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Background/aim: Heavy-ion irradiation seriously perturbs cellular homeostasis and thus damages cells. Vascular endothelial cells (ECs) play an important role in the pathological process of radiation damage. Protecting ECs from heavy-ion radiation is of great significance in the radioprotection of normal tissues. In this study, the radioprotective effect of ß-D-glucan (BG) derived from Saccharomyces cerevisiae on human umbilical vein endothelial cell (EA.hy926) cytotoxicity produced by carbon-ion irradiation was examined and the probable mechanism was established. Materials and methods: EA.hy926 cells were divided into seven groups: a control group; 1, 2, or 4 Gy radiation; and 10 µg/mL BG pretreatment for 24 h before 1, 2, or 4 Gy irradiation. Cell survival was assessed by colony formation assay. Cell cycles, apoptosis, DNA damage, and reactive oxygen species (ROS) levels were measured through flow cytometry. The level of malondialdehyde and antioxidant enzyme activities were analyzed using assay kits. The activation of NF-κB was analyzed using western blotting and a transcription factor assay kit. The expression of downstream target genes was detected by western blotting. Results: BG pretreatment significantly increased the survival of irradiated cells, improved cell cycle progression, and decreased DNA damage and apoptosis. The levels of ROS and malondialdehyde were also decreased by BG. Further study indicated that BG increased the antioxidant enzyme activities, activated Src, and promoted NF-κB activation, especially for the p65, p50, and RelB subunits. The activated NF-κB upregulated the expression of antioxidant protein MnSOD, DNA damage-response and repair-related proteins BRCA2 and Hsp90a, and antiapoptotic protein Bcl-2. Conclusion: Our results demonstrated that BG protects EA.hy926 cells from high linear-energy-transfer carbon-ion irradiation damage through the upregulation of prosurvival signaling triggered by the interaction of BG with its receptor. This confirms that BG is a promising radioprotective agent for heavy-ion exposure. [ABSTRACT FROM AUTHOR]

The functional maturation of neurons is a prolonged process that extends past the mitotic exit and is mediated by the chromatin-dependent orchestration of gene transcription programs. We find that expression of this maturation gene program in mouse cerebellar granule neurons (CGNs) requires dynamic changes in the genomic distribution of histone H3 lysine 27 trimethylation (H3K27me3), demonstrating a function for this chromatin modification beyond its role in cell fate specification. The developmental loss of H3K27me3 at promoters of genes activated as CGNs mature is facilitated by the lysine demethylase and ASD-risk gene, Kdm6b. Interestingly, inhibition of the H3K27 methyltransferase EZH2 in newborn CGNs not only blocks the repression of progenitor genes but also impairs the induction of mature CGN genes, showing the importance of bidirectional H3K27me3 regulation across the genome. These data demonstrate that H3K27me3 turnover in developing postmitotic neurons regulates the temporal coordination of gene expression programs that underlie functional neuronal maturation. [ABSTRACT FROM AUTHOR]

The ability to regulate gene activity spatially and temporally is essential to investigate cell-type- specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish. However, simple and efficient methods for isolation of stable, Cre/lox regulated zebrafish alleles are lacking. Here, we applied our GeneWeld CRISPR-Cas9 targeted integration strategy to generate floxed alleles that provide robust conditional inactivation and rescue. A universal targeting vector, UFlip, with sites for cloning short homology arms flanking a floxed 2A-mRFP gene trap, was integrated into an intron in rbbp4 and rb1. rbbp4off and rb1off integration alleles resulted in strong mRFP expression,>99% reduction of endogenous gene expression, and recapitulated known indel loss-of- function phenotypes. Introduction of Cre led to stable inversion of the floxed cassette, loss of mRFP expression, and phenotypic rescue. rbbp4on and rb1on integration alleles did not cause phenotypes in combination with a loss-of- function mutation. Addition of Cre led to conditional inactivation by stable inversion of the cassette, gene trapping and mRFP expression, and the expected mutant phenotype. Neural progenitor Cre drivers were used for conditional inactivation and phenotypic rescue to showcase how this approach can be used in specific cell populations. Together these results validate a simplified approach for efficient isolation of Cre/lox-responsive conditional alleles in zebrafish. Our strategy provides a new toolkit for generating genetic mosaics and represents a significant advance in zebrafish genetics. [ABSTRACT FROM AUTHOR]

Although plant breeding has traditionally relied on exploiting genetic diversity, epigenetic variations are attracting new attentions in breeding design. Epigenetic modifications play a critical role in regulating gene expression during development and in response to environmental stimulation, and thereby contribute to phenotypic variation. When epigenetic modifications persist during reproduction and are stably transmitted to the next generation, transgenerational inheritance of epigenetic variations has a potential to produce heritable phenotypic diversity. In addition to naturally occurring epialleles, genetic variation due to organization of duplicated genes or transposon polymorphisms are a potential source for inducing epialleles. Furthermore, stress-induced activation and mobilization of transposable elements represent another promising avenue to create new genetic and epigenetic diversity that can be sexually or asexually propagated. Finally, methods for artificially creating epigenetic diversity in experimental models have been developed and applied to select crop species in some cases. With the advance in epigenome profiling techniques, dissection of epigenetic-based complex traits, and the development of molecular tools for locus-specific epigenome editing, the effects of epigenetics for underlying phenotypic traits can be unequivocally elucidated. [ABSTRACT FROM AUTHOR]

Background: MicroRNA-381 (miR-381) has been reported to play suppressive or promoting roles in different malignancies. However, the expression level, biological function, and underlying mechanisms of miR-381 in gastric cancer remain poorly understood. Our previous study indicated that transmembrane protein 16A (TMEM16A) contributed to migration and invasion of gastric cancer and predicted poor prognosis. In this study, we found that miR-381 inhibited the metastasis of gastric cancer through targeting TMEM16A expression. Methods: MiR-381 expression was analyzed using bioinformatic software on open microarray datasets from the Gene Expression Omnibus (GEO) and confirmed by quantitative RT-PCR (qRT-PCR) in human gastric cancer tissues and cell lines. Cell proliferation was investigated using MTT and cell count assays, and cell migration and invasion abilities were evaluated by transwell assay. Xenograft nude mouse models were used to observe tumor growth and pulmonary metastasis. Luciferase reporter assay, western blot, enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were employed to explore the mechanisms of the effect of miR-381 on gastric cancer cells. Results: MiR-381 was significantly down-regulated in gastric cancer tissues and cell lines. Low expression of miR-381 was negatively related to lymph node metastasis, advanced tumor stage and poor prognosis. MiR-381 decreased gastric cancer cell proliferation, migration and invasion in vitro and in vivo. TMEM16A was identified as a direct target of miR-381 and the expression of miR-381 was inversely correlated with TMEM16A expression in gastric cancer tissues. Combination analysis of miR-381 and TMEM16A revealed the improved prognostic accuracy for gastric cancer patients. Moreover, miR-381 inhibited TGF-β signaling pathway and down-regulated epithelial--mesenchymal transition (EMT) phenotype partially by mediating TMEM16A. Conclusions: MiR-381 may function as a tumor suppressor by directly targeting TMEM16A and regulating TGF-β pathway and EMT process in the development of progression of gastric cancer. MiR-381/TMEM16A may be a novel therapeutic candidate target in gastric cancer treatment. [ABSTRACT FROM AUTHOR]

Small non-coding RNAs (sncRNAs), such as microRNAs (miRNAs), small interfering RNAs (siRNAs), PIWIinteracting RNAs (piRNAs), and transfer RNA (tRNA)-derived small RNAs (tsRNAs), play essential roles in regulating various cellular and developmental processes. Over the past three decades, researchers have identified novel sncRNA species from various organisms. These molecules demonstrate dynamic expression and diverse functions, and they are subject to intricate regulation through RNA modifications in both healthy and diseased states. Notably, certain sncRNAs in gametes, particularly sperm, respond to environmental stimuli and facilitate epigenetic inheritance. Collectively, the in-depth understanding of sncRNA functions and mechanisms has accelerated the development of small RNA-based therapeutics. In this review, we present the recent advances in the field, including new sncRNA species and the regulatory influences of RNA modifications. We also discuss the current limitations and challenges associated with using small RNAs as either biomarkers or therapeutic drugs. [ABSTRACT FROM AUTHOR]

