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Data mining on a public domain detected eight potential transcripts which were upregulated in advanced UBUCs, suggesting that they may take part in UC development or/and progression. Retrospectively, immunohistochemistry along with H-score recording was carried out to evaluate the GNB4 protein levels on tissues from UC patients. Correlations between GNB4 H-score and imperative clinicopathological factors, as well as the implication of GNB4 protein level on disease-specific and metastasis-free survivals were assessed. In UTUCs (n = 340) and UBUCs (n = 295), 170 (50.0%) and 148 (50.0%) cases, respectively, were identified to be of high GNB4 expression. The GNB4 protein levels were correlated to numerous clinicopathological features and patients' survivals. Upregulation of the GNB4 protein was significantly associated with primary tumor, nodal metastasis, histological grade, vascular invasion and mitotic rate. High GNB4 protein levels independently and significantly predicted poor disease-specific and metastasis-free in UTUC and UBUC, respectively. Ingenuity pathway analysis furthermore showed that multiple signaling pathways were enriched including 'Communication between Innate and Adaptive Immune Cells' and 'NFκB Signaling'. Our findings demonstrated that the upregulation of the GNB4 protein is an independent unfavorable prognosticator in UC. High GNB4 gene expression plays an important role in UC progression.

NAC TFs play crucial roles in response to abiotic stresses in plants. Here, ZmNAC071 was identified as a nuclear located transcriptional repressor. Overexpression of ZmNAC071 in Arabidopsis enhanced sensitivity of transgenic plants to ABA and osmotic stress. The expression levels of SODs, PODs, P5CSs, and AtMYB61 were inhibited by ZmNAC071, which results in reduced ROS scavenging and proline content, increased ROS level, and water loss. Besides, the expression levels of some ABA or abiotic stress-related genes, like ABIs, RD29A, DREBs, and LEAs were also significantly inhibited by ZmNAC071. Yeast one-hybrid assay demonstrated that ZmNAC071 specifically bound to the cis-acting elements containing CGT[G/A] core sequences in the promoter of stress-related genes, suggesting that ZmNAC071 may participate in the regulation of transcription of these genes through recognizing the core sequences CGT[G/A]. These results will facilitate further studies concerning the cis-elements and downstream genes targeted by ZmNAC071 in maize.

Tetranychus cinnabarinus (Boisduval), i.e., carmine spider mite, is a worldwide pest that can cause serious damage to plants. Problems of resistance have arisen since abamectin usage in the control of T. cinnabarinus. Unfortunately, there are only limited data on the extent of this problem. To understand the development of abamectin resistance in the carmine spider mite, we prokaryotically expressed an intradiol ring-cleavage dioxygenase (ID-RCD) gene sequence, TcID-RCD1, which had a significant upregulated expression of over 7.7 times in an abamectin-resistant strain (AbR) when compared with that of a susceptible strain (SS). The crude enzyme activity also indicated that the AbR had a higher activity than that exhibited in SS. When susceptible individuals were treated with abamectin, TcID-RCD1 was also overexpressed. Furthermore, using the RNA interference (RNAi) technique, TcID-RCD1 was successfully knocked down, with the expression level decreasing significantly to approximately 39% in the SS strain compared with the control. And the mortality of mites feeding on dsTcID-RCD1 increased significantly when treated with abamectin. These results strongly suggest that TcID-RCD1 is involved in abamectin resistance in T. cinnabarinus.

Background Cyflumetofen is an outstanding acaricide with a novel mode of action. Tetranychus cinnabarinus, an important agricultural pest, is notorious for developing resistance to most classes of acaricides rapidly and results in enormous loss for the economy. Our previous study had pointed out glutathione S-transferase (GSTs) significantly contributed to the cyflumetofen-resistance formation in T. cinnabarinus, but the more specific mechanism needed to be further investigated. Results The unstable resistance was observed in cyflumetofen-resistant strain (CyR)under acaricide-free condition. The activity of GSTs increased along with the development of resistance. The expressions of 13 GST genes were detected in CyR and susceptible strain (SS), of which six genes were overexpressed in CyR and the TcGSTm02 was selected as the representative for functional study. The expression of TcGSTm02 changed along with the resistant level of CyR with the same trend. Recombinant protein of TcGSTm02 with high activity was successfully obtained by E. coli expression system, whose activity could be inhibited by cyflumetofen (IC50 = 0.23 mM). Recombinant TcGSTm02 could effectively decompose cyflumetofen, and catalyze GS- to conjugate with cyflumetofen. Conclusion All clues confirmed that GSTs strongly associated with cyflumetofen-resistance and a representative gene, TcGSTm02, showed function on contributing the evolution of cyflumetofen-resistance in T. cinnabarinus. © 2019 Society of Chemical Industry.

The striped flea beetle, Phyllotreta striolata (Fabricius), damages crops in the Brassicaceae. The genetic data for this pest are insufficient to reveal its insecticide resistance mechanisms or to develop molecular markers for resistance monitoring. We used PacBio Iso-Seq technology to sequence the full-length transcriptome of P. striolata. After isoform sequence clustering and removal of redundant transcripts, a total of 41,293 transcripts were obtained, and 35,640 of these were annotated in the database of gene products. Structure analysis uncovered 4,307 alternative splicing events, and 3,836 sequences were recognized as lncRNAs. Transcripts with the complete coding region of important detoxification enzymes were further classified. There were 57 transcripts of P450s distributed in CYP2, CYP3, CYP4, and Mito CYP clades, 29 transcripts of ESTs from 4 functional groups, 17 transcripts of GSTs classified into 5 families, 51 transcripts of ABCs distributed in 6 families, and 19 transcripts of UGTs. Twenty-five lncRNAs were predicted to be regulators of these detoxification genes. Full-length transcriptome sequencing is an efficient method for molecular study of P. striolata and it is also useful for gene function analysis.

