endothelial Cells: Mir-149-5p Restoration Ameliorates the High Glucose-Induced Expression of TNF-α and ER Stress Markers.

BACKGROUND/AIMS: This study aimed to screen microRNAs and their corresponding target genes that are associated with vascular injury in type two diabetes mellitus (T2DM), investigate the effects of differentially expressed miRNAs and their target genes on high glucose-induced vascular injury and establish the mechanism underlying these effects. METHODS: A high-throughput digital gene expression (DGE) sequencing was performed to sequence microRNAs (miRNAs) and messenger RNAs (mRNAs) and determine their differential expression in human umbilical vein endothelial cells (HUVECs) incubated with serum samples from patients with T2DM and healthy volunteers. The HUVECs were transfected with si-TNF-α (tumor necrosis factor α) and a miR-149-5p inhibitor or mimic in vitro and then treated with normal or high glucose. The relative content of nitric oxide (NO) in the cells was detected using the Griess Reagent System. The mRNA and protein expression of endothelial nitric oxide synthase (eNOS) were determined by qRT-PCR and Western blotting. The content of endothelin-1 (ET-1), von Willebrand factor (vWF), and intercellular adhesion molecular-1 (ICAM-1) were detected using an enzyme-linked immunosorbent assay (ELISA) kit. Apoptosis was determined by flow cytometry using the Annexin V/PI apoptosis detection kit. The mRNA and protein expression levels of ER stress (ERS) markers were determined by qRT-PCR and Western blotting. RESULTS: Based on the high-energy sequencing and in vitro pre-experiment studies, we determined that miR-149-5p and TNF-α were a differentially expressed mRNA/miRNA pair in T2DM with vascular injury. The luciferase reporter assay results demonstrated that miR-149-5p could directly target TNF-α. The upregulation of miR-149-5p reduced the high glucose-induced dysfunction in the HUVECs by significantly decreasing the levels of ET-1, vWF, and ICAM-1 and increasing the level of NO and the expression of eNOS. Additionally, we found that miR-149-5p can improve cell injury and reduce apoptosis by restoring the ameliorated high glucose-induced expression of ERS markers. CONCLUSION: TNF-α and miR-149-5p were differentially expressed in T2DM vascular endothelial injury. The over-expression of miR-149-5p ameliorates the high glucose-induced injury in the HUVECs by regulating the expression of TNF-α and ERS markers.

miRNA-1283 Regulates the PERK/ATF4 Pathway in Vascular Injury by Targeting ATF4.

BACKGROUND: In our previous study, we found significant differences in the mRNA and microRNA (miRNA) levels among hypertensive patients with different degrees of vascular endothelial cells damage. These differences were closely associated with endoplasmic reticulum stress (ERS)-related proteins. Moreover, compared to the control group, the expression of transcription factor activating factor 4 (ATF4) was also found to be significantly different in the hypertensive patients with different degrees of vascular endothelial cells damage groups. These results were confirmed using gene prediction software, which showed synergistic effects between ATF4 and miR-1283. ATF4 is a key molecule in ERS. Three ERS pathways exist:protein kinase RNA-like ER kinase (PERK), activating transcription factor 6 (ATF6) and inositol-requiring enzyme-1 (IRE-1)-induced apoptosis. The PERK pathway is the most important and also includes the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) and ATF4. In this report, we studied the regulatory effects of miR-1283 and ATF4 on the PERK-eIF2α-ATF4 signaling pathway using human umbilical vein endothelial cells (HUVECs) and mice. METHODOLOGY/PRINCIPAL FINDINGS: We verified the relationship between miR-1283 and ATF4 using a luciferase activity assay and observed the regulatory effects of miR-1283 and ATF4 on the PERK-eIF2α-ATF4 signaling pathway in vivo and in vitro. CONCLUSIONS/SIGNIFICANCE: ATF4 is a target gene of miR-1283, which regulates the PERK-eIF2α-ATF4 signaling pathway by inhibiting ATF4, and it plays a critical role in inducing injury in HUVECs and mouse heart tissue.