Plant non-specific lipid-transfer proteins (nsLTPs) are small, basic proteins present in abundance in higher plants. They are involved in key processes of plant cytology, such as the stablization of membranes, cell wall organization, and signal transduction. nsLTPs are also known to play important roles in resistance to biotic and abiotic stress, and in plant growth and development, such as sexual reproduction, seed development and germination. The structures of plant nsLTPs contain an eight-cysteine residue conserved motif, linked by four disulfide bonds, and an internal hydrophobic cavity, which comprises the lipid-binding site. This structure endows stability and increases the ability to bind and/or carry hydrophobic molecules. There is growing interest in nsLTPs, due to their critical roles, resulting in the need for a comprehensive review of their form and function. Relevant topics include: nsLTP structure and biochemical features, their classification, identification, and characterization across species, sub-cellular localization, lipid binding and transfer ability, expression profiling, functionality, and evolution. We present advances, as well as limitations and trends, relating to the different topics of the nsLTP gene family. This review collates a large body of research pertaining to the role of nsLTPs across the plant kingdom, which has been integrated as an in depth functional analysis of this group of proteins as a whole, and their activities across multiple biochemical pathways, based on a large number of reports. This review will enhance our understanding of nsLTP activity in planta, prompting further work and insights into the roles of this multifaceted protein family in plants. [ABSTRACT FROM AUTHOR]

The present study aimed to investigate the effects of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway on the Wnt/β-catenin signaling pathway in rats with focal cerebral ischemia-reperfusion injury. A total of 96 rat focal cerebral ischemia-reperfusion models, established according to a modified version of Longa's method, were randomly divided into four groups: Sham-operated (S), cerebral ischemia-reperfusion injury (I), cerebral ischemia-reperfusion + basic fibroblast growth factor (bFGF) post-processing and, finally, cerebral ischemia-reperfusion + bFGF post-processing + PI3K inhibitor LY294002 (LY). Each group consisted of 24 rats and each group was divided into four subgroups according to the indicated reperfusion times of 12, 24, 48 and 72 h. The morphological changes of the cortical tissue and the cellular apoptosis were determined using hematoxylin and eosin staining and the terminal deoxynucleotidyl transferase dUTP nick end labeling method, respectively. The expression levels of phosphorylated (p-)Akt, glycogen synthase kinase-3β (GSK-3β) mRNA and β-catenin in the cortical tissue were detected at different time-points. The number of apoptotic cells and the expression levels of p-Akt, GSK-3β mRNA and β-catenin in the I and LY groups were significantly higher compared with those in the S group (P<0.05). In the bFGF group, the number of apoptotic cells and the mRNA expression levels of GSK-3β were significantly decreased, whereas the expression levels of p-Akt and β-catenin were significantly increased compared with those in the I and LY groups (P<0.05). In cerebral ischemia-reperfusion injury, the PI3K/Akt signaling pathway regulated β-catenin, the main member of the Wnt signaling pathway, via GSK-3β, providing information to assist in further investigation of the mechanism of β-catenin in ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

The TEAD (transcriptional enhancer activator domain) proteins share an evolutionarily conserved DNA-binding TEA domain, which binds to the MCAT cis-acting regulatory element. Previous studies have shown that TEAD proteins are involved in regulating the expression of smooth muscle α-actin. However, it remains undetermined whether TEAD proteins play a broader role in regulating expression of other genes in vascular smooth muscle cells. In this study, we show that the expression of TEAD1 is significantly induced during smooth muscle cell phenotypic modulation and negatively correlates with smooth muscle-specific gene expression. We further demonstrate that TEAD1 plays a novel role in suppressing expression of smooth muscle-specific genes, including smooth muscle α-actin, by abolishing the promyogenic function of myocardin, a key mediator of smooth muscle differentiation. Mechanistically, we found that TEAD1 competes with myocardin for binding to serum response factor (SRF), resulting in disruption of myocardin and SRF interactions and thereby attenuating expression of smooth muscle-specific genes. This study provides the first evidence demonstrating that TEAD1 is a novel general repressor of smooth muscle-specific gene expression through interfering with myocardin binding to SRF. [ABSTRACT FROM AUTHOR]

CD146 has been regarded as a novel potential therapeutic target for multiple cancers. The aim of the study was to investigate the expression of CD146 in gastric cancer and evaluate its clinical-pathological and prognostic significance. The expression of CD146 and three epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, β-catenin and vimentin) was examined in 144 gastric cancers by immunohistochemistry. Fifty-nine cases (41.0%) were defined as positive for CD146 expression. We found that CD146 expression correlated positively with lymph node involvement and a poor prognosis, and retained an independent prognostic factor for gastric cancer patients. Furthermore, positive expression of CD146 was strongly associated with loss of the epithelial marker E-cadherin and acquisition of the expression of the mesenchymal markers nuclear β-catenin and vimentin. These findings suggest that CD146 might promote EMT and progression in gastric cancer, and thus may be a potential therapeutic target for patients with gastric cancers. [ABSTRACT FROM AUTHOR]

Background: Oligoarrays have become an accessible technique for exploring the transcriptome, but it is presently unclear how absolute transcript data from this technique compare to the data achieved with tag-based quantitative techniques, such as massively parallel signature sequencing (MPSS) and serial analysis of gene expression (SAGE). By use of the TransCount method we calculated absolute transcript concentrations from spotted oligoarray intensities, enabling direct comparisons with tag counts obtained with MPSS and SAGE. The tag counts were converted to number of transcripts per cell by assuming that the sum of all transcripts in a single cell was 5·105. Our aim was to investigate whether the less resource demanding and more widespread oligoarray technique could provide data that were correlated to and had the same absolute scale as those obtained with MPSS and SAGE. Results: A number of 1,777 unique transcripts were detected in common for the three technologies and served as the basis for our analyses. The correlations involving the oligoarray data were not weaker than, but, similar to the correlation between the MPSS and SAGE data, both when the entire concentration range was considered and at high concentrations. The data sets were more strongly correlated at high transcript concentrations than at low concentrations. On an absolute scale, the number of transcripts per cell and gene was generally higher based on oligoarrays than on MPSS and SAGE, and ranged from 1.6 to 9,705 for the 1,777 overlapping genes. The MPSS data were on same scale as the SAGE data, ranging from 0.5 to 3,180 (MPSS) and 9 to 1,268 (SAGE) transcripts per cell and gene. The sum of all transcripts per cell for these genes was 3.8·105 (oligoarrays), 1.1·105 (MPSS) and 7.6·104 (SAGE), whereas the corresponding sum for all detected transcripts was 1.1·106 (oligoarrays), 2.8·105 (MPSS) and 3.8·105 (SAGE). Conclusion: The oligoarrays and TransCount provide quantitative transcript concentrations that are correlated to MPSS and SAGE data, but, the absolute scale of the measurements differs across the technologies. The discrepancy questions whether the sum of all transcripts within a single cell might be higher than the number of 5·105 suggested in the literature and used to convert tag counts to transcripts per cell. If so, this may explain the apparent higher transcript detection efficiency of the oligoarrays, and has to be clarified before absolute transcript concentrations can be interchanged across the technologies. The ability to obtain transcript concentrations from oligoarrays opens up the possibility of efficient generation of universal transcript databases with low resource demands. [ABSTRACT FROM AUTHOR]

Background: High-throughput systems for gene expression profiling have been developed and have matured rapidly through the past decade. Broadly, these can be divided into two categories: hybridization-based and sequencing-based approaches. With data from different technologies being accumulated, concerns and challenges are raised about the level of agreement across technologies. As part of an ongoing large-scale cross-platform data comparison framework, we report here a comparison based on identical samples between one-dye DNA microarray platforms and MPSS (Massively Parallel Signature Sequencing). Results: The DNA microarray platforms generally provided highly correlated data, while moderate correlations between microarrays and MPSS were obtained. Disagreements between the two types of technologies can be attributed to limitations inherent to both technologies. The variation found between pooled biological replicates underlines the importance of exercising caution in identification of differential expression, especially for the purposes of biomarker discovery. Conclusion: Based on different principles, hybridization-based and sequencing-based technologies should be considered complementary to each other, rather than competitive alternatives for measuring gene expression, and currently, both are important tools for transcriptome profiling. [ABSTRACT FROM AUTHOR]

Over the last decade, gene expression microarrays have had a profound impact on biomedical research. The diversity of platforms and analytical methods available to researchers have made the comparison of data from multiple platforms challenging. In this study, we describe a framework for comparisons across platforms and laboratories. We have attempted to include nearly all the available commercial and 'in-house' platforms. Using probe sequences matched at the exon level improved consistency of measurements across the different microarray platforms compared to annotation-based matches. Generally, consistency was good for highly expressed genes, and variable for genes with lower expression values as confirmed by quantitative real-time (QRT)-PCR. Concordance of measurements was higher between laboratories on the same platform than across platforms. We demonstrate that, after stringent preprocessing, commercial arrays were more consistent than in-house arrays, and by most measures, one-dye platforms were more consistent than two-dye platforms. [ABSTRACT FROM AUTHOR]