BackgroundAs core units of organ tissues, cells of various types play their harmonious rhythms to maintain the homeostasis of the human body. It is essential to identify the characteristics of cells in human organs and their regulatory networks for understanding the biological mechanisms related to health and disease. However, a systematic and comprehensive single-cell transcriptional profile across multiple organs of a normal human adult is missing.ResultsWe perform single-cell transcriptomes of 84,363 cells derived from 15 tissue organs of one adult donor and generate an adult human cell atlas. The adult human cell atlas depicts 252 subtypes of cells, including major cell types such as T, B, myeloid, epithelial, and stromal cells, as well as novelCOCH+fibroblasts and FibSmo cells, each of which is distinguished by multiple marker genes and transcriptional profiles. These collectively contribute to the heterogeneity of major human organs. Moreover, T cell and B cell receptor repertoire comparisons and trajectory analyses reveal direct clonal sharing of T and B cells with various developmental states among different tissues. Furthermore, novel cell markers, transcription factors, and ligand-receptor pairs are identified with potential functional regulations in maintaining the homeostasis of human cells among tissues.ConclusionsThe adult human cell atlas reveals the inter- and intra-organ heterogeneity of cell characteristics and provides a useful resource in uncovering key events during the development of human diseases in the context of the heterogeneity of cells and organs.

Chitin is an important content in the exoskeletons of arthropods, and its hydrolyzation is catalyzed by chitinases during the process of molting, thus, the chitinases are considered as ideal target to interfere the growth of arthropods. This study intends to clarify the characteristic of the chitinases during the development of Tetranychus cinnabarinus, and screen out important genes as potential control targets. The results showed that the total enzyme concentration of chitinases was significantly higher in larva, the first and second nymph than that in egg and adult. Base on the transcriptome data, six unigenes encoding chitinases were identified and their expression patterns in different developmental stages were detected. The expressions of TcCHIT1 and TcCHIT10 showed high abundance during the molting process and their expression change during the developmental stages was consistent with the enzyme concentration. The full-length of these two genes were further cloned, and the structural characteristics of their proteins were analyzed by constructing the three-dimensional structure model. The results provide basic information to understand the characteristic of chitinases in T. cinnabarinus and might be considered as target for control.

ANAC069 binds to the DNA sequence of C[A/G]CG[T/G] to regulate the expression of genes, resulting in decreased ROS scavenging capability and proline biosynthesis, which contribute to increased sensitivity to salt and osmotic stress. NAM-ATAF1/2 and CUC2 (NAC) proteins are plant-specific transcription factors that play important roles in abiotic stress responses. In the present study, we characterized the physiological and regulatory roles of Arabidopsis thaliana ANAC069 in response to abiotic stresses. Arabidopsis plants overexpressing ANAC069 displayed increased sensitivity to abscisic acid, salt, and osmotic stress. Conversely, ANAC069 knockdown plants showed enhanced tolerance to salt and osmotic stress, but no change in ABA sensitivity. Further studies showed that ANAC069 inhibits the expression of SOD, POD, GST, and P5CS genes. Consequently, the transcript level of ANAC069 correlated negatively with the reactive oxygen species (ROS) scavenging ability and the proline level. The genes regulated by ANAC069 were further studied using a gene chip on a genome-wide scale, and 339 and 226 genes up- and downregulated by ANAC069 were identified. Analysis of the promoters of the genes affected by ANAC069 suggested that ANAC069 regulates the expression of genes mainly through interacting with the DNA sequence C[A/G]CG[T/G] in response to abiotic stresses. Collectively, our data suggest that ANAC069 could recognize C[A/G]CG[T/G] sequences to regulate the expression of genes that negatively regulates salt and osmotic stress tolerance by decreasing ROS scavenging capability and proline biosynthesis.

To investigate chemoreception genes in Conogethes punctiferalis, antennal transcriptome of male and female samples were sequenced and analyzed. A total number of 54,442,128 and 51,132,998 clean reads from male and female samples were obtained respectively. By de novo assembly, a total of 51,240 unigenes for both sexes was generated. Among all unigenes, 26,509 showed similarity to known proteins after alignment to Nr protein database by blastx. Go analysis, KEGG pathway analysis and Protein coding region prediction were also performed subsequently to annotate gene functions. A total of 29 candidate odorant binding proteins (OBPs) including 2 general odorant binding proteins (GOBPs) and 5 pheromone binding protein (PBP), as well as 59 odorant receptors (ORs) were identified. Additionally, 10 chemosensory proteins (CSPs) and 19 ionotropic receptors (IRs) were annotated. With gene expression comparison, 920 up-regulated and 684 down-regulated genes were found in female antennae transcriptome, compared with that of the male. Differently expression levels of 9 chemosensory genes including PBPs up-regulated in the male were detected. Results of this study provide basic information about C. punctiferalis chemoreception genes, which helps to understand pheromone communication in this species and may benefit control of this pest with pheromonal techniques.

