WWP2 is a ubiquitin E3 ligase belonging to the Nedd4-like family. Given that WWP2 target proteins including PTEN that are crucial for regulating cell proliferation or suppressing tumorigenesis, we hav Show more
WWP2 is a ubiquitin E3 ligase belonging to the Nedd4-like family. Given that WWP2 target proteins including PTEN that are crucial for regulating cell proliferation or suppressing tumorigenesis, we have asked whether WWP2 plays a role in controlling cell cycle progression. Here we report that WWP2 is necessary for normal cell cycle progression as its silencing significantly reduces the cell proliferation rate. We have identified that an isoform of WWP2 (WWP2-V4) is highly expressed in the M phase of the cell cycle. Silencing of WWP2 accelerates the turnover of cyclin E, which is accompanied by increased levels of phospho-histone H3 (p-H3) and cyclin B. Moreover, silencing of WWP2 results in compromised phosphorylation of Akt(S473), a residue whose phosphorylation is tightly associated with the activation of the kinase. Combined, these results strongly suggest that WWP2 is an important component in regulating the Akt signaling cascade, as well as cell cycle progression. Show less
Phytochrome-interacting factor 3 (PIF3) activates light-responsive transcriptional network genes in coordination with the circadian clock and plant hormones to modulate plant growth and development. H Show more
Phytochrome-interacting factor 3 (PIF3) activates light-responsive transcriptional network genes in coordination with the circadian clock and plant hormones to modulate plant growth and development. However, little is known of the roles PIF3 plays in the responses to abiotic stresses. In this study, the cloning and functional characterization of the ZmPIF3 gene encoding a maize PIF3 protein is reported. Subcellular localization revealed the presence of ZmPIF3 in the cell nucleus. Expression patterns revealed that ZmPIF3 is expressed strongly in leaves. This expression responds to polyethylene glycol, NaCl stress, and abscisic acid application, but not to cold stress. ZmPIF3 under the control of the ubiquitin promoter was introduced into rice. No difference in growth and development between ZmPIF3 transgenic and wild-type plants was observed under normal growth conditions. However, ZmPIF3 transgenic plants were more tolerant to dehydration and salt stresses. ZmPIF3 transgenic plants had increased relative water content, chlorophyll content, and chlorophyll fluorescence, as well as significantly enhanced cell membrane stability under stress conditions. The over-expression of ZmPIF3 increased the expression of stress-responsive genes, such as Rab16D, DREB2A, OSE2, PP2C, Rab21, BZ8 and P5CS, as detected by real-time PCR analysis. Taken together, these results improve our understanding of the role ZmPIF3 plays in abiotic stresses signaling pathways; our findings also indicate that ZmPIF3 regulates the plant response to drought and salt stresses. Show less
Liver X receptor (LXR) isoforms, LXRα and LXRβ, have similar protein structures and ligands, but diverse tissue distribution. We used two synthetic, non-steroidal LXR agonists, T0901317 and GW3965, to Show more
Liver X receptor (LXR) isoforms, LXRα and LXRβ, have similar protein structures and ligands, but diverse tissue distribution. We used two synthetic, non-steroidal LXR agonists, T0901317 and GW3965, to investigate the effects of LXR agonist modulation on prostate specific antigen (PSA) via the expressions of androgen receptors (AR), LXRα, or LXRβ, in prostate carcinoma cells. LXRα- or LXRβ-knockdown cells were transduced with specific shRNA lentiviral particles. LXRα and LXRβ expressions were assessed by immunoblotting and RT-qPCR assays. Cell proliferation was determined by (3) H-thymidine incorporation assays. The effects of LXR agonists and epigallocatechin gallate (EGCG) on PSA expression were determined by ELISA, immunoblotting, or transient gene expression assays. Treatment with either T0901317 or GW3965 significantly attenuated cell proliferation of LNCaP cells. T0901317 treatment suppressed PSA expression while GW3965 treatment enhanced PSA expression. The increase of PSA promoter activity by GW3965 was dependent on the expression of AR. Either LXRα- or LXRβ-knockdown did not affect the activation of androgen on PSA gene expression. However, as compared with mock knockdown-LNCaP cells, the LXRα-knockdown but not the LXRβ-knockdown attenuated the effects of T0901317 and GW3965 on PSA expressions. The effect of GW3965 on PSA expression was blocked by the addition of EGCG. Our results indicate that T0901317 and GW3965 have divergent effects on PSA expressions. The effects of LXR agonists on PSA expression are LXRα-dependent and AR-dependent. EGCG blocks the inducing effect of GW3965 on PSA expression. Show less
Cryptococcus demonstrates predilection for invasion of the brain, but the mechanism by which Cryptococcus crosses the blood-brain barrier (BBB) to cause brain invasion is largely unknown. In order for Show more
Cryptococcus demonstrates predilection for invasion of the brain, but the mechanism by which Cryptococcus crosses the blood-brain barrier (BBB) to cause brain invasion is largely unknown. In order for Cryptococcus to cross the BBB, there must be a way to either cross human brain microvascular endothelial cells, which are the main constitute of the BBB, or go in between tight junctions. Recent evidence of human brain microvascular endothelial cell responses to transcellular brain invasions includes membrane rearrangements, intracellular signaling pathways and cytoskeletal activations. Several Cryptococcal genes related to the traversal of BBB have been identified, including CPS1, ITR1a, ITR3c, PLB1, MPR1, FNX1 and RUB1. In addition, Cryptococcus neoformans-derived microvesicles may contribute to cryptococcal brain invasion. Paracellularly, Cryptococcus may traverse across BBB using either routes utilizing plasmin, ammonia or macrophages in a Trojan horse mechanism. Show less
RUNX1-RUNX1T1 (formerly AML1-ETO), a transcription factor generated by the t(8;21) translocation in acute myeloid leukemia (AML), dictates a leukemic program by increasing self-renewal and inhibiting Show more