Histone deacetylase 4 (HDAC4) is a class II HDAC implicated in controlling gene expression important for diverse cellular functions, but little is known about how its expression and stability are regulated. We report here that this deacetylase is unusually unstable, with a half-life of less than 8 h. Consistent with the instability of HDAC4 protein, its mRNA was also highly unstable (with a half-life of less than 4 h). The degradation of HDAC4 could be accelerated by exposure of cells to ultraviolet irradiation. HDAC4 degradation was not dependent on proteasome or CRM1-mediated export activity but instead was caspase-dependent and was detectable in diverse human cancer lines. Of two potential caspase consensus motifs in HDAC4, both lying within a region containing proline-, glutamic acid-, serine-, and threonine-rich (PEST) sequences, we identified, by site-directed mutagenesis, Asp-289 as the prime cleavage site. Notably, this residue is not conserved among other class IIa members, HDAC5, -7, and -9. Finally, the induced expression of caspase-cleavable HDAC4 led to markedly increased apoptosis. These results therefore unexpectedly link the regulation of HDAC4 protein stability to caspases, enzymes that are important for controlling cell death and differentiation. [ABSTRACT FROM AUTHOR]

Cotton is a major crop that provides the most important renewable textile fibers in the world. Studies of the taxonomy and evolution of cotton species have received wide attentions, not only due to cotton’s economic value but also due to the fact that Gossypium is an ideal model system to study the origin, evolution, and cultivation of polyploid species. Previous studies suggested the involvement of mitochondrial genome editing sites and copy number as well as mitochondrial functions in cotton fiber elongation. Whereas, with only a few mitogenomes assembled in the cotton genus Gossypium, our knowledge about their roles in cotton evolution and speciation is still scarce. To close this gap, here we assembled 20 mitogenomes from 15 cotton species spanning all the cotton clades (A-G, K, and AD genomes) and 5 cotton relatives using short and long sequencing reads. Systematic analyses uncovered a high level of mitochondrial gene sequence conservation, abundant sequence repeats and many insertions of foreign sequences, as well as extensive structural variations in cotton mitogenomes. The sequence repeats and foreign sequences caused significant mitogenome size inflation in Gossypium and its close relative Kokia in general, while there is no significant difference between the lint and fuzz cotton mitogenomes in terms of gene content, RNA editing, and gene expression level. Interestingly, we further revealed the specific presence and expression of two novel mitochondrial open reading frames (ORFs) in lint-fiber cotton species. Finally, these structural features and novel ORFs help us gain valuable insights into the history of cotton evolution and polyploidization and the origin of species producing long lint fibers from a mitogenomic perspective. [ABSTRACT FROM AUTHOR]

Background: Although the biological insight of acute myeloid leukemia (AML) has increased in the past few years, the discovery of novel discriminative biomarkers remains of utmost value for improving outcome predictions. Systematical studies concerning the clinical implications and genetic correlations of HOXA9 aberrations in patients with AML are relatively promising. Materials and methods: Here, we investigated mutational status and the mRNA levels of the HOXA9 gene in 258 patients with AML. Furthermore, hematological characteristics, chromosome abnormalities, and genetic mutations associated with AML were analyzed, followed by the assessment of clinical survival. Besides, the expression level and mutational status of MEIS1, a cofactor of HOXA9, were also detected in patients with AML with the aim of a deeper understanding about the homeodomain-containing transcription factors associated with hematological characteristics. Results: HOXA9 and MEIS1 mutations were detected in 4.26% and 3.49% AML cases, respectively. No correlations were detected between mutation status and clinical characteristics, cytogenetic and genetic aberrations, and clinical survival. Higher HOXA9 expression levels were correlated with white blood cell count and closely associated with unfavorable karyotype as well as MLL-PTD and EZH2 mutations, whereas, there was an inverse correlation with the French-American-British M3 subtype. Compared with patients with lower HOXA9 expression levels, those with higher HOXA9 expression levels had a lower complete remission rate and inferior survivals in both AML and cytogenetically normal AML. Conclusion: HOXA9 expression may serve as a promising biomarker to ameliorate a prognostic model for predicting clinical outcome and consummating individualized treatment in patients with AML. [ABSTRACT FROM AUTHOR]

Overactivation of Wnt/b-catenin pathway due to dysfunction of retinoid-related orphan receptor a (RORa) is related to cancer development and progression. Diallyl disulfide (DADS), an active component of garlic, has been reported in our previous study for upregulation of RORa expression in gastric cancer (GC) cells. It remains to be elucidated the role and mechanism of RORa in DADS against GC. This study revealed that DADS treatment resulted in reduced expression levels of Wnt1, b-catenin, TCF-4, intranuclear b-catenin and p-b-catenin in GC cells, concomitant with the compromised expression of b-catenin target genes (Axin, c-Jun, and c-Myc). RORa overexpression augmented DADS-induced downregulation of Wnt1/b-catenin pathway, G2/M phase arrest, and cell growth inhibition in vitro and in vivo. Contrarily, knockdown of RORa attenuated these effects of DADS. Interestingly, DADS induced an increase in the binding of RORa to b-catenin, which may lead to reduction of b-catenin phosphorylation and nuclear translocation. This interplay modulated by DADS may affect b-catenin target gene expression for that the opposite results were observed in DADS-treated RORa knockdown and overexpression cells. DADS caused a decrease in vimentin, snail and MMP-9, as well as an increase in E-cadherin and TIMP3 expression, which restricted epithelialemesenchymal transition (EMT), migration, and invasion. The aforementioned effects of DADS were weakened simultaneously when the suppression of DADS on the Wnt1/b-catenin pathway was resisted by knockdown of RORa. In contrast, overexpression of RORa enhanced the effects of DADS. Therefore, RORa-mediated downregulation of Wnt1/b-catenin pathway could undertake an important role in anticancer activity of DADS against GC cell proliferation, EMT, migration, and invasion. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) infection is one of the major causes of hepatocellular carcinoma (HCC). Kinesin family member 4A (KIF4A) is a microtubule-based motor protein, which is upregulated in cervical and lung cancer. However, the expression of KIF4A in HBV-associated HCC, and the effect of HBV on the expression of KIF4A remain to be elucidated. In the present study, the expression profiles of KIF4A were examined in cancerous tissues and paracancerous tissues from patients with HCC, who presented with histories of chronic HBV infection, and the role of HBV in the induction of the expression of KIF4A was investigated. HepG2 cells were transfected with the pHBV1.3, HBV infectious clone and a construct, which contained the luciferase gene under the control of the KIF4A gene promoter. The results demonstrated that the expression of KIF4A was significantly higher in the HCC tissues than in the paracancerous tissues. HBV activated the KIF4A gene promoter and upregulated the mRNA and protein expression of KIF4A. Furthermore, activation of the gene expression of KIF4A increased in a pHBV1.3 concentration-dependent manner. These results provide novel insights into the understanding of HCC oncogenesis caused by HBV. [ABSTRACT FROM AUTHOR]

Aims To determine the use of p40 and laminin 332 (LN332) immunostains for diagnosing metaplastic carcinoma by studying the expression of these and other routine markers in spindle cell metaplastic carcinomas and other malignant spindle cell tumours. Methods We identified cases of spindle cell metaplastic carcinoma (n=36) and other atypical/ malignant spindle cell tumours, including 20 phyllodes tumours (14 borderline, six malignant) and 23 spindle cell sarcomas (three primary to breast). Immunohistochemical staining was performed for p40 and two LN332 chains, β3 (kalinin B1) and γ2 (lamC2). The expression of these markers was compared with p63 and cytokeratins. Results p40 and p63 expression was seen in 21 of 36 (58.3%) and 33 of 36 (91.7%) metaplastic carcinomas, respectively. No phyllodes tumours showed stromal expression of p40 or p63. One of 23 (4.3%) sarcomas showed focal weak p63 staining. LamC2 and kalinin B1 expression was seen in 28 of 36 (77.8%) and 26 of 36 (72.2%) metaplastic carcinomas, respectively. LamC2 and kalinin B1 each showed positive stromal cell expression in two of 20 (10%) phyllodes tumours. No sarcomas showed staining with lamC2. Kalinin B1 staining was seen in 17 of 23 (73.9%) sarcomas, including two of three primary breast sarcomas. Cytokeratin expression was seen in 32 of 36 (88.9%) metaplastic carcinomas and diffuse staining was most often seen in 34βE12 and CK5. Conclusions The diagnostic value of relatively novel markers p40 and LN332 was found to be less than that of routinely used markers (p63 and cytokeratins). p40 proved to be a specific marker but lacked the sensitivity of p63, while LN332 showed staining in a significant proportion of phyllodes tumours and sarcomas. [ABSTRACT FROM AUTHOR]