NAC (NAM, ATAF1/2, and CUC2) transcription factors play important roles in many biological processes, and mainly bind to the NACRS with core sequences &ldquo, CACG&rdquo, or &ldquo, CATGTG&rdquo, to regulate gene expression. However, whether NAC proteins can bind to other motifs without these core sequences remains unknown. In this study, we employed a Transcription Factor-Centered Yeast one Hybrid (TF-Centered Y1H) screen to study the motifs recognized by ANAC074. In addition to the NACRS core cis-element, we identified that ANAC074 could bind to MybSt1, NRS1, and NRS2. Y1H and GUS assays showed that ANAC074 could bind the promoters of ethylene responsive genes and stress responsive genes via the NRS1, NRS2, or MybSt1 element. ChIP study further confirmed that the bindings of ANAC074 to MybSt1, NRS1, and NRS2 actually occurred in Arabidopsis. Furthermore, ten NAC proteins from different NAC subfamilies in Arabidopsis thaliana were selected and confirmed to bind to the MybSt1, NRS1, and NRS2 motifs, indicating that they are recognized commonly by NACs. These findings will help us to further reveal the functions of NAC proteins.

Selenium is a trace element necessary for living organisms. It exists mainly in the form of selenite in nature. In plants, selenium can enhance defenses against pests. In this study, transcriptome sequencing technology was used to analyze the response mechanism of Tetranychus cinnabarinus to plant-mediated selenium treatment. We tested four sodium selenite treatments (5, 20, 50, and 200 μM) that were the same for short (2 d) and long (30 d) treatment durations. The results showed that the number of differentially expressed genes (DEGs) in the short-term treatment was greater than in the long-term treatment. This indicated that the gene expression of spider mites gradually stabilized during the selenium treatment. Regardless of the long-term and short-term conditions, spider mites had the largest response to the 20 μM sodium selenite treatment. The functional annotation classification of DEGs showed no significant difference under different concentrations and treatment durations. A total of 25 genes were differentially expressed in all eight treatments, including four down-regulated cytochrome P450 genes and one up-regulated chitinase gene. We speculate that selenium may have the potential to enhance the activity of chemical acaricides. Transcriptome sequencing of sodium selenite treatment at different concentrations and different times revealed the response mechanism of spider mites under plant-mediated selenium treatment. At the same time, it also provides new clues for the development of methods for preventing and controlling spider mites with selenium.

Cytochrome P450 monooxygenases (P450s), as an important family of detoxification enzymes, participate in the metabolism of agrochemicals in almost all agricultural pests and play important roles in the development of insecticide resistance. Two P450 genes (CYP389B1 and CYP392A26) were identified and their expression patterns were investigated in our previous study. In this study, four more P450 gene sequences (CYP391A1, CYP384A1, CYP392D11 and CYP392A28) from the Clan 2, Clan 3 and Clan 4 families were identified and characterized. Quantitative PCR analysis showed that these four P450 genes were highly expressed in a fenpropathrin-resistant (FeR) strain of Tetranychus cinnabarinus. In addition, their expressions were much more sensitive to fenpropathrin induction in the FeR strain than the susceptible strain. Gene-silencing experiments via double-stranded RNA feeding were carried out. The results showed that mRNA levels of these six P450 genes were reduced in the FeR strain and the activities of P450s were decreased. Consequently mite susceptibilities to fenpropathrin were increased. Interestingly, silencing all six P450 genes simultaneously had an even greater effect on resistance than silencing them individually. This study increases our understanding of the molecular mechanisms of insecticide detoxification, suggesting that the overexpression of these six P450 genes might play important roles in fenpropathrin resistance in T. cinnabarinus collaboratively.

Diacylglycerol acyltransferase (DGAT) catalyzes the only rate-limiting step in the pathway of plant oil (TAG) biosynthesis and is involved in plant development. In this study, five DGAT family members were identified from maize genome database. Phylogenetic analysis classified the ZmDGATs into type-I, II, and III clusters. Conserved functional domain analysis revealed that the proteins encoded by ZmDGAT1 contained conserved MBOAT domains, while two ZmDGAT2-encoding proteins harbored LPLAT domains. qRT-PCR analysis showed that ZmDGAT genes exhibited very high relative expression in developing seeds, especially at the early stage of seed development. Under various abiotic stress conditions, differential responses of ZmDGAT genes were observed. An overall significant induction of ZmDGAT genes under cold stress in leaves and a quick and strong response to osmotic stresses in roots were highlighted. This study provides useful information for understanding the roles of DGATs in oil accumulation and stress responses in higher plants.

Insect body pigmentation and coloration are critical to adaption to the environment. To explore the mechanisms that drive pigmentation, we used the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) genome editing system to target the ebony gene in the non-model insect Spodoptera litura. Ebony is crucial to melanin synthesis in insects. By directly injecting Cas9 messenger RNA and ebony-specific guide RNAs into S. litura embryos, we successfully induced a typical ebony-deficient phenotype of deep coloration of the puparium and induction of melanin formation during the pupal stage. Polymerase chain reaction-based genotype analysis demonstrated that various mutations had occurred at the sites targeted in ebony. Our study clearly demonstrates the function of ebony in the puparium coloration and also provides a potentially useful marker gene for functional studies in S. litura as well as other lepidopteran pests.