RUNX1-RUNX1T1 (formerly AML1-ETO), a transcription factor generated by the t(8;21) translocation in acute myeloid leukemia (AML), dictates a leukemic program by increasing self-renewal and inhibiting differentiation. Here we demonstrate that the histone demethylase JMJD1C functions as a coactivator for RUNX1-RUNX1T1 and is required for its transcriptional program. JMJD1C is directly recruited by RUNX1-RUNX1T1 to its target genes and regulates their expression by maintaining low H3K9 dimethyl (H3K9me2) levels. Analyses in JMJD1C knockout mice also establish a JMJD1C requirement for RUNX1-RUNX1T1's ability to increase proliferation. We also show a critical role for JMJD1C in the survival of multiple human AML cell lines, suggesting that it is required for leukemic programs in different AML cell types through its association with key transcription factors. Show less
Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cor Show more
Etomidate is a rapid hypnotic intravenous anesthetic agent. The major side effect of etomidate is the reduced plasma concentration of corticosteroids, leading to the abnormal reaction of adrenals. Cortisol and testosterone biosynthesis has similar biosynthetic pathway, and shares several common steroidogenic enzymes, such as P450 side chain cleavage enzyme (CYP11A1) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). The effect of etomidate on Leydig cell steroidogenesis during the cell maturation process is not well established. Immature Leydig cells isolated from 35 day-old rats were cultured with 30 μM etomidate for 3 hours in combination with LH, 8Br-cAMP, 25R-OH-cholesterol, pregnenolone, progesterone, androstenedione, testosterone and dihydrotestosterone, respectively. The concentrations of 5α-androstanediol and testosterone in the media were measured by radioimmunoassay. Leydig cells were cultured with various concentrations of etomidate (0.3-30 μM) for 3 hours, and total RNAs were extracted. Q-PCR was used to measure the mRNA levels of following genes: Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Srd5a1, and Akr1c14. The testis mitochondria and microsomes from 35-day-old rat testes were prepared and used to detect the direct action of etomidate on CYP11A1 and HSD3B1 activity. In intact Leydig cells, 30 μM etomidate significantly inhibited androgen synthesis. Further studies showed that etomidate also inhibited the LH- stimulated androgen production. On purified testicular mitochondria and ER fractions, etomidate competitively inhibited both CYP11A1 and HSD3B1 activities, with the half maximal inhibitory concentration (IC50) values of 12.62 and 2.75 μM, respectively. In addition, etomidate inhibited steroidogenesis-related gene expression. At about 0.3 μM, etomidate significantly inhibited the expression of Akr1C14. At the higher concentration (30 μM), it also reduced the expression levels of Cyp11a1, Hsd17b3 and Srd5a1. In conclusion, etomidate directly inhibits the activities of CYP11A1 and HSD3B1, and the expression levels of Cyp11a1 and Hsd17b3, leading to the lower production of androgen by Leydig cells. Show less
Whether polymorphisms rs11856808 and rs9652490 of the Leucine-rich repeat and Ig domain containing, Nogo receptor-interacting protein-1 (LINGO1) gene, as well as rs10968280, rs13362909 and rs7033345 o Show more
Whether polymorphisms rs11856808 and rs9652490 of the Leucine-rich repeat and Ig domain containing, Nogo receptor-interacting protein-1 (LINGO1) gene, as well as rs10968280, rs13362909 and rs7033345 of the LINGO2 gene, increase the risk for Parkinson's disease (PD) is controversial. Considering the overlap of the clinical and pathological characteristics among PD and multiple system atrophy (MSA), we explored the associations between these five polymorphisms and PD and MSA in a Chinese population. A total of 1055 PD patients, 320 MSA patients, and 810 healthy controls (HCs) were genotyped for these five polymorphisms in LINGO1 and LINGO 2 using Sequenom iPLEX Assay technology. Moreover, after combining our results with available published data, a meta-analysis was conducted to investigate the associations between LINGO 1 rs11856808 and rs9652490 and the risk of PD. The frequency of the minor alleles "T" of LINGO1 rs11856808 was significantly lower in PD than that in HCs (p = 0.011, OR 0.89, 95 % CI 0.81-0.97), but not in MSA. Moreover, there were no significant differences in the minor allele frequency distributions of the other four polymorphisms between PD and HCs, and between MSA and HCs. The meta-analysis showed a lack of association of rs9652490 and PD, regardless of the genetic model or ethnic origin. However, the rs11856808 allele decreased the risk of PD in patients of Asian origin in a dominant genetic model. Our findings suggest that rs11856808 plays a protective role by decreasing the risk for PD, but not for MSA, in Asian population, the other four polymorphisms do not contribute to the risk for PD and MSA. Show less
Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator. The present study aimed to investigate the effect of FGF21 on cholesterol efflux and the expression of ATP binding cassette (ABC) A1 Show more
Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator. The present study aimed to investigate the effect of FGF21 on cholesterol efflux and the expression of ATP binding cassette (ABC) A1 and G1 in human THP-1 macrophage-derived foam cells. Furthermore, the present study aimed to investigate the role of the liver X receptor (LXR) α in this process. A model of oxidized low-density lipoprotein-induced foam cells from human THP-1 cells was established. The effect of FGF21 on cholesterol efflux was analyzed using a liquid scintillation counter. The expression of ABCA1 and ABCG1 was determined using quantitative polymerase chain reaction and western blot analyses. FGF21 was found to enhance apolipoprotein A1- and high-density lipoprotein-mediated cholesterol efflux. FGF21 was also observed to increase the mRNA and protein expression of ABCA1 and ABCG1. Furthermore, LXRα-short interfering RNA attenuated the stimulatory effects induced by FGF21. These findings suggest that FGF21 may have a protective effect against atherosclerosis by enhancing cholesterol efflux through the induction of LXRα-dependent ABCA1 and ABCG1 expression. Show less
Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic c Show more
Microtubule actin crosslinking factor 1 (MACF1), a widely expressed cytoskeletal linker, plays important roles in various cells by regulating cytoskeleton dynamics. However, its role in osteoblastic cells is not well understood. Based on our previous findings that the association of MACF1 with F-actin and microtubules in osteoblast-like cells was altered under magnetic force conditions, here, by adopting a stable MACF1-knockdown MC3T3-E1 osteoblastic cell line, we found that MACF1 knockdown induced large cells with a binuclear/multinuclear structure. Further, immunofluorescence staining showed disorganization of F-actin and microtubules in MACF1-knockdown cells. Cell counting revealed significant decrease of cell proliferation and cell cycle analysis showed an S phase cell cycle arrest in MACF1-knockdown cells. Moreover and interestingly, MACF1 knockdown showed a potential effect on cellular MTT reduction activity and mitochondrial content, suggesting an impact on cellular metabolic activity. These results together indicate an important role of MACF1 in regulating osteoblastic cell morphology and function. Show less
The cell cycle is composed of bistable molecular switches that govern the transitions between gap phases (G1 and G2) and the phases in which DNA is replicated (S) and partitioned between daughter cell Show more
The cell cycle is composed of bistable molecular switches that govern the transitions between gap phases (G1 and G2) and the phases in which DNA is replicated (S) and partitioned between daughter cells (M). Many molecular details of the budding yeast G1-S transition (Start) have been elucidated in recent years, especially with regard to its switch-like behavior due to positive feedback mechanisms. These results led us to reevaluate and expand a previous mathematical model of the yeast cell cycle. The new model incorporates Whi3 inhibition of Cln3 activity, Whi5 inhibition of SBF and MBF transcription factors, and feedback inhibition of Whi5 by G1-S cyclins. We tested the accuracy of the model by simulating various mutants not described in the literature. We then constructed these novel mutant strains and compared their observed phenotypes to the model's simulations. The experimental results reported here led to further changes of the model, which will be fully described in a later article. Our study demonstrates the advantages of combining model design, simulation, and testing in a coordinated effort to better understand a complex biological network. Show less
Fourteen novel compounds were prepared and their antagonistic activities against liver X receptors (LXR) α/β were tested in vitro. Compound 26 had an IC50 value of 6.4 µM against LXRα and an IC50 valu Show more
Fourteen novel compounds were prepared and their antagonistic activities against liver X receptors (LXR) α/β were tested in vitro. Compound 26 had an IC50 value of 6.4 µM against LXRα and an IC50 value of 5.6 µM against LXRβ. Docking studies and the results of structure-activity relationships support the further development of this chemical series as LXRα/β antagonists. Show less
Dickkopf1 (DKK1), a canonical Wnt/β-catenin pathway antagonist, is closely associated with cardiovascular disease and adipogenesis. We performed an in vitro study to determine whether oxidized low-den Show more
Dickkopf1 (DKK1), a canonical Wnt/β-catenin pathway antagonist, is closely associated with cardiovascular disease and adipogenesis. We performed an in vitro study to determine whether oxidized low-density lipoprotein (ox-LDL) increased the expression of DKK1 in macrophages and whether β-catenin and liver X receptor α (LXRα) were involved in this regulation. Induction of DKK1 expression by ox-LDL decreased the level of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) via a Wnt/β-catenin pathway and increased ATP-binding cassette transporter A/G1 (ABCA/G1) levels via a signal transducer and activator of transcription 3 (STAT3) pathway. Lower LOX-1 and higher ABCA/G1 levels inhibited cholesterol loading in macrophages. In conclusion, ox-LDL may induce DKK1 expression in macrophages to inhibit the accumulation of lipids through a mechanism that involves downregulation of LOX-1-mediated lipid uptake and upregulation of ABCA/G1-dependent cholesterol efflux. Show less
To improve an asialoglycoprotein receptor (ASGPR)-based enrichment method for detection of circulating tumor cells (CTCs) of hepatocellular carcinoma (HCC). Peripheral blood samples were collected fro Show more
To improve an asialoglycoprotein receptor (ASGPR)-based enrichment method for detection of circulating tumor cells (CTCs) of hepatocellular carcinoma (HCC). Peripheral blood samples were collected from healthy subjects, patients with HCC or various other cancers, and patients with hepatic lesions or hepatitis. CTCs were enriched from whole blood by extracting CD45-expressing leukocytes with monoclonal antibody coated-beads following density gradient centrifugation. The remaining cells were cytocentrifuged on polylysine-coated slides. Isolated cells were treated by triple immunofluorescence staining with CD45 antibody and a combination of antibodies against ASGPR and carbamoyl phosphate synthetase 1 (CPS1), used as liver-specific markers, and costained with DAPI. The cell slide was imaged and stained tumor cells that met preset criteria were counted. Recovery, sensitivity and specificity of the detection methods were determined and compared by spiking experiments with various types of cultured human tumor cell lines. Expression of ASGPR and CPS1 in cultured tumor cells and tumor tissue specimens was analyzed by flow cytometry and triple immunofluorescence staining, respectively. CD45 depletion of leukocytes resulted in a significantly greater recovery of multiple amounts of spiked HCC cells than the ASGPR(+) selection (Ps < 0.05). The expression rates of either ASGPR or CPS1 were different in various liver cancer cell lines, ranging between 18% and 99% for ASGPR and between 9% and 98% for CPS1. In both human HCC tissues and liver cancer cell lines, there were a few HCC cells that did not stain positive for ASGPR or CPS1. The mixture of monoclonal antibodies against ASGPR and CPS1 identified more HCC cells than either antibody alone. However, these antibodies did not detect any tumor cells in blood samples spiked with the human breast cancer cell line MCF-7 and the human renal cancer cell line A498. ASGPR(+) or/and CPS1(+) CTCs were detected in 29/32 (91%) patients with HCC, but not in patients with any other kind of cancer or any of the other test subjects. Furthermore, the improved method detected a higher CTC count in all patients examined than did the previous method (P = 0.001), and consistently achieved 12%-21% higher sensitivity of CTC detection in all seven HCC patients with more than 40 CTCs. Negative depletion enrichment combined with identification using a mixture of antibodies against ASGPR and CPS1 improves sensitivity and specificity for detecting circulating HCC cells. Show less
