The dual-luciferase reporter assay is widely used for microRNA target identification and the functional validation of predicted targets. To determine whether curcumin regulates expression of the histone methyltransferase enhancer of zeste homolog 2 (EZH2) by targeting its 3'untranslated region (3'UTR), two luciferase reporter systems containing exactly the same sequence of the EZH2 3'UTR were used to perform dual-luciferase reporter assays. Surprisingly, there were certain discrepancies between the luciferase activities derived from these two reporter constructs. We normalized luciferase activity to an internal control to determine the amount of the reporter construct successfully transfected into cells, induced a transcriptional block with flavopiridol, quantified renilla luciferase mRNA levels, and compared the absolute luciferase activity among the different groups. The results suggested that curcumin promoted the transcription of the luciferase genes located downstream of the simian vacuolating virus 40 (SV40) early enhancer/promoter, but not those located downstream of the human cytomegalovirus (CMV) immediate-early or the herpes simplex virus thymidine kinase (HSV-TK) promoters. These results explain the discrepancies between the two luciferase reporter systems. The current study underscores the importance of taking caution when interpreting the results of dual-luciferase reporter assays and provides strategies to overcome the potential pitfall accompanying dual-luciferase reporter systems.

Liver X receptor (LXR) plays an important role in reverse cholesterol transport (RCT), and activation of LXR could reduce atherosclerosis. In the present study we used a cell-based screening method to identify new potential LXRβ agonists. A novel benzofuran-2-carboxylate derivative was identified with LXRβ agonist activity: E17110 showed a significant activation effect on LXRβ with an EC50 value of 0.72 μmol/L. E17110 also increased the expression of ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) in RAW264.7 macrophages. Moreover, E17110 significantly reduced cellular lipid accumulation and promoted cholesterol efflux in RAW264.7 macrophages. Interestingly, we found that the key amino acids in the LXRβ ligand-binding domain had distinct interactions with E17110 as compared to TO901317. These results suggest that E17110 was identified as a novel compound with LXRβ agonist activity in vitro via screening, and could be developed as a potential anti-atherosclerotic lead compound.

Pig shows multiple superior characteristics in anatomy, physiology, and genome that have made this species to be more suitable models for human diseases, especially for neurodegenerative diseases, because they have similar cerebral convolutions compared with human neocortex. Recently, CRISPR/Cas9 system shows enormous potential for engineering the pig genome. In this study, we expect to generate human Parkinson's disease pig model using CRISPR/Cas9 system by simultaneously targeting three distinct genomic loci, parkin/DJ-1/PINK1, in Bama miniature pigs. By co-injection of Cas9 mRNA and multiplexing single guide RNAs (sgRNAs) targeting parkin, DJ-1, and PINK1 genes, respectively, into in vivo derived pronuclear embryos, we simultaneously targeted three distinct genomic loci. The gene modified piglets remain healthy and display normal behavior at the age of 10 months. In addition, despite the high number of sgRNAs were employed in the present study, our trio-based whole-genome sequencing analysis suggested that the incidence of off-target events is low. Our results demonstrate that the simplicity, efficiency, and power of the CRISPR/Cas9 system to allow for the modification of multiple genes in pigs and yield results of high medical value.

Progranulin (PGRN) is an autocrine growth factor with tumorigenic roles in various tumors including cervical cancer. In this study, we investigated mammalian target of rapamycin (mTOR) signaling in response to PGRN induction and the contribution of the PGRN-stimulated PI3K/Akt/mTOR signaling pathway in the transformation and progression of cervical cancer. Here we identified a strong linkage between PGRN and phosphorylated-mTOR in cervical cancer tissues. PGRN promoted the phosphorylation of mTOR and activated mTOR signaling in human cervical mucosa epithelial cells and cervical cancer cells, and TNFR2 was needed for PGRN-stimulated mTOR signaling. Inhibition of mTOR signaling with rapamycin decreased PGRN-stimulated protein synthesis, transformation and proliferation of cervical cells in vitro, and tumor formation and growth in vivo. Thus, our findings update the signal transduction pathways of PGRN by suggesting that mTOR signaling contributes to PGRN-stimulated carcinogenesis of cervical cancer. Inhibition of PGRN/PI3K/Akt/mTOR signaling may be targeted in treatment of cervical cancer.