Background Carboxylesterases (CarEs) are important in pesticide resistance. Four overexpressed CarE genes with inducible character were screened out in fenpropathrin-resistant Tetranychus cinnabarinus, but their functional roles remained to be further analyzed by RNAi and protein expression. Results Feeding a single double-stranded (ds)RNA of each of four genes led to gene-specific downregulation of mRNA, decreased esterase activity and diminished resistance in T. cinnabarinus. More interestingly, feeding four dsRNAs simultaneously led to a more significant decrease in enzymatic activity and fold resistance than feeding a single dsRNA individually, suggesting that these CarE genes were involved in fenpropathrin-resistance and had cooperative roles. The gene CarE6 was regarded as the primary and representative candidate to be functionally expressed, because silencing of CarE6 led to the most significant decrease in resistance level. The activity of CarE6 protein was competitively inhibited by fenpropathrin. It could effectively decompose 41.7 ± 0.09% of fenpropathrin within 3 h, proving that CarE6 protein was capable of metabolizing fenpropathrin effectively in T. cinnabarinus. Conclusion The results confirm that four CarE genes are cooperatively involved in fenpropathrin resistance and the metabolic enzymes encoded by these overexpressed genes do indeed metabolize acaricide in resistant T. cinnabarinus in the evolution of acaricide resistance. © 2018 Society of Chemical Industry.

Diacylglycerol kinase (DGK) is a kind of phosphokinase that catalyzes the formation of signaling molecule phosphatidic acid. In this study, seven maize (Zea mays) DGK gene family members were identified by an exploration of maize genome via multiple online databases, and designated as ZmDGK1-7, respectively. The proteins encoded by ZmDGKs ranged from 487 to 716 amino acids, and had a molecular weight (MWs) between 54.6 and 80.2 kDa. Phylogenetic analysis revealed that ZmDGKs grouped into three clusters as described for known plant DGK families: Cluster I was composed of three maize DGKs, ZmDGK1, ZmDGK4 and ZmDGK5, cluster II contained ZmDGK6, and the isoforms ZmDGK2, ZmDGK3 and ZmDGK7 fell into cluster III. ZmDGK proteins featured the typical functional domains, while all seven ZmDGKs have a conserved catalytic domain DGKc, only the cluster I ZmDGKs have the DAG/PE binding domain. Most ZmDGK genes showed ubiquitous expression profiles at various developmental stages, while a high relative expression was observed at the tasseling stage. ZmDGK genes exhibited differential expression patterns in response to abiotic stresses including cold, salinity and drought, and all ZmDGK genes were found obviously up-regulated by cold. The distinct roles of ZmDGKs in cold response was also supported by the finding that an accumulation of DGK products–PA under low temperature. This study will help to better understand the roles of DGKs in the development and abiotic stress responses in major crops.

By switching position of the N and S atom in the thiazole ring which were similar to the previously reported agent 5-(4-ethoxyphenyl)-4-(3',4',5'-trimethoxyphenyl)thiazol-2-amine, a series of 4,5-diarylthiazole derivatives were synthesized using Friedel-Crafts reaction based on chemical modification of Combrestatatin A-4 (CA-4). Their antiproliferative activities were evaluated and identified as new microtubule destabilizing agents. Structure-activity relationship study indicated that compound 8a with 3,4,5-trimethoxyphenyl group at the C-4 position and 4-ethoxyphenyl group at the C-5 position of 2-amino substituted thiazole was of the most potent inhibitory activity in this series. 8a was found to exhibit the IC50 values of 8.4-26.4nM in five human cancer cell lines, with comparable inhibition effects to CA-4. Moreover, 8a showed potency as a tubulin polymerization inhibitor, with colchicine site binding ability and comparable extent of inhibition against the growth of P-glycoprotein over-expressing multidrug resistant cell lines. Mechanism studies revealed that 8a could block the progression of cell cycle in the G2/M phase and result in cellular apoptosis in cancer cells. As a new tubulin destabilizing agent, 8a was also found high antivascular activity as it concentration-dependently reduced the cell migration and disrupted capillary like tube formation of HUVEC cells. Furthermore, 8a significantly suppressed the tumor growth in HCT116 and SK-OV-3 xenograft models with tumor growth inhibitory rate of 55.12% and 72.7%, respectively. Our studies highlighted that 8a was a promising microtubule targeting antitumor agent.

Mitogen-activated protein kinases (MAPKs), also termed extracellular signal-regulated kinases (ERKs), are cytoplasmic and nuclear serine/threonine kinases involved in signal transduction of several extracellular effectors. In mammals, ERKs participate in the regulation of spermatogenesis, mature spermatozoa motility, hyperactivation, and the acrosome reaction. To investigate ERK functions in Eriocheir sinensis reproduction, we successfully cloned the full-length ERK from the testis of E. sinensis (ES-ERK). The 1098-nucleotide open reading frame encodes a 365-amino-acid protein with a predicted molecular weight of 42 kDa. Expressions of ES-ERK in different tissues and testis development stages were detected by the quantitative RT-PCR and Western blotting. ES-ERK is expressed relatively highly in the testis. The expression of ES-ERK protein gradually increased in the spermatid stage, reaching a peak in sperm stage. Western blotting showed a similar expression pattern for the total ES-ERK protein, but phospho-ERK (p-ERK) showed the higher expression in spermatid than sperm stage. We also used trypan blue and hematoxylin and eosin staining to identify structural changes in E. sinensis spermatozoa during the process of acrosome reaction (AR). After stimulating the process of AR, the ES-ERK has translocated from the nucleus to the acrosomal tubule. This result suggested that the ERK MAPK might be involved in the regulation of the E. sinensis acrosome reaction.