Endothelial dysfunction, common in chronic kidney disease (CKD), significantly increases cardiovascular disease risk in CKD patients. This study investigates whether different vitamin D receptor agoni Show more
Endothelial dysfunction, common in chronic kidney disease (CKD), significantly increases cardiovascular disease risk in CKD patients. This study investigates whether different vitamin D receptor agonists exhibit different effects on endothelial function and on aortic gene expression in an animal CKD model. The 5/6 nephrectomized (NX) rat was treated with or without alfacalcidol (0.02, 0.04 and 0.08μg/kg), paricalcitol (0.04 and 0.08μg/kg), or VS-105 (0.004, 0.01 and 0.16μg/kg). All three compounds at the test doses suppressed serum parathyroid hormone effectively. Alfacalcidol at 0.08μg/kg raised serum calcium significantly. Endothelial function was assessed by pre-contracting thoracic aortic rings with phenylephrine, followed by treatment with acetylcholine or sodium nitroprusside. Uremia significantly affected endothelial-dependent aortic relaxation, which was improved by all three compounds in a dose-dependent manner with alfacalcidol and paricalcitol exhibiting a lesser effect. DNA microarray analysis of aorta samples revealed that uremia impacted the expression of numerous aortic genes, many of which were normalized by the vitamin D analogs. Real-time RT-PCR analysis confirmed that selected genes such as Abra, Apoa4, Fabp2, Hsd17b2, and Hspa1b affected by uremia were normalized by the vitamin D analogs with alfacalcidol exhibiting less of an effect. These results demonstrate that different vitamin D analogs exhibit different effects on endothelial function and aortic gene expression in 5/6 NX rats. This article is part of a Special Issue entitled '17th Vitamin D Workshop'. Show less
During development, scaffold proteins serve as important platforms for orchestrating signaling complexes to transduce extracellular stimuli into intracellular responses that regulate dendritic spine m Show more
During development, scaffold proteins serve as important platforms for orchestrating signaling complexes to transduce extracellular stimuli into intracellular responses that regulate dendritic spine morphology and function. Axin ("axis inhibitor") is a key scaffold protein in canonical Wnt signaling that interacts with specific synaptic proteins. However, the cellular functions of these protein-protein interactions in dendritic spine morphology and synaptic regulation are unclear. Here, we report that Axin protein is enriched in synaptic fractions, colocalizes with the postsynaptic marker PSD-95 in cultured hippocampal neurons, and interacts with a signaling protein Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in synaptosomal fractions. Axin depletion by shRNA in cultured neurons or intact hippocampal CA1 regions significantly reduced dendritic spine density. Intriguingly, the defective dendritic spine morphogenesis in Axin-knockdown neurons could be restored by overexpression of the small Rho-GTPase Cdc42, whose activity is regulated by CaMKII. Moreover, pharmacological stabilization of Axin resulted in increased dendritic spine number and spontaneous neurotransmission, while Axin stabilization in hippocampal neurons reduced the elimination of dendritic spines. Taken together, our findings suggest that Axin promotes dendritic spine stabilization through Cdc42-dependent cytoskeletal reorganization. Show less
Persistent neuroadaptations following chronic psychostimulant exposure include reduced striatal dopamine D2 receptor (D2R) levels. The signaling of D2Rs is initiated by Gαi/o proteins and terminated b Show more
Persistent neuroadaptations following chronic psychostimulant exposure include reduced striatal dopamine D2 receptor (D2R) levels. The signaling of D2Rs is initiated by Gαi/o proteins and terminated by regulator of G protein signaling (RGS) proteins. The purpose of this study is to examine the association of the drug taking behavior and gene expression profile of D2/D3Rs, and their associated signaling proteins in the ventral tegmental area (VTA) and nucleus accumbens (NAc) using a rodent model of amphetamine (AMPH) self-administration. Rats were allowed to self-administer AMPH (0.187 mg/kg/infusion for a maximum of 40 injections in 6h daily sessions) for 5 days during which rats showed an escalated rate of AMPH intake across days. AMPH self-administration induced profound brain region-dependent alterations of the targeted genes. There was a positive correlation of the messenger ribonucleic acid (mRNA) levels of RGS10 between the VTA and the NAc in the control animals, which was abolished by AMPH self-administration. AMPH self-administration also produced a negative correlation of the mRNA levels of RGS7 and RGS19 between the two brain regions, which was not present in the control group. Furthermore, AMPH taking behavior was associated with changes in certain gene expression levels. The mRNA levels of RGS2 and RGS4 in both the VTA and NAc were positively correlated with the rate of AMPH intake. Additionally, the rate of AMPH intake was also positively correlated with RGS10 and negatively correlated with RGS17 and the short form of D2Rs mRNA level in the VTA. Although there were significant changes in the mRNA levels of RGS7 and RGS8 in the NAc, none of these measures were correlated with the rate of AMPH intake. The present study suggested that short-term AMPH self-administration produced pronounced changes in the VTA that were more associated with AMPH taking behavior than changes in the NAc. Show less