A study was conducted to determine the effects of dietary non-protein energy sources on growth, tissue lipid accumulation and lipid metabolism-related genes expression of grass carp. Triplicate groups of fish were fed for 9 weeks on four isonitrogenous (300 g kg−1) experimental diets with four levels of non-protein energy (6.52 kJ g−1 control diet, 5.32 kJ g−1 high- CEL diet, 8.46 kJ g−1 high- CHO diet and 8.53 kJ g−1 high- LIP diet respectively). Increasing dietary non-protein energy source levels did not improve the growth, and the high- CEL diet reduced the growth of grass carp. The high- CHO diet tended to induce high hepatosomatic index, with high fat and glycogen content of liver. However, the high- LIP diet caused the high mesenteric fat index, but did not increase liver fat. The m RNA abundance and activities of hepatic lipogenic enzymes were significantly increased in the high- CHO diet group, whereas the opposite tendencies were observed in the high- LIP diet group. Peroxisome proliferator-actived receptor-α ( PPARα) in liver and PPARγ in mesenteric adipose tissue were up-regulated in the high- CEL diet group. Lipoprotein lipase ( LPL) gene expression was significantly increased both in liver and mesenteric adipose tissue of fish fed the high- LIP diet, while the LPL gene expression was up-regulated in liver but down-regulated in mesenteric adipose tissue of fish fed the high- CEL diet. These findings suggest that an increase in dietary non-protein energy sources alters the genes expression of lipid metabolism and increased lipid deposition. [ABSTRACT FROM AUTHOR]

Aim: To clone the hemocyanin gene from Macrobrachium nipponense, and to do the bioinformatics and spatial and temporal expressions analysis of the gene. Material and methods: The hemocyanin gene (MNHc) was cloned from hepatopancreas of Macrobrachium nipponense using the rapid amplification of cDNA ends method and its sequence was analyzed with biological software. Spatial and temporal expressions of the gene were detected by real-time PCR. Results: The full-length cDNA of hemocyanin was 2151 bp, containing a 14 bp of 5'-untranslated region, a 148 bp of 3'-untranslated region and a 1989 bp of open reading frame. This gene encoded a protein of 663 amino acids with a calculated molecular mass of 76.65 kDa and isoelectric point of 5.42. The deduced amino acid sequence contained three hemocyanin domains and two conserved copper-binding sites, and exhibited higher similarity with that of Pacifastacus leniusculus (70%) and Litopenaeus vannamei (63%). This gene was expressed highly in the hepatopancreas, weakly in the muscles and haemocytes, and hardly in the mandibular organ, epidermis and ovarian. Its transcript derived from hepatopancreas was varied at different stages during the molt cycle. The transcript level of gene increased significantly 3 h after injection of pathogenic bacteria Aeromonas hydrophila. Conclusion: Like other crustaceans, the hemocyanin gene of Macrobrachium nipponense contained the typical hemocyanin domains and conserved copper-binding sites, and was highly expressed in hepatopancreas during stage C, as well as acted as an important molecule involved in immune defence of Aeromonas hydrophila. [ABSTRACT FROM AUTHOR]

The mouse Flk1 gene is expressed in various mesodermal progenitor cells of developing embryos. Recent studies have shown that Flk1 expression marks multipotent mesodermal progenitors, giving rise to various hemato-cardiovascular cell lineages during development. Flk1 expression also marks hemato-cardiovascular cell lineages in differentiating embryonic stem (ES) cells, which may be used in transplantation decisions to treat cardiovascular diseases. Despite its developmental and clinical importance in cardiovascular tissues, the transcriptional regulatory system of Flk1 has remained unclear. Here, we report a novel enhancer of the mouse Flk1 gene directing early mesodermal expression during development as well as ES differentiation. The enhancer enriches various mesodermal progenitors, such as primitive erythropoietic progenitors, hemangioblast (BL-CFC) and cardiovascular progenitors (CV-CFC). The enhancer is activated by Bmp, Wnt and Fgf, and it contains Gata-, Cdx-, Tcf/Lef-, ER71/Etv2- and Fox-binding sites, some of which are bound specifically by each of these transcription factors. As these transcription factors are known to act under the control of the Bmp, Wnt and Fgf families, early Flk1 expression may be induced by cooperative interactions between Gata, Tcf/Lef, Cdx and ER71/Etv2 under the control of Bmp, Wnt and Fgf signaling. The enhancer is required for early Flk1 expression and for hemangioblast development during ES differentiation. [ABSTRACT FROM AUTHOR]

Bmi-1 and SALL4 are putative oncogenes that modulate stem cell pluripotency and play a role in leukemogenesis. Murine Sail4 also has been shown to play an essential role in maintaining the properties of ES cells and governing the fate of the primitive inner cell mass. Here, we demonstrate that transcription from the Bmi-1 promoter is strikingly activated by SALL4 in a close-dependent manner by using a luciferase reporter gene assay. Both promoter deletion construct studies and ChIP from a myeloid stem cell line, 32D, demonstrate that SALL4 binds to a specific region of the Bmi-1 promoter. Deletion of one copy of Sall4 by gene targeting in mouse bone marrow significantly reduced Bmi-1 expression. Reducing SALL4 expression by siRNA in the HL-60 leukemia cell line also results in significant down-regulation of Bmi-1. Furthermore, Bmi-1 expression is up-regulated in transgenic mice that constitutively overexpress human SALL4, and the levels of Bmi-1 in these mice increase as they progress from normal to preleukemic (myelodysplastic syndrome) and leukemic (acute myeloid leukemia) stages. High levels of H3-K4 trimethylation and H3-K79 dimethylation were observed in the SALL4 binding region of the Bmi-1 promoter. These findings suggest a novel link between SALL4 and Bmi-1 in regulating self-renewal of normal and leukemic stem cells. An increase in histone H3-K4 and H3-K79 methylation within the Bmi-1 promoter provides an epigenetic mechanism for histone modifications in SALL4-mediated Bmi-1 gene deregulation. [ABSTRACT FROM AUTHOR]

Qizhen capsule (QZC) is a traditional Chinese medicine (TCM) preparation that has been widely used in clinical practice and exerts promising therapeutic effects against breast, lung, and gastric cancers. However, studies have not reported whether QZC inhibits colorectal cancer (CRC) development and progression. Meanwhile, the underlying molecular mechanisms of its anticancer activity have not been studied. To investigate the anticancer effects of QZC on CRC and the possible underlying molecular mechanisms of QZC in vitro and in vivo. The MTT assay and flow cytometry were used to determine the viability and apoptosis of HCT116 and HT-29 cancer cells. A xenograft nude mouse model was used to study the antitumor effects of QZC in vivo. Western blotting was performed to determine the expression of key proteins responsible for the molecular mechanisms elicited by QZC. Immunofluorescence staining was performed to detect the expression of nonsteroidal anti-inflammatory drug (NSAID)-activated gene-1 or growth differentiation factor-15 (NAG-1/GDF15). Small interfering RNAs (siRNAs) were used to silence NAG-1/GDF15 in cells. In this study, QZC significantly reduced the viability of HCT116 and HT-29 cells and induced apoptosis in dose- and time-dependent manners, but displayed much less toxicity toward normal cells. QZC-induced apoptosis in HCT116 cells was accompanied by the deregulation of the expression of the Bcl-2, Bax, PARP, caspase-3, and caspase-9 proteins. Furthermore, QZC induced NAG-1/GDF15 expression in HCT116 cells, while silencing of NAG-1/GDF15 attenuated QZC-induced apoptosis and cell death. Next, QZC increased the phosphorylation of mTOR, AMPK, p38, and MAPK/ERK in HCT116 cells. We then demonstrated that QZC-induced apoptosis and NAG-1/GDF15 upregulation were mediated by MAPK/ERK activation. Moreover, QZC significantly inhibited HCT116 xenograft tumor growth in nude mice, which was accompanied by NAG/GDF15 upregulation and MAPK/ERK activation. QZC also prevented 5-FU-induced weight loss or cachexia in tumor-bearing mice. The expression of Ki67 and PCNA was suppressed, while cleaved caspase-3 level and TUNEL staining were increased in the tumor sections from QZC-treated mice compared to the control. QZC is a novel anticancer agent for CRC that targets NAG-1/GDF15 via the MAPK/ERK signaling pathway. [Display omitted] • Qizhen capsule (QZC) preferably inhibits cell viability in colorectal cancer cells. • NAG/GDF15 is responsible for QZC-induced apoptosis in colorectal cancer cells. • QZC induces apoptosis through activating MAPK/ERK signaling pathway. • QZC inhibits colorectal cancer tumorigenesis in xenograft tumor mice. • QZC prevents 5-FU-induced weight loss in tumor-bearing mice. [ABSTRACT FROM AUTHOR]