DDX6 belongs to a family of DEAD-box RNA helicases, which are RNA splicing proteins that ensure the correct folding and structure of mature RNA. Gametogenesis requires the participation of many kinds of RNA. To explore its functions during Eriocheir sinensis gametogenesis, we cloned a full-length DDX6 cDNA sequence from E. sinensis (Es-DDX6) which contains a 1536-nucleotide open reading frame encoding a 512-amino acid protein. Multiple sequence alignments showed that Es-DDX6 has ten conservative DEAD-box family motifs. Tissue expression analysis of Es-DDX6 mRNA and protein levels showed that Es-DDX6 was highly expressed in both the ovary and testis. qRT-PCR analysis revealed the widespread expression of Es-DDX6 mRNA during various stages of gonad development peaking in October. In addition, immunohistochemical studies showed that oocytes and the spermatogonium and primary spermatocytes of testes contained high levels of cytoplasmic Es-DDX6 and decreased expression levels in spermatids. Interestingly, there was no expression of Es-DDX6 in these cells as they matured along the male reproductive system. Since oocytes and spermatocytes are active in meiosis and oocytes undergo rapid growth in October, these results provide preliminary evidence that Es-DDX6 plays a role in E. sinensis gametogenesis and oocyte growth processes.

To construct a Bacillus subtilis strain for improved uridine production. The AAG2846–2848 fragment of the pyrAB gene, encoding carbamoylphosphate synthetase, was deleted in B. subtilis TD246 leading to a 245% increase of uridine production and the conversion from glucose to uridine increased by 10.5%. Overexpression of the pyr operon increased the production of uridine by a further 31% and the conversion rate of glucose to uridine was increased by 18%. In addition, the blocking of arginine synthesis or disabling of glutamate dehydrogenase significantly enhanced the uridine production. The highest-producing strain, B. subtilis TD297, accumulated 11 g uridine/l with a yield of 240 mg uridine/g glucose in shake-flask cultivation. This is the first report of engineered B. subtilis strains which can produce more than 11 g uridine/l, with a yield reaching 240 mg uridine/g glucose in shake-flask cultivation.

Endometriosis is a benign disease, with malignant properties. A necessary step in the progression of endometriosis is tissue remodeling, which is coordinated by the activities of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs). This study evaluated the regulation of abnormal MMP and TIMP gene expression during endometriosis. Among the two genes families, promoter regions of MMP2, MMP3, MMP7, TIMP3, and TIMP4 were significantly altered in proliferative-phase endometriotic lesions compared to menstrual cycle-matched eutopic tissue from endometriosis-free women. In addition, a negative correlation was found between the DNA methylation status of the promoter region and transcript abundance of MMP2. Our findings suggest that changes in DNA methylation at the promoter region of MMP2 could underlie the changes in its expression in the ectopic endometria from patients with endometriosis.

Nogo-A has been identified as an inhibitor of neurite outgrowth specific to the central nervous system. However, little is known about the role of Nogo-A in hepatocellular carcinoma (HCC), the most common primary malignant tumor with a high mortality rate. This study aimed to investigate the role of endogenous Nogo-A in human liver cancer cells. Reverse transcription polymerase chain reaction was used to detect the expression of Nogo-A in four liver cancer cell lines. A lentivirus vector was then constructed to mediate RNA interference (RNAi) targeting of Nogo‑A (LV‑Nogo-A‑siRNA) and was confirmed to successfully suppress the expression of the Nogo-A gene in SMMC-7721 cells. Furthermore, Nogo-A was observed to be highly expressed in liver cancer cell lines. RNAi of Nogo-A using the LV‑Nogo-A‑siRNA construct significantly decreased Nogo-A protein expression and specifically inhibited the growth of SMMC-7721 cells. This growth inhibitory effect may be attributed to an increase in G2/M phase arrest and apoptosis in SMMC-7721 cells containing Nogo-A‑siRNA. The results of this study demonstrate that Nogo-A may represent a novel therapeutic target for the treatment of liver cancer, in addition to its potent roles in neural systems.