Recent genome-wide association studies have identified several loci influencing lipid levels. The present study focused on the triglycerides (TG)-associated locus, the APOA4-APOA5-ZNF259-BUD13 gene cl Show more
Recent genome-wide association studies have identified several loci influencing lipid levels. The present study focused on the triglycerides (TG)-associated locus, the APOA4-APOA5-ZNF259-BUD13 gene cluster on chromosome 11, to explore the role of genetic variants in this gene cluster in the development of increasing TG levels and coronary heart disease (CHD). Six single nucleotide polymorphisms (SNPs), rs4417316, rs651821, rs6589566, rs7396835, rs964184 and rs17119975, in the APOA4-APOA5-ZNF259-BUD13 gene cluster were selected and genotyped in 5374 healthy Chinese subjects. There were strong significant associations between the six SNPs and TG levels (P < 1.0 × 10(-8)). Moreover, a weighted genotype score was found to be associated with TG levels (P = 3.28 × 10(-13)). The frequencies of three common haplotypes were observed to be significantly different between the high TG group and the low TG group (P < 0.05). However, no significant effects were found for the SNPs regarding susceptibility to CHD in the Chinese case-control populations. This study highlights the genotypes, genotype scores and haplotypes of the APOA4-APOA5-ZNF259-BUD13 gene cluster that were associated with TG levels in a Chinese population; however, the genetic variants in this gene cluster did not increase the risk of CHD in the Chinese population. Show less
The additional mutational complexity associated with copy number variation (CNV) can provide important clues as to the underlying mechanisms of CNV formation. Correct annotation of the additional muta Show more
The additional mutational complexity associated with copy number variation (CNV) can provide important clues as to the underlying mechanisms of CNV formation. Correct annotation of the additional mutational complexity is, however, a prerequisite for establishing the mutational mechanism. We illustrate this point through the characterization of a novel ∼230 kb EXT1 duplication CNV causing autosomal dominant hereditary multiple osteochondromas. Whole-genome sequencing initially identified the CNV as having a 22-bp insertion at the breakpoint junction and, unprecedentedly, multiple breakpoint-flanking micromutations on both sides of the duplication. Further investigation revealed that this genomic rearrangement had a duplication-inverted triplication-duplication structure, the inverted triplication being a 41-bp sequence synthesized from a nearby template. This permitted the identification of the sequence determinants of both the initiation (an inverted Alu repeat) and termination (a triplex-forming sequence) of break-induced replication and suggested a possible model for the repair of replication-associated double-strand breaks. Show less
Familial hypertrophic cardiomyopathy (HCM) is attributed to mutations in genes that encode for the sarcomere proteins, especially Mybpc3 and Myh7. Genotype-phenotype correlation studies show significa Show more
Familial hypertrophic cardiomyopathy (HCM) is attributed to mutations in genes that encode for the sarcomere proteins, especially Mybpc3 and Myh7. Genotype-phenotype correlation studies show significant variability in HCM phenotypes among affected individuals with identical causal mutations. Morphological changes and clinical expression of HCM are the result of interactions with modifier genes. With the exceptions of angiotensin converting enzyme, these modifiers have not been identified. Although mouse models have been used to investigate the genetics of many complex diseases, natural murine models for HCM are still lacking. In this study we show that the DBA/2J (D2) strain of mouse has sequence variants in Mybpc3 and Myh7, relative to widely used C57BL/6J (B6) reference strain and the key features of human HCM. Four-month-old of male D2 mice exhibit hallmarks of HCM including increased heart weight and cardiomyocyte size relative to B6 mice, as well as elevated markers for cardiac hypertrophy including β-myosin heavy chain (MHC), atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and skeletal muscle alpha actin (α1-actin). Furthermore, cardiac interstitial fibrosis, another feature of HCM, is also evident in the D2 strain, and is accompanied by up-regulation of type I collagen and α-smooth muscle actin (SMA)-markers of fibrosis. Of great interest, blood pressure and cardiac function are within the normal range in the D2 strain, demonstrating that cardiac hypertrophy and fibrosis are not secondary to hypertension, myocardial infarction, or heart failure. Because D2 and B6 strains have been used to generate a large family of recombinant inbred strains, the BXD cohort, the D2 model can be effectively exploited for in-depth genetic analysis of HCM susceptibility and modifier screens. Show less
The unwinding of nucleic acid secondary structures within cells is crucial to maintain genomic integrity and prevent abortive transcription and translation initiation. DHX36, also known as RHAU or G4R Show more
The unwinding of nucleic acid secondary structures within cells is crucial to maintain genomic integrity and prevent abortive transcription and translation initiation. DHX36, also known as RHAU or G4R1, is a DEAH-box ATP-dependent helicase highly specific for DNA and RNA G-quadruplexes (G4s). A fundamental mechanistic understanding of the interaction between helicases and their G4 substrates is important to elucidate G4 biology and pave the way toward G4-targeted therapies. Here we analyze how the thermodynamic stability of G4 substrates affects binding and unwinding by DHX36. We modulated the stability of the G4 substrates by varying the sequence and the number of G-tetrads and by using small, G4-stabilizing molecules. We found an inverse correlation between the thermodynamic stability of the G4 substrates and rates of unwinding by DHX36. In stark contrast, the ATPase activity of the helicase was largely independent of substrate stability pointing toward a decoupling mechanism akin to what has been observed for many double-stranded DEAD-box RNA helicases. Our study provides the first evidence that DHX36 uses a local, non-processive mechanism to unwind G4 substrates, reminiscent of that of eukaryotic initiation factor 4A (eIF4A) on double-stranded substrates. Show less