Previously, a di-D-fructofuranose 1,2':2,3' dianhydride (DFA III)-producing strain, Arthrobacter aurescens SK8.001, was isolated from soil, and the gene cloning and characterization of the DFA III-forming enzyme was studied. In this study, a DFA III hydrolysis enzyme (DFA IIIase)-encoding gene was obtained from the same strain, and the DFA IIIase gene was cloned and expressed in Escherichia coli. The SDS-PAGE and gel filtration results indicated that the purified enzyme was a homotrimer holoenzyme of 145 kDa composed of subunits of 49 kDa. The enzyme displayed the highest catalytic activity for DFA III at pH 5.5 and 55°C, with specific activity of 232 U mg-1. Km and Vmax for DFA III were 30.7 ± 4.3 mM and 1.2 ± 0.1 mM min-1, respectively. Interestingly, DFA III-forming enzymes and DFA IIIases are highly homologous in amino acid sequence. The molecular modeling and docking of DFA IIIase were first studied, using DFA III-forming enzyme from Bacillus sp. snu-7 as a template. It was suggested that A. aurescens DFA IIIase shared a similar three-dimensional structure with the reported DFA III-forming enzyme from Bacillus sp. snu-7. Furthermore, their catalytic sites may occupy the same position on the proteins. Based on molecular docking analysis and site-directed mutagenesis, it was shown that D207 and E218 were two potential critical residues for the catalysis of A. aurescens DFA IIIase.

The distribution patterns of total protein and protein components in different layers of wheat grain were investigated using the pearling technique, and the sources of different protein components and pearling fractions were identified using (15)N isotope tracing methods. It was found that N absorbed from jointing to anthesis (JA) and remobilized to the grain after anthesis was the principal source of grain N, especially in the outer layer. For albumin and globulin, the amount of N absorbed during different stages all showed a decreasing trend from the surface layer to the center part. Whereas, for globulin and glutenin, the N absorbed after anthesis accounted for the main part indicating that for storage protein, the utilization of N assimilated after anthesis is greater than that of the stored N assimilated before anthesis. It is concluded that manipulation of the N application rate during different growth stages could be an effective approach to modulate the distribution of protein fractions in pearled grains for specific end-uses.

The domestic pig Sus scrofa domesticus originated from the wild boar S. scrofa about 10,000 years ago. During domestication, drastic morphological, physiological, and behavioral changes developed between domestic pigs and wild boars through artificial and natural selection. The long non-coding RNA (lncRNA) H19, which is located within the imprinting gene cluster H19-IGF2, plays an important role in regulating muscle development in humans and mice. This study systematically analyzed the molecular evolution of H19 and its possible epigenetic changes during pig domestication and breeding to explore the genetic and epigenetic contributions of H19 to pig domestication.

SNX-2112 is a promising anticancer agent but has poor solubility in both water and oil. In the study reported here, we aimed to develop a nanocrystal formulation for SNX-2112 and to determine the pharmacokinetic behaviors of the prepared nanocrystals.

Fructans are the polymers of fructose molecules, normally having a sucrose unit at what would otherwise be the reducing terminus. Inulin and levan are two basic types of simple fructan, which contain β-(2, 1) and β-(2, 6) fructosyl-fructose linkage, respectively. Fructans not only can serve as soluble dietary fibers for food industry, but also may be biologically converted into high-value products, especially high-fructose syrup and fructo-oligosaccharides. In recent years, much attention has been focused on production of difructose dianhydrides (DFAs) from fructans. DFAs are cyclic disaccharides consisting of two fructose units with formation of two reciprocal glycosidic linkages. They are expected to have promising properties and beneficial effects on human health. DFAs can be produced from fructans by fructan fructotransferases. Inulin fructotransferase (IFTase) (DFA III-forming) and IFTase (DFA I-forming) catalyze the DFA III and DFA I production from inulin, respectively, and levan fructotransferase (LFTase) (DFA IV-forming) catalyzes the production of DFA IV from levan. In this article, the DFA-producing microorganisms are summarized, relevant studies on various DFAs-producing enzymes are reviewed, and especially, the comparisons of the enzymes are presented in detail.

The aim of this study was to confirm the association of RHOB and FAM167A-BLK gene polymorphisms with susceptibility to systemic sclerosis (SSc) in a Chinese Han population.