Despite the crucial importance of Hox genes functions during animal development, the mechanisms that control their transcription in time and space are not yet fully understood. In this context, it was proposed that Hotair, a lncRNA transcribed from within the HoxC cluster regulates Hoxd gene expression in trans, through the targeting of Polycomb and consecutive transcriptional repression. This activity was recently supported by the skeletal phenotype of mice lacking Hotair function. However, other loss of function alleles at this locus did not elicit the same effects. Here, we re-analyze the molecular and phenotypic consequences of deleting the Hotair locus in vivo. In contrast with previous findings, we show that deleting Hotair has no detectable effect on Hoxd genes expression in vivo. In addition, we were unable to observe any significant morphological alteration in mice lacking the Hotair transcript. However, we find a subtle impact of deleting the Hotair locus upon the expression of the neighboring Hoxc11 and Hoxc12 genes in cis. Our results do not support any substantial role for Hotair during mammalian development in vivo. Instead, they argue in favor of a DNA-dependent effect of the Hotair deletion upon the transcriptional landscape in cis., Author Summary During mammalian embryonic development, Hox genes must be tightly regulated. It was proposed earlier that part of this regulation relies upon Hotair, a long non-coding RNA that recruits repressive protein complexes onto the HoxD gene cluster to keep these genes silent before they become activated. A genetic deletion of Hotair in mice induced homeotic transformations, thus supporting this hypothesis. However, other alleles involving this locus gave controversial results and hence we re-assessed the effect of the full deletion of Hotair in vivo. In our genetic background and using our analytical conditions, we could not confirm the reported morphological alterations, nor could we detect any mis-regulation of Hoxd genes in those fetal tissues where Hotair is detected in control animals. However, the genomic deletion induces the mis-regulation in-cis of the neighboring Hoxc11 and Hoxc12 genes, a side-effect which may underlie a weakly penetrant alteration observed in the shape of some tail vertebrae.

Schizophrenia (SCZ) may cause tuberculosis, the treatments for which can induce anti-tuberculosis drug-induced hepatotoxicity (ATDH) and SCZ-like disorders. To date, the causal genes of both SCZ and ATDH are unknown. To identify them, we proposed a new network-based method by integrating network random walk with restart algorithm, gene set enrichment analysis, and hypergeometric test; using this method, we identified 500 common causal genes. For gene validation, we created a regularly updated online database ATDH-SCZgenes and conducted a systematic meta-analysis of the association of each gene with either disease. Till now, only GSTM1 and GSTT1 have been well studied with respect to both diseases; and a total of 23 high-quality association studies were collected for the current meta-analysis validation. Finally, the GSTM1 present genotype was confirmed to be significantly associated with both ATDH [Odds Ratio (OR): 0.71, 95% confidence interval (CI): 0.56–0.90, P = 0.005] and SCZ (OR: 0.78, 95% CI: 0.66–0.92, P = 0.004) according to the random-effect model. Furthermore, these significant results were supported by “moderate” evidence according to the Venice criteria. Our findings indicate that GSTM1 may be a causal gene of both ATDH and SCZ, although further validation pertaining to other genes, such as CYP2E1 or DRD2, is necessary.

Neural stem cells (NSCs) play essential roles in nervous system development and postnatal neuroregeneration and their deregulation underlies the development of neurodegenerative disorders. Yet how NSC proliferation and differentiation are controlled is not fully understood. Here we present evidence that tumor suppressor p53 regulates NSC proliferation and differentiation via the bone morphogenetic proteins (BMP)-Smad1 pathway and its target gene inhibitor of DNA binding 1 (Id1). p53 deficiency led to increased neurogenesis in vivo, and biased neuronal differentiation and augmented NSC proliferation of ex vivo NSCs. This is accompanied by elevated Smad1 expression/activation in the brain and NSC, which contributes to accelerated neuronal differentiation of p53(-/-) NSCs. p53 deficiency also leads to upregulation of Id1, whose expression is repressed by p53 in BMP-Smad1-dependent and -independent manners. Elevated Id1 expression contributes to augmented proliferation and, unexpectedly, accelerated neuronal differentiation of p53(-/-) NSCs as well. This study reveals a molecular mechanism by which tumor suppressor p53 controls NSC proliferation and differentiation and establishes a connection between p53 and Id1.

The 14-3-3 family, which is composed of seven distinct members in humans, plays important roles in the cell cycle, apoptosis, synaptic plasticity and neuronal differentiation and migration. Previous genetic and post-mortem gene expression studies have linked this family to schizophrenia. However, the direction of gene expression changes in these studies has been inconsistent, and reports of 14-3-3 gene expression in living schizophrenic patients are still lacking. Here, we assessed 14-3-3 gene and protein expression levels in peripheral blood leukocytes from drug-naïve first-episode schizophrenic patients and matched controls. mRNA and protein expression levels were quantified by qRT-PCR and UPLC-MRM/MS, respectively. Expression analysis revealed four downregulated and one upregulated mRNA transcripts as well as five downregulated protein levels of 14-3-3 isoforms in schizophrenia. Moreover, significant positive correlations between 14-3-3 mRNA and protein expression levels were found in schizophrenia, and we also identified negative correlations between ε, θ and ζ isoform expression levels and positive symptoms of schizophrenia. Our results suggest that gene and protein expression levels for the 14-3-3 family are dysregulated in schizophrenia, perhaps owing to specific regulatory mechanisms, and we also suggest that expression of the 14-3-3ε, θ and ζ isoform genes could be useful indicators of disease severity.

article i nfo Heat shock protein 70 (HSP70) is a member of molecular chaperones and is important for reproductive bio- logical processes in eukaryotes. In this regard, a full length HSP70 comprised of 2366 nucleotides from the red claw crayfish Cherax quadricarinatus (CqHSP70) was characterized, which with an open-reading frame of 1959 bp encoded 652 amino acid residues. Its mRNA transcript expression in the testes was measured by real-time quantitative PCR. CqHSP70 mRNA transcripts were detected in all organs especially high in the testes. Furthermore, the greatest transcript levels were found during the spermatogonial preparation phase, while the lowest levels were found during the resting stage of the reproductive cycle. In addition, its distribution in the testes was determined by Western blot and immunohistochemistry. Western blot results revealed a single immunoreactive band with an estimated molecular mass of 70 kDa in different phases of testes development. The strongest immunolabeling positive signal was found in spermatogonia, with lower positive staining in secondary spermatocytes, and weak or absent level in the mature sperm. Interestingly, CqHSP70 was mainly located in the cytoplasm of sperm cells. Thus, our results indicate that CqHSP70 is an essential protein in spermatogenesis, with a crucial reproductive function during sperm maturation in invertebrates.