To evaluate whether the Chinese Han population harbors genetic markers associated with risk of acute myocardial infarction (MI), which have previously been identified in other ethnic populations. Acco Show more
To evaluate whether the Chinese Han population harbors genetic markers associated with risk of acute myocardial infarction (MI), which have previously been identified in other ethnic populations. According to predefined criteria, 549 Chinese patients with acute MI and 551 Chinese subjects (controls) without a history of coronary artery disease (CAD) were selected for the study. Three prevalent single nucleotide polymorphisms (SNPs; rs1412444(LIPA), rs662799(APOA5) and rs964184(ZNF259)) associated with CAD and MI in other ethnic populations, were selected for sequence and association analyses within blood DNA of the Chinese Han population. Only two SNPs, rs662799 (APOA5) and rs964184 (ZNF259) found at two independent loci, were associated with risk of MI in the Chinese Han population. Using Bonferroni correction methods, significant differences in the association of these two SNPs (rs662799 (p = 0.0228) and rs964184 (p = 0.0060)) between Chinese patients with MI versus controls were revealed. We identified a significant association between two SNPs (rs964184 and rs662799) on chromosome 11q23.3 and MI risk in the Chinese Han population, which extends their clinical relevance to predicting the risk of MI in diverse ethnic populations. Show less
FADS1 (fatty acid desaturase 1) plays a crucial role in fatty acid metabolism, and it was recently reported to be involved in tumorigenesis. However, the role of FADS1 expression in esophageal squamou Show more
FADS1 (fatty acid desaturase 1) plays a crucial role in fatty acid metabolism, and it was recently reported to be involved in tumorigenesis. However, the role of FADS1 expression in esophageal squamous cell carcinoma (ESCC) remains unknown. In the current study, we investigated the expression and clinical pathologic and prognostic significance of FADS1 in ESCC. Immunohistochemical analyses revealed that 58.2% (146/251) of the ESCC tissues had low levels of FADS1 expression, whereas 41.8% (105/251) exhibited high levels of FADS1 expression. In positive cases, FADS1 expression was detected in the cytoplasm of cells. Correlation analyses demonstrated that FADS1 expression was significantly correlated with tumor location (p=0.025) but not with age, gender, histological grade, tumor status, nodal status or TNM staging. Furthermore, patients with tumors expressing high levels of FADS1had a longer disease-free survival time (p<0.001) and overall survival time (p<0.001). Univariate and multivariate analyses revealed that, along with nodal status, FADS1 expression was an independent and significant predictive factor (p<0.001). In conclusion, our study suggested that FADS1 might be a valuable biomarker and potential therapeutic target for ESCC. Show less
Ammonia detoxification is essential for physiological well-being, and the urea cycle in liver plays a predominant role in ammonia disposal. Nobiletin (NOB), a natural dietary flavonoid, is known to ex Show more
Ammonia detoxification is essential for physiological well-being, and the urea cycle in liver plays a predominant role in ammonia disposal. Nobiletin (NOB), a natural dietary flavonoid, is known to exhibit various physiological efficacies. In the current study, we investigated a potential role of NOB in ammonia control and the underlying cellular mechanism. C57BL/6 mice were fed with regular chow (RC), high-fat (HFD) or high-protein diet (HPD) and treated with either vehicle or NOB. Serum and/or urine levels of ammonia and urea were measured. Liver expression of genes encoding urea cycle enzymes and C/EBP transcription factors was determined over the circadian cycle. Luciferase reporter assays were carried out to investigate function of CCAAT consensus elements on the carbamoyl phosphate synthetase (Cps1) gene promoter. A circadian clock-deficient mouse mutant, Clock (Δ19/Δ19) , was utilized to examine a requisite role of the circadian clock in mediating NOB induction of Cps1. NOB was able to lower serum ammonia levels in mice fed with RC, HFD or HPD. Compared with RC, HFD repressed the mRNA and protein expression of Cps1, encoding the rate-limiting enzyme of the urea cycle. Interestingly, NOB rescued CPS1 protein levels under the HFD condition via induction of the transcription factors C/EBPα and C/EBPβ. Expression of other urea cycle genes was also decreased by HFD relative to RC and again restored by NOB to varying degrees, which, in conjunction with Cps1 promoter reporter analysis, suggested a C/EBP-dependent mechanism for the co-induction of urea cycle genes by NOB. In comparison, HPD markedly increased CPS1 levels relative to RC, yet NOB did not further enrich CPS1 to a significant extent. Using the circadian mouse mutant Clock (Δ19/Δ19) , we also showed that a functional circadian clock, known to modulate C/EBP and CPS1 expression, was required for NOB induction of CPS1 under the HFD condition. NOB, a dietary flavonoid, exhibits a broad activity in ammonia control across varying diets, and regulates urea cycle function via C/EBP-and clock-dependent regulatory mechanisms. Show less
WW domains harbor substrates containing proline-rich motifs, but the substrate specificity and binding mechanism remain elusive for those WW domains less amenable for structural studies, such as human Show more