Background: Although the functional redundancy of catechol 1,2-dioxygenase (C12O) genes has been reported in several microorganisms, limited enzymes were characterised, let alone the advantage of the coexistence of the multiple copies of C12O genes. Results: In this study, four novel C12O genes, designated catA, catAI, catAII and catAIII, in the naphthalene-degrading strain Pseudomonas putida ND6, were cloned and characterised. Phylogenetic analysis of their deduced amino acid sequences revealed that the four C12O isozymes each formed independent subtrees, together with homologues from other organisms. All four enzymes exhibited maximum activity at pH 7.4 and higher activity in alkaline than in acidic conditions. Furthermore, CatA, CatAI and CatAIII were maximally active at a temperature of 45°C, whereas a higher optimum temperature was observed for CatAII at a temperature of 50°C. CatAI exhibited superior temperature stability compared with the other three C12O isozymes, and kinetic analysis indicated similar enzyme activities for CatA, CatAI and CatAII, whereas that of CatAIII was lower. Significantly, among metal ions tested, only Cu2+ substantially inhibited the activity of these C12O isozymes, thus indicating that they have potential to facilitate bioremediation in environments polluted with aromatics in the presence of metals. Moreover, gene expression analysis at the mRNA level and determination of enzyme activity clearly indicated that the redundancy of the catA genes has increased the levels of C12O. Conclusion: The results clearly imply that the redundancy of catA genes increases the available amount of C12O in P. putida ND6, which would be beneficial for survival in challenging environments. [ABSTRACT FROM AUTHOR]

Host innate immunity is crucial for cellular responses against viral infection sensed by distinct pattern recognition receptors and endoplasmic reticulum (ER) stress. Enterovirus 71 (EV71) is a causative agent of hand, foot, and mouth disease and neurological diseases. However, the exact mechanism underlying the link between ER stress induced by EV71 infection and host innate immunity is largely unknown. In this study, we demonstrated that EV71 infection induces the homocysteine-induced ER protein (HERP), a modulator of the ER stress response which is dependent on the participation of MAVS. Virus-induced HERP subsequently stimulates host innate immunity to repress viral replication by promoting type-I IFNs (IFN-a and IFN-b) and type-III IFN (IFN-l1) expression. Through interacting with TANK-binding kinase 1, HERP amplifies the MAVS signaling and facilitates the phosphorylation and nuclear translocation of IFN regulatory factor 3 and NF-kB to enhance the expression of IFNs, which leads to a broad inhibition of the replication of RNA viruses, including EV71, Sendai virus, influenza A virus, and vesicular stomatitis virus. Therefore, we demonstrated that HERP plays an important role in the regulation of host innate immunity in response to ER stress during the infection of RNAviruses. These findings provide new insights into the mechanism underlying the replication of RNAviruses and the production of IFNs, and also demonstrate a new role of HERP in the regulation of host innate immunity in response to viral infection. [ABSTRACT FROM AUTHOR]

The present study aimed at investigating the selective enrichment of surface marker and functional microRNA (miRNA) profiles of cluster of differentiation (CD)34+ blast-derived microvesicles (MVs) from parental cells in chronic myelogenous leukemia (CML), thus providing an experimental basis for MVs to be used to predict characteristics of CD34+ blasts. Magnetic activated cell sorting and continuous differential centrifugation were used to isolate primary CML CD34+ blasts and MVs, in addition to utilizing flow cytometry to identify surface markers of CD34+ blasts and blast-derived MVs. Microarray analysis and the reverse transcription-quantitative polymerase chain reaction were performed to analyze miRNA profiles of CD34+ blasts and MVs. The results of the present study indicated that primary CML CD34+ blasts were able to release MVs, which were selectively enriched with the surface markers CD34 and CD123, and functional miRNAs from parental cells. A total of 15 miRNAs were upregulated in CD34+ blast derived-MVs compared with in CD34+ cells. Distinct Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology terms characterized by altered gene expression and potentially associated miRNA were identified. Upregulated miRNAs in MVs were associated with cell development, tumorigenesis and signaling pathways involving ErbB and phosphoinositide 3-kinase/protein kinase B. The present study provides evidence, which increases the understanding of physiological functions of cancer-derived MVs, and aids the understanding of the roles of CD34+ blast-derived MVs in CML-associated processes. [ABSTRACT FROM AUTHOR]

In mammals, interferon regulatory factor 4 (IRF4) plays an important role in the process of development and differentiation of B cells, T cells and dendritic cells. It can regulate immune pathway through IRF5, MyD88, IL21, PGC1α, and NOD2. In the present study, we investigated the expression pattern of IRF4 paralogues and these related genes for the first time in teleosts. The results showed that these genes were all expressed predominantly in known immune tissues while IRF5 was also relatively highly expressed in muscle. IRF4b, IL21, MyD88, IRF5 and NOD2 showed maternal expression in the oocyte and the higher expression of IRF4a, Mx and PGC1α before hatching might be involved in the embryonic innate defense system. Zebrafish embryonic fibroblast (ZF4) cells were infected with GCRV and SVCV. During GCRV infection, the expression of Mx was significantly up-regulated from 3 h to 24 h, reaching the highest level at 12 h (101.5-fold over the controls, P < 0.001). And the expression of IRF4a was significantly up-regulated from 3 h to 48 h, reaching the highest level at 12 h (13.75-fold over the controls, P < 0.001). While the expression of IRF4b was only slightly up-regulated at 12 h and 24 h (3.39-fold, 1.93-fold) above control levels, respectively. Whereas the expression of Mx was significantly up-regulated during SVCV infection from 1 h to 48 h, reaching the highest level at 24 h (11.49-fold over the controls, P < 0.001). IRF4a transcripts were significantly up-regulated from 6 h to 24 h, reaching the highest level at 24 h (41-fold over the controls, P < 0.01). IRF4b only showed a slightly up-regulation by SVCV at 24 h (3.2-fold over the controls, P < 0.01). IRF4a and IRF4b displayed a distinct tissue expression pattern, embryonic stages expression and inducible expression in vivo and in vitro, suggesting that IRF4 paralogues might play different roles in immune system. [ABSTRACT FROM AUTHOR]

Steroidogenic acute regulatory (StAR) protein plays a pivotal role in steroidogenesis. Previously, we have demonstrated that prenatal nicotine exposure suppressed fetal adrenal steroidogenesis via steroidogenic factor 1 deacetylation. This study further explored the potential role of the transcriptional repressor Yin Yang 1 (YY1) in nicotine-mediated StAR inhibition. Nicotine was subcutaneously administered (1.0 mg/kg) to pregnant rats twice per day and NCI-H295A cells were treated with nicotine. StAR and YY1 expression were analyzed by real-time PCR, immunohistochemistry, and Western blotting. Histone modifications and the interactions between the YY1 and StAR promoter were assessed using chromatin immunoprecipitation (ChIP). Prenatal nicotine exposure increased YY1 expression and suppressed StAR expression. ChIP assay showed that there was a decreasing trend for histone acetylation at the StAR promoter in fetal adrenal glands, whereas H3 acetyl-K14 at the YY1 promoter presented an increasing trend following nicotine exposure. Furthermore, in nicotine-treated NCI-H295A cells, nicotine enhanced YY1 expression and inhibited StAR expression. ChIP assay showed that histone acetylation decreased at the StAR promoter in NCI-H295A cells and that the interaction between the YY1 and StAR promoter increased. These data indicated that YY1-medicated histone deacetylation modification in StAR promoters might play an important role in the inhibitory effect of nicotine on StAR expression. [ABSTRACT FROM AUTHOR]

Genome-wide association studies (GWAS) of bladder cancer have identified a number of susceptibility loci in European populations but have yet to uncover the genetic determinants underlying bladder cancer incidence among other ethnicities. Therefore, we performed the first GWAS in a Chinese cohort comprising 3,406 cases of bladder cancer and 4,645 controls. We identified a new susceptibility locus for bladder cancer at 5q12.3, located in the intron of CWC27 (rs2042329), that was significantly associated with disease risk (OR = 1.40; P = 4.61 × 10-11). However, rs2042329 was not associated with bladder cancer risk in patients of European descent. The rs2042329 risk allele was also related to significantly increased expression levels of CWC27 mRNA and protein in bladder cancer tissues from Chinese patients. Additional functional analyses suggested that CWC27 played an oncogenic role in bladder cancer by inducing cell proliferation and suppressing apoptosis. In conclusion, the identification of a risk-associated locus at 5q12.3 provides new insights into the inherited susceptibility to bladder cancer in Chinese populations and may help to identify high-risk individuals. [ABSTRACT FROM AUTHOR]