Rhodopsin-like G protein-coupled receptors (GPCRs) are known to be involved in the GPCR signal transduction system and regulate many essential physiological processes in organisms. This study, for the first time, revealed that knockdown of the rhodopsin-like GPCR gene in resistant mosquitoes resulted in a reduction of mosquitoes’ resistance to permethrin, simultaneously reducing the expression of two cAMP-dependent protein kinase A genes (PKAs) and four resistance related cytochrome P450 genes. The function of rhodopsin-like GPCR was further confirmed using transgenic lines of Drosophila melanogaster, in which the tolerance to permethrin and the expression of Drosophila resistance P450 genes were both increased. The roles of GPCR signaling pathway second messenger cyclic adenosine monophosphate (cAMP) and downstream effectors PKAs in resistance were investigated using cAMP production inhibitor Bupivacaine HCl and the RNAi technique. Inhibition of cAMP production led to significant decreases in both the expression of four resistance P450 genes and two PKA genes and mosquito resistance to permethrin. Knockdown of the PKA genes had shown the similar effects on permethrin resistance and P450 gene expression. Taken together, our studies revealed, for the first time, the role of the GPCR/cAMP/PKA-mediated regulatory pathway governing P450 gene expression and P450-mediated resistance in Culex mosquitoes.

Supplemental Digital Content is available in the text, Identification of cancer-associated genes by genomic profiling contributes to the elucidation of tumor development and progression. The methylthioadenosine phosphorylase (MTAP) gene, located at chromosome 9p21, plays a critical role in tumorigenicity and disease progression in a wide variety of cancers. However, the prognostic impact of MTAP in patients with nasopharyngeal carcinoma (NPC) remains obscured. Through data mining from published transcriptomic database, MTAP was first identified as a differentially downregulated gene in NPC. In this study, our aim was to evaluate the expression of MTAP in NPC and to clarify its prognostic significance. MTAP immunohistochemistry was retrospectively performed and analyzed in biopsy specimens from 124 NPC patients who received standard treatment without distant metastasis at initial diagnosis. The immunoexpression status was correlated with the clinicopathological variables, disease-specific survival (DSS), distant metastasis-free survival (DMFS), and local recurrence-free survival (LRFS). Real-time quantitative polymerase chain reaction (PCR) was used to measure MTAP gene dosage. In some cases, we also performed methylation-specific PCR and pyrosequencing to assess the status of promoter methylation. MTAP deficiency was significantly associated with advanced tumor stages (P = 0.023) and univariately predictive of adverse outcomes for DSS, DMFS, and LRFS. In the multivariate comparison, MTAP deficiency still remained prognostically independent to portend worse DSS (P = 0.021, hazard ratio = 1.870) and DMFS (P = 0.009, hazard ratio = 2.154), together with advanced AJCC stages III to IV. Homozygous deletion or promoter methylation of MTAP gene were identified to be significantly associated with MTAP protein deficiency (P

Carboxylesterases (CarEs) play important roles in metabolism and detoxification of dietary and environmental xenobiotics in insects and mites. On the basis of the Tetranychuscinnabarinus transcriptome dataset, 23 CarE genes (6 genes are full sequence and 17 genes are partial sequence) were identified. Synergist bioassay showed that CarEs were involved in acaricide detoxification and resistance in fenpropathrin- (FeR) and cyflumetofen-resistant (CyR) strains. In order to further reveal the relationship between CarE gene's expression and acaricide-resistance in T. cinnabarinus, we profiled their expression in susceptible (SS) and resistant strains (FeR, and CyR). There were 8 and 4 over-expressed carboxylesterase genes in FeR and CyR, respectively, from which the over-expressions were detected at mRNA level, but not DNA level. Pesticide induction experiment elucidated that 4 of 8 and 2 of 4 up-regulated genes were inducible with significance in FeR and CyR strains, respectively, but they could not be induced in SS strain, which indicated that these genes became more enhanced and effective to withstand the pesticides' stress in resistant T. cinnabarinus. Most expression-changed and all inducible genes possess the Abhydrolase_3 motif, which is a catalytic domain for hydrolyzing. As a whole, these findings in current study provide clues for further elucidating the function and regulation mechanism of these carboxylesterase genes in T. cinnabarinus' resistance formation.