Mild traumatic brain injury (mTBI) is a significant public health care burden in the United States. However, we lack a detailed understanding of the pathophysiology following mTBI and its relation to symptoms and recovery. With advanced magnetic resonance imaging (MRI), we can investigate brain perfusion and oxygenation in regions known to be implicated in symptoms, including cortical gray matter and subcortical structures. In this study, we assessed 14 mTBI patients and 18 controls with susceptibility weighted imaging and mapping (SWIM) for blood oxygenation quantification. In addition to SWIM, 7 patients and 12 controls had cerebral perfusion measured with arterial spin labeling (ASL). We found increases in regional cerebral blood flow (CBF) in the left striatum, and in frontal and occipital lobes in patients as compared to controls (p = 0.01, 0.03, 0.03 respectively). We also found decreases in venous susceptibility, indicating increases in venous oxygenation, in the left thalamostriate vein and right basal vein of Rosenthal (p = 0.04 in both). mTBI patients had significantly lower delayed recall scores on the standardized assessment of concussion, but neither susceptibility nor CBF measures were found to correlate with symptoms as assessed by neuropsychological testing. The increased CBF combined with increased venous oxygenation suggests an increase in cerebral blood flow that exceeds the oxygen demand of the tissue, in contrast to the regional hypoxia seen in more severe TBI. This may represent a neuroprotective response following mTBI, which warrants further investigation.

Bengazoles A⁻G from the marine sponge Jaspis sp. exhibit potent in vitro antifungal activity against Candida spp. and other pathogenic fungi. The mechanism of action (MOA) of bengazole A was explored in Candida albicans under both liquid culture and surface culture on Mueller-Hinton agar. Pronounced dose-dependent synergistic antifungal activity was observed with bengazole A in the presence of bengamide A, which is also a natural product from Jaspis sp. The MOA of bengazole A was further explored by monitoring the sterol composition of C. albicans in the presence of sub-lethal concentrations of bengazole A. The GCMS of solvent extracts prepared from liquid cultures of C. albicans in the presence of clotrimazole-a clinically approved azole antifungal drug that suppresses ergosterol biosynthesis by the inhibition of 14α-demethylase-showed reduced cellular ergosterol content and increased concentrations of lanosterol and 24-methylenedihydrolanosterol (a shunt metabolite of ergosterol biosynthesis). No change in relative sterol composition was observed when C. albicans was cultured with bengazole A. These results eliminate an azole-like MOA for the bengazoles, and suggest that another as-yet unidentified mechanism is operative.

Patients with unprovoked first venous thromboembolism (VTE) are at a high risk of recurrence. Although circulating microRNAs (miRNAs) have been found to be associated with VTE and are markers of hypercoagulability, this study is the first to examine whether circulating miRNAs are associated with the risk of VTE recurrence.

Seven diterpene lactones, andrographolide (1), isoandrographolide (2), neo-andrographolide (3), 14-deoxy-11,12-didehydroandrographolide (4), 14-deoxyandrographiside (5), 14-deoxy-11,12-didehydroandrographiside (6), 3,14-dideoxyandrographolide (10), and three flavones, andrographidine C (7), andrographidine A (8), 5-hydroxy-7,8-dimethoxyflavanone (9) have been successfully and efficiently isolated from A. paniculata using an off-line two dimensional (2D) high-speed counter-current chromatography (HSCCC) method for the first time. For the first dimension HSCCC separation, petroleum ether-ethyl acetate-methanol-water 3:7:5:5 (v/v) was employed to isolate 14.4 mg of compound 1, 3.1 mg of compound 2, 7.8 mg of compound 3, and 18.0 mg of compound 4 from 200 mg of the A. paniculata extract. For the second dimension HSCCC separation, petroleum ether-ethyl acetate-methanol-water 2:8:1:9 (v/v) and 5:5:6:4 (v/v) were employed to isolate the collected fractions ranged from 55 to 79 min and the flow out fraction, respectively, which led to 5.1 mg of compound 5, 4.4 mg of compound 6, 2.4 mg of compound 7, 3.3 mg of compound 8, 4.0 mg of compound 9, 7.0 mg of compound 10. The structures of these diterpene lactones and flavones were elucidated by extensive spectroscopic methods.