Transcriptome analysis of blood stasis syndrome in subjects with hypertension.

OBJECTIVE: To screen for mRNAs associated with blood stasis syndrome and to explore the genetic mechanisms of blood stasis syndrome in hypertension. METHODS: This study involved groups of patients with hypertension and blood stasis, including those with Qi deficiency, Qi stagnation, cold retention and heat retention; as well as hypertensive patients without blood stasis and healthy individuals. Human umbilical vein endothelial cells were co-cultured with the sera of these healthy individuals and patients with blood stasis syndrome. Total RNA was extracted from these cells and assessed by a high-throughput sequencing method (Solexa) and digital gene expression. Differentially expressed genes among these six groups were compared using whole genome sequences, and mRNAs associated with blood stasis syndrome identified. Differences in gene use and gene ontology function were analyzed. Genes enriched significantly and their pathways were determined, as were network interactions, and encoded proteins. Gene identities were confirmed by real-time polymerase chain reactions. RESULTS: Compared with cells cultured in sera of the blood stasis groups, those culture in sera of healthy individuals and of the non-blood stasis group showed 11 and 301 differences, respectively in stasis-related genes. Genes identified as differing between the blood stasis and healthy groups included activating transcription factor 4, activating transcription factor 3, DNA-damage inducible transcription factor 3, Tribbles homolog 3, CCAAT/enhancer binding protein-ß, and Jun proto-oncogene (JUN). Pathway and protein interaction network analyses showed that these genes were associated with endoplasmic reticulum stress. Cells cultured in sera of patients with blood stasis and Qi deficiency, Qi stagnation, heat retention, and cold retention were compared with cells cultured in sera of patients with the other types blood stasis syndrome. The comparison showed differences in expression of 28, 28, 34, and 32 specific genes, respectively. CONCLUSION: The pathogenesis of blood stasis syndrome in hypertension is related to endoplasmic reticulum stress and involves the differential expression of the activating transcription factor 4, activating transcription factor 3, DNA-damage inducible transcription factor 3, Tribbles homolog 3, CCAAT/enhancer binding protein-ß, and JUN genes.

The Long Intron 1 of Growth Hormone Gene from Reeves' Turtle (Chinemys reevesii) Correlates with Negatively Regulated GH Expression in Four Cell Lines.

Turtles grow slowly and have a long lifespan. Ultrastructural studies of the pituitary gland in Reeves' turtle (Chinemys reevesii) have revealed that the species possesses a higher nucleoplasmic ratio and fewer secretory granules in growth hormone (GH) cells than other animal species in summer and winter. C. reevesii GH gene was cloned and species-specific similarities and differences were investigated. The full GH gene sequence in C. reevesii contains 8517 base pairs (bp), comprising five exons and four introns. Intron 1 was found to be much longer in C. reevesii than in other species. The coding sequence (CDS) of the turtle's GH gene, with and without the inclusion of intron 1, was transfected into four cell lines, including DF-1 chicken embryo fibroblasts, Chinese hamster ovary (CHO) cells, human embryonic kidney 293FT cells, and GH4C1 rat pituitary cells; the turtle growth hormone (tGH) gene mRNA and protein expression levels decreased significantly in the intron-containing CDS in these cell lines, compared with that of the corresponding intronless CDS. Thus, the long intron 1 of GH gene in Reeves' turtle might correlate with downregulated gene expression.

Comparison of miRNA expression profiles in pituitary-adrenal axis between Beagle and Chinese Field dogs after chronic stress exposure.