To identify potential key microRNAs (miRNAs) and their target genes for colorectal cancer (CRC). High-throughput sequencing data of miRNA expression and gene expression (ID: GSE46622) were downloaded from Gene Expression Omnibus, including matched colon tumor, normal colon epithelium, and liver metastasis tissues from eight CRC patients. Paired t-test and NOISeq separately were utilized to identify differentially expressed miRNAs (DE-miRNAs) and genes. Then, target genes with differential expression and opposite expression trends were identified for DE-miRNAs. Combined with tumor suppressor gene, tumor-associated gene, and TRANSFAC databases, CRC-restricted miRNAs were screened out based on miRNA-target pairs. Compared with normal tissues, there were 56 up- and 37 downregulated miRNAs in metastasis tissues, as well as eight up- and 30 downregulated miRNAs in tumor tissues. miRNA-1 was downregulated in tumor and metastasis tissues, while its target oncogenes TWIST1 and GATA4 were upregulated. Besides, miRNA-let-7f-1-3p was downregulated in tumor tissues, which also targeted TWIST1. In addition, miRNA-133b and miRNA-4458 were downregulated in tumor tissues, while their common target gene DUSP9 was upregulated. Conversely, miRNA-450- b-3p was upregulated in metastasis tissues, while its target tumor suppressor gene CEACAM7 showed downregulation. The identified CRC-restricted miRNAs might be implicated in cancer progression via their target genes, suggesting their potential usage in CRC treatment. [ABSTRACT FROM AUTHOR]

Background: Adipose tissue-derived stromal stem cells (ASCs) represent a promising regenerative resource for soft tissue reconstruction. Although autologous grafting of whole fat has long been practiced, a major clinical limitation of this technique is inconsistent long-term graft retention. To understand the changes in cell function during the transition of ASCs into fully mature fat cells, we compared the transcriptome profiles of cultured undifferentiated human primary ASCs under conditions leading to acquisition of a mature adipocyte phenotype. Methods: Microarray analysis was performed on total RNA extracted from separate ACS isolates of six human adult females before and after 7 days (7 days: early stage) and 21 days (21 days: late stage) of adipocyte differentiation in vitro. Differential gene expression profiles were determined using Partek Genomics Suite Version 6.4 for analysis of variance (ANOVA) based on time in culture. We also performed unsupervised hierarchical clustering to test for gene expression patterns among the three cell populations. Ingenuity Pathway Analysis was used to determine biologically significant networks and canonical pathways relevant to adipogenesis. Results: Cells at each stage showed remarkable intra-group consistency of expression profiles while abundant differences were detected across stages and groups. More than 14,000 transcripts were significantly altered during differentiation while ~6000 transcripts were affected between 7 days and 21 days cultures. Setting a cutoff of +/-two-fold change, 1350 transcripts were elevated while 2929 genes were significantly decreased by 7 days. Comparison of early and late stage cultures revealed increased expression of 1107 transcripts while 606 genes showed significantly reduced expression. In addition to confirming differential expression of known markers of adipogenesis (e.g., FABP4, ADIPOQ, PLIN4), multiple genes and signaling pathways not previously known to be involved in regulating adipogenesis were identified (e.g. POSTN, PPP1R1A, FGF11) as potential novel mediators of adipogenesis. Quantitative RT-PCR validated the microarray results. Conclusions: ASC maturation into an adipocyte phenotype proceeds from a gene expression program that involves thousands of genes. This is the first study to compare mRNA expression profiles during early and late stage adipogenesis using cultured human primary ASCs from multiple patients. [ABSTRACT FROM AUTHOR]

Background: Adipose tissue-derived stromal stem cells (ASCs) represent a promising regenerative resource for soft tissue reconstruction. Although autologous grafting of whole fat has long been practiced, a major clinical limitation of this technique is inconsistent long-term graft retention. To understand the changes in cell function during the transition of ASCs into fully mature fat cells, we compared the transcriptome profiles of cultured undifferentiated human primary ASCs under conditions leading to acquisition of a mature adipocyte phenotype. Methods: Microarray analysis was performed on total RNA extracted from separate ACS isolates of six human adult females before and after 7 days (7 days: early stage) and 21 days (21 days: late stage) of adipocyte differentiation in vitro. Differential gene expression profiles were determined using Partek Genomics Suite Version 6.4 for analysis of variance (ANOVA) based on time in culture. We also performed unsupervised hierarchical clustering to test for gene expression patterns among the three cell populations. Ingenuity Pathway Analysis was used to determine biologically significant networks and canonical pathways relevant to adipogenesis. Results: Cells at each stage showed remarkable intra-group consistency of expression profiles while abundant differences were detected across stages and groups. More than 14,000 transcripts were significantly altered during differentiation while ~6000 transcripts were affected between 7 days and 21 days cultures. Setting a cutoff of +/-two-fold change, 1350 transcripts were elevated while 2929 genes were significantly decreased by 7 days. Comparison of early and late stage cultures revealed increased expression of 1107 transcripts while 606 genes showed significantly reduced expression. In addition to confirming differential expression of known markers of adipogenesis (e.g., FABP4, ADIPOQ, PLIN4), multiple genes and signaling pathways not previously known to be involved in regulating adipogenesis were identified (e.g. POSTN, PPP1R1A, FGF11) as potential novel mediators of adipogenesis. Quantitative RT-PCR validated the microarray results. Conclusions: ASC maturation into an adipocyte phenotype proceeds from a gene expression program that involves thousands of genes. This is the first study to compare mRNA expression profiles during early and late stage adipogenesis using cultured human primary ASCs from multiple patients. [ABSTRACT FROM AUTHOR]

It is known that thyroid hormone (TH) is a major determinant of muscle fiber composition, but the molecular mechanism by which it does so remains unclear. Here, we demonstrated that miR-133a1 is a direct target gene of TH in muscle. Intriguingly, miR-133a, which is enriched in fast-twitch muscle, regulates slow-to-fast muscle fiber type conversion by targeting TEA domain family member 1 (TEAD1), a key regulator of slow muscle gene expression. Inhibition of miR-133a in vivo abrogated TH action on muscle fiber type conversion. Moreover, TEAD1 overexpression antagonized the effect of miR-133a as well as TH on muscle fiber type switch. Additionally, we demonstrate that TH negatively regulates the transcription of myosin heavy chain I indirectly via miR-133a/TEAD1. Collectively, we propose that TH inhibits the slow muscle phenotype through a novel epigenetic mechanism involving repression of TEAD1 expression via targeting by miR-133a1. This identification of a TH-regulated microRNA therefore sheds new light on how TH achieves its diverse biological activities. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is a group of hematological malignancies with high heterogeneity. There is an increasing need to improve the risk stratification of AML patients, including those with normal cytogenetics, using molecular biomarkers. Here, we report a metabolomics study that identified a distinct glucose metabolism signature with 400 AML patients and 446 healthy controls. The glucose metabolism signature comprises a panel of 6 serum metabolite markers, which demonstrated prognostic value in cytogenetically normal AML patients. We generated a prognosis risk score (PRS) with 6 metabolite markers for each patient using principal component analysis. A low PRS was able to predict patients with poor survival independently of well-established markers. We further compared the gene expression patterns of AML blast cells between low and high PRS groups, which correlated well to the metabolic pathways involving the 6 metabolite markers, with enhanced glycolysis and trichloracetic acid cycle at gene expression level in low PRS group. In vitro results demonstrated enhanced glycolysis contributed to decreased sensitivity to antileukemic agent arabinofuranosyl cytidine (Ara-C), whereas inhibition of glycolysis suppressed AML cell proliferation and potentiated cytotoxicity of Ara-C. Our study provides strong evidence for the use of serum metabolites and metabolic pathways as novel prognostic markers and potential therapeutic targets for AML. [ABSTRACT FROM AUTHOR]

Abstract: The R/L type pyruvate kinase (PKLR) gene was cloned from liver of grass carp (Ctenopharyngodon idella) by degenerate oligonucleotide primed PCR, including a complete open reading frame (ORF) of 1617bp. Homological protein analysis showed that the PKLR in grass carp shared a high degree of sequence identity with other known PK sequences. In addition, tissue distribution analysis showed that the highest expression of the PKLR was detected in liver. Then triplicate groups of grass carp (9 tanks) were fed twice daily to satiation with 91.8, 192.9, and 402.6gkg−1 carbohydrate diets for 8weeks, respectively. Dietary 402.6gkg−1 carbohydrate decreased growth performance, feed intake and feed utilization. With increasing dietary carbohydrates, the hepatic PK activity and mRNA expression of grass carp were up-regulated significantly. The results indicated that the PKLR play a modulation role in the adaptation of hepatic glycolysis to dietary high carbohydrates in grass carp. [Copyright &y& Elsevier]