WW domains harbor substrates containing proline-rich motifs, but the substrate specificity and binding mechanism remain elusive for those WW domains less amenable for structural studies, such as human WWP2 (hWWP2). Herein we have employed multiple techniques to investigate the second WW domain (WW2) in hWWP2. Our results show that hWWP2 is a specialized E3 for PPxY motif-containing substrates only and does not recognize other amino acids and phospho-residues. The strongest binding affinity of WW2, and the incompatibility between each WW domain, imply a novel relationship, and our SPR experiment reveals a dynamic binding mode in Class-I WW domains for the first time. The results from alanine-scanning mutagenesis and modeling further point to functionally conserved residues in WW2. Show less
Glucose-dependent insulinotropic polypeptide (GIP) is a gut hormone secreted in response to dietary fat and glucose. The blood GIP level is elevated in obesity and diabetes. GIP stimulates proinflamma Show more
Glucose-dependent insulinotropic polypeptide (GIP) is a gut hormone secreted in response to dietary fat and glucose. The blood GIP level is elevated in obesity and diabetes. GIP stimulates proinflammatory gene expression and impairs insulin sensitivity in cultured adipocytes. In obesity, hypoxia within adipose tissue can induce inflammation. The aims of this study were 1) to examine the proinflammatory effect of increased GIP signaling in adipose tissues in vivo and 2) to clarify the association between GIP and hypoxic signaling in adipose tissue inflammation. We administered GIP intraperitoneally to misty (lean) and db/db (obese) mice and examined adipose tissue inflammation and insulin sensitivity. We also examined the effects of GIP and hypoxia on expression of the GIP receptor (GIPR) gene and proinflammatory genes in 3T3-L1 adipocytes. GIP administration increased monocyte chemoattractant protein-1 (MCP-1) expression and macrophage infiltration into adipose tissue and increased blood glucose in db/db mice. GIPR and hypoxia-inducible factor-1α (HIF-1α) expressions were positively correlated in the adipose tissue in mice. GIPR expression increased dramatically in differentiated adipocytes. GIP treatment of adipocytes increased MCP-1 and interleukin-6 (IL-6) production. Adipocytes cultured either with RAW 264 macrophages or under hypoxia expressed more GIPR and HIF-1α, and GIP treatment increased gene expression of plasminogen activator inhibitor 1 and IL-6. HIF-1α gene silencing diminished both macrophage- and hypoxia-induced GIPR expression and GIP-induced IL-6 expression in adipocytes. Thus, increased GIP signaling plays a significant role in adipose tissue inflammation and thereby insulin resistance in obese mice, and HIF-1α may contribute to this process. Show less
MYBPC3 dysfunctions have been proven to induce dilated cardiomyopathy, hypertrophic cardiomyopathy, and/or left ventricular noncompaction; however, the genotype-phenotype correlation between MYBPC3 an Show more
MYBPC3 dysfunctions have been proven to induce dilated cardiomyopathy, hypertrophic cardiomyopathy, and/or left ventricular noncompaction; however, the genotype-phenotype correlation between MYBPC3 and restrictive cardiomyopathy (RCM) has not been established. The newly developed next-generation sequencing method is capable of broad genomic DNA sequencing with high throughput and can help explore novel correlations between genetic variants and cardiomyopathies. A proband from a multigenerational family with 3 live patients and 1 unrelated patient with clinical diagnoses of RCM underwent a next-generation sequencing workflow based on a custom AmpliSeq panel, including 64 candidate pathogenic genes for cardiomyopathies, on the Ion Personal Genome Machine high-throughput sequencing benchtop instrument. The selected panel contained a total of 64 genes that were reportedly associated with inherited cardiomyopathies. All patients fulfilled strict criteria for RCM with clinical characteristics, echocardiography, and/or cardiac magnetic resonance findings. The multigenerational family with 3 adult RCM patients carried an identical nonsense MYBPC3 mutation, and the unrelated patient carried a missense mutation in the MYBPC3 gene. All of these results were confirmed by the Sanger sequencing method. This study demonstrated that MYBPC3 gene mutations, revealed by next-generation sequencing, were associated with familial and sporadic RCM patients. It is suggested that the next-generation sequencing platform with a selected panel provides a highly efficient approach for molecular diagnosis of hereditary and idiopathic RCM and helps build new genotype-phenotype correlations. Show less
To comprehensively understand the underlying molecular events accounting for aberrant Wnt signaling activation in hepatocellular carcinoma (HCC). This study was retrospective. The HCC tissue specimens Show more
To comprehensively understand the underlying molecular events accounting for aberrant Wnt signaling activation in hepatocellular carcinoma (HCC). This study was retrospective. The HCC tissue specimens used in this research were obtained from patients who underwent liver surgery. The Catalogue of Somatic Mutations in Cancer (COSMIC) database was searched for the mutation statuses of CTNNB1, TP53, and protein degradation regulator genes of CTNNB1. Dual-luciferase reporter assay was performed with TOP/FOP reporters to detect whether TP53 gain-of-function (GOF) mutations could enhance the transcriptional activity of Wnt signaling. Methylation sensitive restriction enzyme-quantitative PCR was used to explore the methylation status of CpG islands located in the promoters of APC, SFRP1, and SFRP5 in HCCs with different risk factors. Finally, nested-reverse transcription PCR was performed to examine the integration of HBx in front of LINE1 element and the existence of HBx-LINE1 chimeric transcript in Hepatitis B virus-related HCC. All results in this article were analyzed with the software SPSS version 19.0 for Windows, and different groups were compared by χ(2) test as appropriate. Based on the data from COSMIC database, compared with other solid tumors, mutation frequency of CTNNB1 was significantly higher in HCC (P < 0.01). The rate of CTNNB1 mutation was significantly less frequent in Hepatitis B virus-related HCC than in other etiologies (P < 0.01). Dual-luciferase reporter system and TOP/FOP reporter assays confirmed that TP53 GOF mutants were able to enhance the transcriptional ability of Wnt signaling. An