MicoRNAs (miRNAs), usually as gene regulators, participate in various biological processes, including stress responses. The hypothalamus-pituitary-adrenal axis (HPA axis) is an important pathway in regulating stress response. Although the mechanism that HPA axis regulates stress response has been basically revealed, the knowledge that miRNAs regulate stress response within HPA axis, still remains poor. The object of this study was to investigate the miRNAs in the pituitary and adrenal cortex that regulate chronic stress response with high-throughput sequencing. The pituitary and adrenal cortex of beagles and Chinese Field dogs (CFD) from a stress exposure group (including beagle pituitary 1 (BP1), CFD pituitary 1 (CFDP1), beagle adrenal cortex 1 (BAC1), CFD adrenal cortex 1 (CFDAC1)) and a control group (including beagle pituitary 2 (BP2), CFD pituitary 2 (CFDP2), beagle adrenal cortex 2 (BAC2), CFD adrenal cortex 2 (CFDAC2)), were selected for miRNA-seq comparisons. Comparisons, that were made in pituitary (including BP1 vs. BP2, CFDP1 vs. CFDP2, BP1 vs. CFDP1 and BP2 vs. CFDP2) and adrenal cortex (including BAC1 vs. BAC2, CFDAC1 vs. CFDAC2, BAC1 vs. CFDAC1 and BAC2 vs. CFDAC2), showed that a total of 39 and 18 common differentially expressed miRNAs (DE-miRNAs) (Total read counts > 1,000, Fold change > 2 & p-value < 0.001), that shared in at least two pituitary comparisons and at least two adrenal cortex comparisons, were detected separately. These identified DE-miRNAs were predicted for target genes, thus resulting in 3,959 and 4,010 target genes in pituitary and adrenal cortex, respectively. Further, 105 and 10 differentially expressed genes (DEGs) (Fold change > 2 & p-value < 0.05) from those target genes in pituitary and adrenal cortex were obtained separately, in combination with our previous corresponding transcriptome study. Meanwhile, in line with that miRNAs usually negatively regulated their target genes and the dual luciferase reporter assay, we finally identified cfa-miR-205 might play an important role by upregulating MMD in pituitary and hippocampus, thus enhancing the immune response, under chronic stress exposure. Our results shed light on the miRNA expression profiles in the pituitary and adrenal cortex with and without chronic stress exposure, and provide a new insight into miR-205 with its feasible role in regulating chronic stress in the pituitary and hippocampus through targeting MMD.

Transcriptome comparison in the pituitary-adrenal axis between Beagle and Chinese Field dogs after chronic stress exposure.

Chronic stress can induce a series of maladjustments, and the response to stress is partly regulated by the hypothalamus-pituitary-adrenal axis. The aim of this study was to investigate the genetic mechanisms of this axis regulating stress responsiveness. The pituitary and adrenal cortex of Beagle and Chinese Field Dog (CFD) from a stress exposure group [including Beagle pituitary 1 (BP1), CFD pituitary 1 (CFDP1), Beagle adrenal cortex 1 (BAC1), CFD adrenal cortex 1 (CFDAC1)] and a control group [including Beagle pituitary 2 (BP2), CFD pituitary 2 (CFDP2), Beagle adrenal cortex 2 (BAC2), CFD adrenal cortex 2 (CFDAC2)], selected to perform RNA-seq transcriptome comparisons, showed that 40, 346, 376, 69, 70, 38, 57 and 71 differentially expressed genes were detected in BP1 vs. BP2, CFDP1 vs. CFDP2, BP1 vs. CFDP1, BP2 vs. CFDP2, BAC1 vs. BAC2, CFDAC1 vs. CFDAC2, BAC1 vs. CFDAC1 and BAC2 vs. CFDAC2 respectively. NPB was a gene common to BAC1 vs. BAC2 and CFDAC1 vs. CFDAC2, indicating it was a potential gene affecting response to chronic stress, regardless of the extent of chronic stress induced. PLP1 was a gene common to BP1 vs. CFDP1 and BP2 vs. CFDP2, suggesting its important roles in affecting the stress-tolerance difference between the two breeds, regardless of whether there was stress exposure or not. Pathway analysis found 12, 4, 11 and 1 enriched pathway in the comparisons of BP1 vs. CFDP1, BP2 vs. CFDP2, CFDP1 vs. CFDP2 and BAC1 vs. BAC2 respectively. Glutamatergic synapse, neuroactive ligand-receptor interaction, retrograde endocannabinoid signaling, GABAergic synapse, calcium signaling pathway and dopaminergic synapse were the most significantly enriched pathways in both CFDP1 vs. CFDP2 and BP1 vs. CFDP1. GO, KEGG pathway and gene network analysis demonstrated that GRIA3, GRIN2A, GRIN2B and NPY were important in regulating the stress response in CFD. Nevertheless, ADORA1, CAMK2A, GRM1, GRM7 and NR4A1 might be critical genes contributing to the stress-tolerance difference between CFD and Beagle when subjected to stress exposure. In addition, RGS4 and SYN1 might play important roles both in regulating the stress response in CFD and in affecting the stress-tolerance difference in different breeds. These observations clearly showed that some genes in the adrenal cortex and pituitary could regulate the stress response in Beagle and CFDs, whereas some others could affect the stress-tolerance difference between these two breeds. Our results can contribute to a more comprehensive understanding of the genetic mechanisms of response to chronic stress.