Previous studies have shown that P2Y12 receptor inhibitors might prevent ventricular arrhythmias and cardiac dysfunction in patients with coronary artery disease. However, few studies have focused on comparison of the efficacy of novel oral potent P2Y12 receptor inhibitors with clopidogrel on these outcomes.

Pigs share many similarities with humans in terms of anatomy, physiology and genetics, and have long been recognized as important experimental animals in biomedical research. Using an N-ethyl-N-nitrosourea (ENU) mutagenesis screen, we previously identified a large number of pig mutants, which could be further established as human disease models. However, the identification of causative mutations in large animals with great heterogeneity remains a challenging endeavor. Here, we select one pig mutant, showing congenital nude skin and thyroid deficiency in a recessive inheritance pattern. We were able to efficiently map the causative mutation using family-based genome-wide association studies combined with whole-exome sequencing and a small sample size. A loss-of-function variant (c.1226 A>G) that resulted in a highly conserved amino acid substitution (D409G) was identified in the DUOX2 gene. This mutation, located within an exonic splicing enhancer motif, caused aberrant splicing of DUOX2 transcripts and resulted in lower H2O2 production, which might cause a severe defect in thyroid hormone production. Our findings suggest that exome sequencing is an efficient way to map causative mutations and that DUOX2D409G/D409G mutant pigs could be a potential large animal model for human congenital hypothyroidism.

Enhanced hepatic gluconeogenesis is an important cause of hyperglycemia in type 2 diabetes. However, the regulatory mechanisms underlying disordered hepatic gluconeogenesis remains largely unclear. In the present study, we investigated the potential role of hepatic neuregulin 4 (Nrg4) in the regulation of gluconeogenesis in mice.

How the human brain differs from those of non-human primates is largely unknown and the complex drivers underlying such differences at the genomic level remain unclear. In this study, we selected 243 brain-related genes, based on Gene Ontology, and identified 184,113 DNaseI hypersensitive sites (DHSs) within their regulatory regions. To performed comprehensive evolutionary analyses, we set strict filtering criteria for alignment quality and filtered 39,132 DHSs for inclusion in the investigation and found that 2,397 (~6%) exhibited evidence of accelerated evolution (aceDHSs), which was a much higher proportion that DHSs genome-wide. Target genes predicted to be regulated by brain-aceDHSs were functionally enriched for brain development and exhibited differential expression between human and chimpanzee. Alignments indicated 61 potential human-specific transcription factor binding sites in brain-aceDHSs, including for CTCF, FOXH1, and FOXQ1. Furthermore, based on GWAS, Hi-C, and eQTL data, 16 GWAS SNPs, and 82 eQTL SNPs were in brain-aceDHSs that regulate genes related to brain development or disease. Among these brain-aceDHSs, we confirmed that one enhanced the expression of GPR133, using CRISPR-Cas9 and western blotting. The GPR133 gene is associated with glioblastoma, indicating that SNPs within DHSs could be related to brain disorders. These findings suggest that brain-related gene regulatory regions are under adaptive evolution and contribute to the differential expression profiles among primates, providing new insights into the genetic basis of brain phenotypes or disorders between humans and other primates.

The arginine vasopressin (AVP) system has been postulated to play a role in glucose homeostasis, insulin resistance, and diabetes mellitus in human and animal studies. The aim of this study was to evaluate the role of plasma copeptin in Chinese patients with type 2 diabetes mellitus (T2DM) with and without diabetic retinopathy (DR).

Ubiquitin-specific protease 22 (USP22), a novel deubiquitinating enzyme, belongs to an extended family of proteins that have ubiquitin hydrolase activity. Recently, USP22 has attracted widespread attention because of its implication in carcinogenesis. However, there have been no studies, to our knowledge, investigating the expression of USP22 in osteosarcoma (OS) and its association with OS progression. In this study, we explored the role of USP22 in OS. We demonstrated that USP22 was highly expressed in OS tissue and cell lines. Downregulation of USP22 inhibited OS cell proliferation, invasion, and epithelial-mesenchymal transition (EMT) in vitro. In addition, downregulation of USP22 suppressed OS tumor growth and metastasis in vivo. We also found that the PI3K/Akt signaling pathway was involved in the tumor-promoting effect of USP22 on OS progression. Taken together, we suggest USP22 as a novel therapeutic target for OS.