exclusive relationship between the status of TP53 and CTNNB1 mutations was observed. However, according to the COSMIC database, TP53 GOF mutation is rare in HCC, which indicates that TP53 GOF mutation is not a reason for the aberrant activation of Wnt signaling in HCC. APC and AXIN1 were mutated in HCC. By using methylation sensitive restriction enzyme-quantitative PCR, hypermethylation of APC was detected in HCC with different risk factors, whereas SFRP1 and SFRP5 were not hypermethylated in any of the HCC etiologies, which indicates that the mutation of APC and AXIN1, together with the methylation of APC could take part in the overactivation of Wnt signaling. Nested-reverse transcription PCR failed to detect the integration of HBx before the LINE1 element, or the existence of an HBx-LINE1 chimeric transcript, suggesting that integration could not play a role in the aberrant activation of Wnt signaling in HCC. In HCC, genetic/epigenetic aberration of CTNNB1 and its protein degradation regulators are the major cause of Wnt signaling overactivation. Show less
GADD45A is a TP53-regulated and DNA damage-inducible tumor suppressor protein, which regulates cell cycle arrest, apoptosis, and DNA repair, and inhibits tumor growth and angiogenesis. However, the fu Show more
GADD45A is a TP53-regulated and DNA damage-inducible tumor suppressor protein, which regulates cell cycle arrest, apoptosis, and DNA repair, and inhibits tumor growth and angiogenesis. However, the function of GADD45A in autophagy remains unknown. In this report, we demonstrate that GADD45A plays an important role in regulating the process of autophagy. GADD45A is able to decrease LC3-II expression and numbers of autophagosomes in mouse tissues and different cancer cell lines. Using bafilomycin A1 treatment, we have observed that GADD45A regulates autophagosome initiation. Likely, GADD45A inhibition of autophagy is through its influence on the interaction between BECN1 and PIK3C3. Immunoprecipitation and GST affinity isolation assays exhibit that GADD45A directly interacts with BECN1, and in turn dissociates the BECN1-PIK3C3 complex. Furthermore, we have mapped the 71 to 81 amino acids of the GADD45A protein that are necessary for the GADD45A interaction with BECN1. Knockdown of BECN1 can abolish autophagy alterations induced by GADD45A. Taken together, these findings provide the novel evidence that GADD45A inhibits autophagy via impairing the BECN1-PIK3C3 complex formation. Show less
Hereditary multiple osteochondromas (HMO) is an autosomal dominant bone disorder characterised by the presence of multiple benign cartilage-capped tumours. Exostosin-1 (EXT1) and EXT2 are the major mo Show more
Hereditary multiple osteochondromas (HMO) is an autosomal dominant bone disorder characterised by the presence of multiple benign cartilage-capped tumours. Exostosin-1 (EXT1) and EXT2 are the major morbigenous genes associated with HMO, mutations in which are responsible for 90% of all HMO cases. In patients with HMO, osteochondromas arise adjacent to the metaphysis and typically remain in the metaphyseal region of the long bones. Therefore, it is rare for osteochondromas to be identified intra-articularly, although they may manifest as loose bodies. The present study describes a rare case of HMO manifesting as limited flexing range in the right knee joint of a 27-year-old male patient. Computed tomography and magnetic resonance imaging (MRI) revealed three intra-articular osteochondromas located in the intercondylar fossa of the patient's right knee. The intra-articular osteochondromas and protuberant extra-articular osteochondromas around the right knee were resected, resulting in improved right knee function and no postoperative recurrence. Pathological analysis revealed that the intra-articular osteochondromas had a thinner cartilage cap layer than the extra-articular osteochondromas. In addition, genetic analysis of the patient and the patient's mother was conducted. From this, it was determined that a nonsense mutation, c.115G>T (p.E39X) in exon 1 of the EXT1 gene, was the cause of HMO in this case. Thus, it is proposed that osteochondromas with a pedicle within the knee, may tear and become loose intra-articular bodies, resulting in limited joint function and thereby contributing to the progression of HMO. Show less
Smooth muscle 22α (SM22α) is involved in stress fiber formation and enhances contractility in vascular smooth muscle cells (VSMCs). In many cases, SM22α acts as an adapter protein to assemble signalin Show more
Smooth muscle 22α (SM22α) is involved in stress fiber formation and enhances contractility in vascular smooth muscle cells (VSMCs). In many cases, SM22α acts as an adapter protein to assemble signaling complexes and regulate signaling, but whether SM22α regulates contractile signaling induced by angiotensin II (AngII) remains unclear. To address this issue, we established a hypertension model of Sm22α(-/-) mice, and demonstrated that hypertension induced by AngII was attenuated in Sm22α(-/-) mice. A decreased vasoconstriction was observed in aortic rings from Sm22α(-/-) mice. Furthermore, loss of SM22α resulted in a reduced contractile response to AngII in VSMCs in vitro. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) induced by AngII was impaired following depletion of SM22α, in parallel with a reduced contractility. The decay of ERK1/2 activity was associated with increased expression of mitogen-activated protein kinase phosphatase 3 (MKP3). Inhibition of MKP3 activity rescued ERK1/2 activity. SM22α depletion caused an enhanced interaction of MKP3 with ERK1/2, and a reduced ubiquitination and degradation of MKP3. Knockdown of SM22α extended the half-life of MKP3. In conclusion, SM22α promotes AngII-induced contraction by maintenance of ERK1/2 signaling cascades through facilitating ubiquitination and degradation of MKP3. The vasoconstriction is attenuated in aortic rings from Sm22α(-/-) mice. MKP3 mediates dephosphorylation of ERK1/2 in AngII-induced VSMC contraction. SM22α inhibits the interaction of ERK1/2 with MKP3. SM22α promotes ubiquitination and degradation of MKP3. SM22α facilitates AngII-induced contraction by maintenance of ERK1/2 signaling. Show less