Establishment of the tree shrew as an alcohol-induced Fatty liver model for the study of alcoholic liver diseases.

Currently, the pathogenesis of alcoholic liver diseases (ALDs) is not clear. As a result, there is no effective treatment for ALDs. One limitation is the lack of a suitable animal model for use in studying ALDs. The tree shrew is a lower primate animal, characterized by a high-alcohol diet. This work aimed to establish a fatty liver model using tree shrews and to assess the animals' suitability for the study of ALDs. Tree shrews were treated with alcohol solutions (10% and 20%) for two weeks. Hemophysiology, blood alcohol concentrations (BACs), oxidative stress factors, alcohol metabolic enzymes and hepatic pathology were checked and assayed with an automatic biochemical analyzer, enzyme-linked immunosorbent assay (ELISA), western blot, hematoxylin-eosin (HE) staining and oil red O staining, and magnetic resonance imaging (MRI). Compared with the normal group, the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), total cholesterol (TC), triglyceride (TG), reactive oxygen species (ROS), and malondialdehyde (MDA) were significantly enhanced in alcohol-treated tree shrews. However, the activity of reduced glutathione hormone (GSH) and superoxide dismutase (SOD) declined. Notable changes in alcohol dehydrogenase(ADH1), aldehyde dehydrogenase(ALDH2), CYP2E1, UDP-glucuronosyl transferase 1A1 (UGT1A1) and nuclear factor erythroid-related factor 2 (Nrf2) were observed. HE and oil red O staining showed that hepatocyte swelling, hydropic degeneration, and adipohepatic syndrome occurred in the tree shrews. Alcohol can induce fatty liver-like pathological changes and result in alterations in liver function, oxidative stress factors, alcohol metabolism enzymes and Nrf2. Therefore, the established fatty liver model of tree shrews induced by alcohol should be a promising tool for the study of ALDs.

Differential expression of microRNA in endothelial cells incubated with serum of hypertension patients with blood-stasis syndrome.

OBJECTIVE: To screen out blood-stasis syndrome (BSS)-associated microRNA and therefore determine the possible target for treating hypertension. METHODS: A high-energy sequencing method and digital gene expression sequencing theory were adopted to sequence microRNA (miRNA) and messenger RNA (mRNA), and to determine differential expression in human umbilical vein endothelial cells incubated with serum samples from hypertension patients with or without BSS, and healthy controls. The results were confirmed using gene prediction software. RESULTS: A total of 13 miRNAs and 11 mRNAs showed statistical difference both in the BSS/normal groups and BSS/non-BSS groups, respectively. Four pairs of target mRNA/miRNA were identified: FRMD4A/hsa-miR-34a, MAP3K14/hsa-miR-34a, PER1/hsa-miR-34a, and FGF2/hsa-miR-132. CONCLUSION: Four mRNA/miRNA pairs mentioned above seem to be involved in pathogenesis and maintenance of hypertension with BSS.

Constructing protein-protein interaction network of hypertension with blood stasis syndrome via digital gene expression sequencing and database mining.