MicroRNAs (miRNAs) are small, single-stranded, noncoding RNAs that function as negative regulators of gene expression. They are transcribed from endogenous DNA and form hairpin structures (termed as pre-miRNAs) that are processed to form mature miRNAs. It remains largely unknown as to the molecular consequences of the natural genetic variation in pre-miRNAs. Here, we report that an A → G polymorphism (rs71428439) is located in Homo sapiens miR-149 stem-loop region. This polymorphism results in a change in the structure of the miR-149 precursor. Our results showed that the genotype distribution of this polymorphism in myocardial infarction cases was significantly different from that in the control subjects. We examined the biological significance of this polymorphism on the production ofmaturemiR-149, and we observed that the G-allelic miR-149 precursor displayed a lower production of mature miR-149 compared with the A-allelic one. Further investigations disclosed that miR-149 could withstand mitochondrial fission and apoptosis through targeting the pro-apoptotic factor p53-upregulated modulator of apoptosis (Puma). Enforced expression ofmiR-149 promoted cell survival, whereas knockdown of miR-149 rendered cells to be sensitive to apoptotic stimulation. Intriguingly, the A to G variation led pre-miR-149 to elicit an attenuated effect on the inhibition of mitochondrial fission and apoptosis. Finally, this polymorphism exerts its influence on cardiac function in the mouse model of myocardial infarction. These data suggest that this polymorphism in the miR-149 precursor may result in important phenotypic traits of myocardial infarction. Our findings warrant further investigations on the relationship between miR-149 polymorphism and myocardial infarction. [ABSTRACT FROM AUTHOR]

Background: Polyunsaturated fatty acids (PUFAs) have positive effect on the regulation of plasma lipids. But the mechanism for them to modulate lipid homeostasis in macrophage is still unclear. In this study, we employed PUFA to pretreat macrophages and evaluated the variations of lipid droplet (LD) content, lipid composition, and expressions of LD-associated genes in macrophage-derived foam cells. Method: THP-1-derived macrophages or human peripheral blood monocyte-derived macrophages were pre-treated with four non-esterified fatty acids (NEFAs) separately: saturated fatty acid (SFA)-palmitic acid (PA), monounsaturated fatty acids (MUFAs)-oleic acid (OA), PUFAs-linoleic acid (LA) and eicosapentaenoic acid (EPA). Intracellular lipid content and cholesterol efflux were analyzed in THP-1 macrophage-derived foam cells. Related gene expressions were detected by quantitative real-time PCR. Results: PUFA pre-treatment reduced cholesterol content in foam cells and increased cholesterol efflux to lipid-free apoAI in conditioned medium compared with PA or OA group. Cell death-inducing DFF45 like effector (CIDE) and Perilipin-Adipophilin-TIP47 (PAT) family members, as LD-associated proteins, showed specific gene expression profiles after PUFA pre-treatment. These results may help to explain the process of lipid metabolism within foam cells. Conclusion: PUFA (LA or EPA) had a potential protective effect against cholesterol accumulation. The specific expressions of CIDE and PAT genes may provide clues to explore the protective mechanism of PUFA in foam cells. [ABSTRACT FROM AUTHOR]

Mitochondrial uncoupling protein-4 (UCP4) protects against Complex I deficiency as induced by 1-methyl-4- phenylpyridinium (MPP+), but how UCP4 affects mitochondrial function is unclear. Here we investigated how UCP4 affects mitochondrial bioenergetics in SH-SY5Y cells. Cells stably overexpressing UCP4 exhibited higher oxygen consumption (10.1%, p<0.01), with 20% greater proton leak than vector controls (p<0.01). Increased ATP supply was observed in UCP4- overexpressing cells compared to controls (p<0.05). Although state 4 and state 3 respiration rates of UCP4-overexpressing and control cells were similar, Complex II activity in UCP4-overexpressing cells was 30% higher (p<0.05), associated with protein binding between UCP4 and Complex II, but not that of either Complex I or IV. Mitochondrial ADP consumption by succinate-induced respiration was 26% higher in UCP4-overexpressing cells, with 20% higher ADP:O ratio (p<0.05). ADP/ ATP exchange rate was not altered by UCP4 overexpression, as shown by unchanged mitochondrial ADP uptake activity. UCP4 overexpression retained normal mitochondrial morphology in situ, with similar mitochondrial membrane potential compared to controls. Our findings elucidate how UCP4 overexpression increases ATP synthesis by specifically interacting with Complex II. This highlights a unique role of UCP4 as a potential regulatory target to modulate mitochondrial Complex II and ATP output in preserving existing neurons against energy crisis. [ABSTRACT FROM AUTHOR]

Background: The morphogenesis of the cerebral cortex depends on the precise control of gene expression during development. Small non-coding RNAs, including microRNAs and other groups of small RNAs, play profound roles in various physiological and pathological processes via their regulation of gene expression. A systematic analysis of the expression profile of small non-coding RNAs in developing cortical tissues is important for clarifying the gene regulation networks mediating key developmental events during cortical morphogenesis. Results: Global profiling of the small RNA transcriptome was carried out in rat cerebral cortex from E10 till P28 using next-generation sequencing technique. We found an extraordinary degree of developmental stage-specific expression of a large group of microRNAs. A group of novel microRNAs with functional hints were identified, and brain-enriched expression and Dicer-dependent production of high-abundant novel microRNAs were validated. Profound editing of known microRNAs at "seed" sequence and flanking sequence was observed, with much higher editing events detected at late postnatal stages than embryonic stages, suggesting the necessity of microRNA editing for the fine tuning of gene expression during the formation of complicated synaptic connections at postnatal stages. Conclusion: Our analysis reveals extensive regulation of microRNAs during cortical development. The dataset described here will be a valuable resource for clarifying new regulatory mechanisms for cortical development and diseases and will greatly contribute to our understanding of the divergence, modification, and function of microRNAs [ABSTRACT FROM AUTHOR]

Background: Human peripheral blood is a promising material for biomedical research. However, various kinds of biological and technological factors result in a large degree of variation in blood gene expression profiles. Methodology/Principal Findings: Human peripheral blood samples were drawn from healthy volunteers and analysed using the Human Genome U133Plus2 Microarray. We applied a novel approach using the Principle Component Analysis and Eigen-R2 methods to dissect the overall variation of blood gene expression profiles with respect to the interested biological and technological factors. The results indicated that the predominating sources of the variation could be traced to the individual heterogeneity of the relative proportions of different blood cell types (leukocyte subsets and erythrocytes). The physiological factors like age, gender and BMI were demonstrated to be associated with 5.3% to 9.2% of the total variation in the blood gene expression profiles. We investigated the gene expression profiles of samples from the same donors but with different levels of RNA quality. Although the proportion of variation associated to the RNA Integrity Number was mild (2.1%), the significant impact of RNA quality on the expression of individual genes was observed. Conclusions: By characterizing the major sources of variation in blood gene expression profiles, such variability can be minimized by modifications to study designs. Increasing sample size, balancing confounding factors between study groups, using rigorous selection criteria for sample quality, and well controlled experimental processes will significantly improve the accuracy and reproducibility of blood transcriptome study. [ABSTRACT FROM AUTHOR]

The induction of a strong mucosal immune response is essential to building successful HIV vaccines. Highly attenuated recombinant HIV vaccinia virus can be administered mucosally, but even high doses of immunization have been found unable to induce strong mucosal antibody responses. In order to solve this problem, we studied the interactions of recombinant HIV vaccinia virus Tiantan strain (rVTT-gagpol) in mucosal epithelial cells (specifically Caco-2 cell layers) and in BALB/c mice. We evaluated the impact of this virus on HIV antigen delivery and specific immune responses. The results demonstrated that rVTT-gagpol was able to infect Caco-2 cell layers and both the nasal and lung epithelia in BALB/c mice. The progeny viruses and expressed p24 were released mainly from apical surfaces. In BALB/c mice, the infection was limited to the respiratory system and was not observed in the blood. This showed that polarized distribution limited antigen delivery into the whole body and thus limited immune response. To see if this could be improved upon, we stimulated unpolarized budding of the virus and HIV antigens by treating both Caco-2 cells and BALB/c mice with colchicine. We found that, in BALB/c mice, the degree of infection and antigen expression in the epithelia went up. As a result, specific immune responses increased correspondingly. Together, these data suggest that polarized budding limits antigen delivery and immune responses, but unpolarized distribution can increase antigen expression and delivery and thus enhance specific immune responses. This conclusion can be used to optimize mucosal HIV vaccine strategies. [ABSTRACT FROM AUTHOR]