OBJECTIVE: To construct a protein-protein interaction (PPI) network in hypertension patients with blood-stasis syndrome (BSS) by using digital gene expression (DGE) sequencing and database mining techniques. METHODS: DGE analysis based on the Solexa Genome Analyzer platform was performed on vascular endothelial cells incubated with serum of hypertension patients with BSS. The differentially expressed genes were filtered by comparing the expression levels between the different experimental groups. Then functional categories and enriched pathways of the unique genes for BSS were analyzed using Database for Annotation, Visualization and Integrated Discovery (DAVID) to select those in the enrichment pathways. Interologous Interaction Database (I2D) was used to construct PPI networks with the selected genes for hypertension patients with BSS. The potential candidate genes related to BSS were identified by comparing the number of relationships among genes. Confirmed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), gene ontology (GO) analysis was used to infer the functional annotations of the potential candidate genes for BSS. RESULTS: With gene enrichment analysis using DAVID, a list of 58 genes was chosen from the unique genes. The selected 58 genes were analyzed using I2D, and a PPI network was constructed. Based on the network analysis results, candidate genes for BSS were identified: DDIT3, JUN, HSPA8, NFIL3, HSPA5, HIST2H2BE, H3F3B, CEBPB, SAT1 and GADD45A. Verified through qRT-PCR and analyzed by GO, the functional annotations of the potential candidate genes were explored. CONCLUSION: Compared with previous methodologies reported in the literature, the present DGE analysis and data mining method have shown a great improvement in analyzing BSS.

Identification of TDRP1 gene and its association with pig reproduction traits.

This study was performed to identify and characterize the pig TDRP1 gene and to investigate its association with reproduction traits. The obtained pig TDRP1 cDNA (713 base pair [bp]) comprises a 561-bp open reading frame, which encodes a peptide of 187 amino acids. The identities of pig TDRP1 cDNA were 84.6%, 75.7%, and 77.4% with its counterparts in human, rat, and mice, respectively. Real-time polymerase chain reaction indicated that pig TDRP1 gene was highly expressed in pituitary of male and uterus of female animals. The pig TDRP1 gene contains three exons and two introns. A total of 13 single-nucleotide polymorphisms (SNPs) and 1 indel were identified in the screened partial genomic sequence, with most polymorphisms in introns. Allelic frequencies of five SNPs among eight pig breeds were further investigated, and it indicated that Landrace had the lowest genetic diversity. In Yorkshire, three SNPs (c.215+144T>C, c.215+249A>G, and c.215+672T>C) exhibited complete linkage disequilibrium in one haplotype block, and association analyses showed that all of them were significantly associated with number born alive of first parity (NBA1) (p<0.05). c.215+672T>C was also significantly associated with NBA6 (p<0.05). In addition, these three SNPs and two other ones (c.215+1001G>A and c.215+1026C>T) were associated with total born alive of second parity (TBA2) and TBA6 at the suggestive level (0.05

Analysis of muscle and ovary transcriptome of Sus scrofa: assembly, annotation and marker discovery.

Pig (Sus scrofa) is an important organism for both agricultural and medical purpose. This study aims to investigate the S. scrofa transcriptome by the use of Roche 454 pyrosequencing. We obtained a total of 558 743 and 528 260 reads for the back-leg muscle and ovary tissue each. The overall 1 087 003 reads give rise to 421 767 341 bp total residues averaging 388 bp per read. The de novo assemblies yielded 11 057 contigs and 60 270 singletons for the back-leg muscle, 12 204 contigs and 70 192 singletons for the ovary and 18 938 contigs and 102 361 singletons for combined tissues. The overall GC content of S. scrofa transcriptome is 42.3% for assembled contigs. Alternative splicing was found within 4394 contigs, giving rise to 1267 isogroups or genes. A total of 56 589 transcripts are involved in molecular function (40 916), biological process (38 563), cellular component (35 787) by further gene ontology analyses. Comparison analyses showed that 336 and 553 genes had significant higher expression in the back-leg muscle and ovary each. In addition, we obtained a total of 24 214 single-nucleotide polymorphisms and 11 928 simple sequence repeats. These results contribute to the understanding of the genetic makeup of S. scrofa transcriptome and provide useful information for functional genomic research in future.