Bacterial ghosts that are generated using the regulated PhiX174 lysis gene E offer a new avenue for the study of inactivated vaccines. Here, we constructed a library of mutant gene E using a gene-shuffling technique. After screening and recombination with the prokaryotic non-fusion expression vector pBV220, two lysis plasmids were selected. Among which, a novel mutant E gene (named mE), consisting of a 74-bp non-encoding sequence at 5'-end and a 201-bp gene ΔE, significantly increased the lysis effect on prokaryotic Escherichia coli and Salmonella enteritidis. Moreover, lysis efficiency, as measured by the OD600 value, reached 1.0 (109 CFU), avoiding the bottleneck problem observed with other bacterial lysis procedures, which results in a low concentration of bacteria in suspension, and consequent low production of bacterial ghosts. Our results may provide a promising avenue for the development of bacterial ghost vaccines. [ABSTRACT FROM AUTHOR]

AbstractThe purpose of this study was to test the hypothesis that overexpression of angiotensin-converting enzyme 2 (ACE2) may favorably affect left ventricular (LV) remodeling and function after myocardial infarction (MI). The left anterior descending coronary artery was ligated to produce anterior MI in 100 Wistar–Kyoto rats that were randomly divided into Ad-ACE2, Ad-ACE2+A779, Ad-EGFP, model, and sham groups. Two weeks later, rats in the Ad-ACE2 and Ad-EGFP groups received direct intramyocardial injection of Ad-ACE2 and Ad-EGFP, respectively. Rats in the Ad-ACE2+A779 group received both intramyocardial injection of Ad-ACE2 and a continuous intravenous infusion of A779 for 15 days. LV volume and systolic function, the extent of myocardial fibrosis, and levels of ACE2, angiotensin II (Ang II), and collagen I protein expression were evaluated. Four weeks after ACE2 gene transfer, the Ad-ACE2 group showed reduced LV volume, extent of myocardial fibrosis, and expression levels of ACE, Ang II, and collagen I in the myocardium, and increased LV ejection fraction and levels of ACE2 activity and expression in comparison with the Ad-EGFP and model groups. These results suggest that ACE2 overexpression attenuated LV fibrosis and improved LV remodeling and systolic function. In conclusion, overexpression of ACE2 favorably affected the pathological process of LV remodeling after MI by inhibiting ACE activity, reducing AngII levels, and up-regulating Ang-(1–7) expression, thus providing a potential therapeutic target in the treatment of heart failure. [ABSTRACT FROM AUTHOR]

Background: Selenium has been shown to inhibit cancer development and growth through the mediation of selenium-binding proteins. Decreased expression of selenium-binding protein 1 has been reported in cancers of the prostate, stomach, colon, and lungs. No information, however, is available concerning the roles of selenium-binding protein 1 in uterine leiomyoma. Methods: Using Western Blot analysis and immunohistochemistry, we examined the expression of selenium-binding protein 1 in uterine leiomyoma and normal myometrium in 20 patients who had undergone hysterectomy for uterine leiomyoma. Results and Discussion: The patient age ranged from 34 to 58 years with a mean of 44.3 years. Proliferative endometrium was seen in 8 patients, secretory endometrium in 7 patients, and atrophic endometrium in 5 patients. Two patients showed solitary leiomyoma, and eighteen patients revealed 2 to 5 tumors. Tumor size ranged from 1 to 15.5 cm with a mean of 4.3 cm. Both Western Blot analysis and immunohistochemistry showed a significant lower level of selenium-binding protein 1 in leiomyoma than in normal myometrium. Larger tumors had a tendency to show a lower level of selenium-binding protein 1 than smaller ones, but the difference did not reach a statistical significance. The expression of selenium-binding protein 1 was the same among patients with proliferative, secretory, and atrophic endometrium in either leiomyoma or normal myometrium. Also, we did not find a difference of selenium-binding protein 1 level between patients younger than 45 years and older patients in either leiomyoma or normal myometrium. Conclusions: Decreased expression of selenium-binding protein 1 in uterine leiomyoma may indicate a role of the protein in tumorigenesis. Our findings may provide a basis for future studies concerning the molecular mechanisms of selenium-binding protein 1 in tumorigenesis as well as the possible use of selenium in prevention and treatment of uterine leiomyoma. [ABSTRACT FROM AUTHOR]

In this study, we applied RT-PCR and cDNA cloning techniques to clone myosin heavy chain (MYH) cDNA from muscle tissues of the mandarin fish Siniperca kneri. The cDNA was determined to be of 6987 base pairs in length, encoding a peptide of 1937 amino acids (Genbank accession no. EF446616). A search of encoded protein sequences in the NCBI conserved domain database indicated the presence of all known protein domains for MYH proteins, i.e. the myosin motor domain in the N-terminal region, the DIL domain at the C-terminus, and the ATPase domain. The MYH gene and its protein were expressed predominantly in muscle tissues and weakly in cardiac tissues. Developmentally, the MYH gene was first expressed in the muscle formation stage and continued later on. Our work provided a novel mypsin heavy chain gene sequence in fish biology and the results indicate that the MYH gene and the protein it encodes are important for the growth and development of the mandarin fish, as well as its muscle characterization. [ABSTRACT FROM AUTHOR]

A well-known observation with respect to cancer biology is that transformed cells display a disturbed cytoskeleton. The underlying mechanisms, however, remain only partly understood. In an effort to identify possible mechanisms, we compared the proteome of pancreatic cancer with matched normal pancreas and observed diminished protein levels of gelsolin-an actin filament severing and capping protein of crucial importance for maintaining cytoskeletal integrity-in pancreatic cancer. Additionally, pancreatic ductal adenocarcinomas displayed substantially decreased levels of gelsolin as judged by Western blot and immunohistochemical analyses of tissue micoarrays, when compared with cancerous and untransformed tissue from the same patients (P < 0.05). Importantly, no marked downregulation of gelsolin mRNA was observed (P > 0.05), suggesting that post-transcriptional mechanisms mediate low gelsolin protein levels. In apparent agreement, high activity ubiquitin-proteasome pathway in both patient samples and the BxPC-3 pancreatic cancer cell line was detected, and inhibition of the 26s proteasome system quickly restored gelsolin protein levels in the latter cell line. The status of ubiquitinated gelsolin is related to lymph node metastasis of pancreatic cancer. In conclusion, gelsolin levels are actively downregulated in pancreatic cancer and enhanced targeting of gelsolin to the ubiquitin-proteasome pathway is an important contributing factor for this effect. [ABSTRACT FROM AUTHOR]

The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10) is a lipid and protein phosphatase. We report here that PTEN physically associates with the NR1 and NR2B subunits of NMDA receptors (NMDARs) in rat hippocampus. Downregulating the protein expression of PTEN inhibits the function of extrasynaptic NMDARs and decreases NMDAR surface expression, suggesting a crucial role for endogenous PTEN in the modulation of NMDAR-mediated neuronal function. Reducing PTEN expression also enhances Akt/Bad phosphorylation in hippocampal neurons. Importantly, suppressing lipid and protein phosphatase activity of PTEN, respectively, activates Akt and inhibits extrasynaptic NMDAR activity and thereby protects against ischemic neuronal death in vitro and in vivo. Thus, our study reveals a dual neuroprotective mechanism by which Akt/Bad and extrasynaptic NMDARs are regulated via downregulation of two distinct PTEN phosphatase activities and present the possibility of PTEN as a potential therapeutic target for stroke treatment. [ABSTRACT FROM AUTHOR]

Background: A limiting factor of cDNA microarray technology is the need for a substantial amount of RNA per labeling reaction. Thus, 20-200 micro-grams total RNA or 0.5-2 micro-grams poly (A) RNA is typically required for monitoring gene expression. In addition, gene expression profiles from large, heterogeneous cell populations provide complex patterns from which biological data for the target cells may be difficult to extract. In this study, we chose to investigate a widely used mRNA amplification protocol that allows gene expression studies to be performed on samples with limited starting material. We present a quantitative study of the variation and noise present in our data set obtained from experiments with either amplified or non-amplified material. Results: Using analysis of variance (ANOVA) and multiple hypothesis testing, we estimated the impact of amplification on the preservation of gene expression ratios. Both methods showed that the gene expression ratios were not completely preserved between amplified and non-amplified material. We also compared the expression ratios between the two cell lines for the amplified material with expression ratios between the two cell lines for the non-amplified material for each gene. With the aid of multiple t-testing with a false discovery rate of 5%, we found that 10% of the genes investigated showed significantly different expression ratios. Conclusion: Although the ratios were not fully preserved, amplification may prove to be extremely useful with respect to characterizing low expressing genes. [ABSTRACT FROM AUTHOR]