The carmine spider mite Tetranychus cinnabarinus is the most serious of crop mite pests in China. Their ability to rapidly develop resistance to acaricides has caused difficulty in controlling this mite. In this study, the molecular mechanism of acaricide resistance associated with esterase genes TCE1 and TCE2 was investigated in susceptible and acaricide-resistant strains of T. cinnabarinus. The quantitative real-time PCR (qrtPCR) method was adopted to compare the expression level of two esterase genes TCE1 and TCE2 among four different strains (abamectin-resistant, AbR; fenpropathrin-resistant, FeR; omethoate-resistant, OmR and susceptible strains, S) of T. cinnabarinus. The relative expression level of TCE2 was 1.39–2.47 fold in the three resistant strains compared with the S strain. And after inducing with abamectin, fenpropathrin, and omethoate the highest expression level of TCE2 in the S was 1.64-, 2.92- and 2.24-fold compared with the control, respectively, and this difference was found to be significant. However, there was no obvious difference of the mRNA relative expression levels of TCE1 genes among the four strains, and those of TCE1 were not higher than the control throughout the study. Furthermore, the expression modes of TCE1 and TCE2 in AbR and FeR were similar with that in the S after being treated with abamectin and fenpropathrin, respectively. These results indicated that the enhanced expression of esterase gene TCE2 was associated with acaricide-resistance in T. cinnabarinus.

MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs (ncRNAs) that function to regulate gene expression at the post-transcriptional level. Although their functions were originally described during normal development, miRNAs have emerged as integral components of the oncogenic and tumor suppressor network, regulating nearly all cellular processes altered during tumor formation. In particular, mir-17-92, a miRNA polycistron also known as oncomir-1, is among the most potent oncogenic miRNAs. Genomic amplification and elevated expression of mir-17-92 were both found in several human B-cell lymphomas, and its enforced expression exhibits strong tumorigenic activity in multiple mouse tumor models. mir-17-92 carries out pleiotropic functions during both normal development and malignant transformation, as it acts to promote proliferation, inhibit differentiation, increase angiogenesis and sustain cell survival. Unlike most protein coding genes, mir-17-92 is a polycistronic miRNA cluster that contains multiple miRNA components, each of which has a potential to regulate hundreds of target mRNAs. This unique gene structure of mir-17-92 may underlie the molecular basis for its pleiotropic functions in a cell type and context dependent manner. Here we review the recent literature on the functional studies of mir-17-92, and highlight its potential impacts on the oncogene network. These findings on mir-17-92 indicate that miRNAs, together with protein coding genes, are integrated components of the molecular pathways that regulate tumor development and tumor maintenance.

Three heat shock protein 70 (Hsp70) cDNAs were isolated from the carmine spider mite, Tetranychus cinnabarinus. They were tentatively named as TCHsp70-1, TCHsp70-2 and TCHsp70-3. Structural analyses showed that all of the three TCHsp70 cDNAs held the full open reading frame (ORF). Putative protein sequences and a phylogenetic tree suggested that TCHsp70-1 and TCHsp70-3 were cytoplasm HSP70 and TCHsp70-2 was endoplasmic reticulum HSP70. Comparison of deduced amino acid sequences of TCHsp70-1 and TCHsp70-3 showed 84.78% identity, TCHsp70-1 and TCHsp70-2 showed 57.33% identity, TCHsp70-2 and TCHsp70-3 showed 58.26% identity. Real-time comparative quantitative PCR revealed that the relative expression of TCHsp70-2 was lower than TCHsp70-1 and TCHsp70-3 at each temperature tested. TCHsp70-1 and TCHsp70-3 shared a similar expression pattern after cold and heat shock compared with their expression at normal temperature (26 degrees C), but the mRNA expression of TCHsp70-1 was significantly higher and lower than that of TCHsp70-3 at cold and heat shock temperatures (except for 34 degrees C), respectively. This result possibly indicated the expression patterns of TCHsp70 were affected by their location in different cellular compartments. The results also indicated that three TCHsp70s, especially TCHsp70-1 and TCHsp70-3, may play an important role in mediating tolerance to cold, thermal stress for Tetranychus cinnabarinus.

Background A number of studies have pointed to the involvement of AKT signaling pathways in the etiology of schizophrenia. The purpose of this study was to determine whether the AKT1 gene is involved in the etiology of schizophrenia and whether it affects therapeutic outcomes in the Chinese population. Method Five single nucleotide polymorphisms (SNPs) were genotyped among 384 schizophrenic patients (DSM-IV criteria) and 384 healthy controls from the Chinese population. We systematically analyzed the association of the AKT1 gene with schizophrenia on the basis of sex, age at onset, therapeutic response to typical antipsychotics and atypical antipsychotics, and presence or absence of extrapyramidal syndrome. The study was conducted from May 2004 to June 2006. Results We found a positive association of the G allele of the SNP marker rs3803300 with schizophrenia (p = .003), both in early-onset and late-onset subjects, and that a haplotype A-G-C-G-A constructed by the 5 SNPs showed significant association (p = .00004886). However, we found no relationship between any of the 5 SNP markers and therapeutic response to typical and atypical antipsychotics and chlorpromazine-induced extrapyramidal syndrome. Conclusions Our study suggests that AKT1 is a susceptibility gene for schizophrenia in the Chinese population and that the AKT1 gene may play no major role in the therapeutic response to antipsychotics or in chlorpromazine-induced extrapyramidal syndrome.

Background Lines of evidence have suggested that the brain-derived neurotrophic factor (BDNF) gene may be involved in the pathogenesis of schizophrenia. Two common functional polymorphisms C-270T and Val66Met within the BDNF gene were first reported by Kunugi et al. [Kunugi, H., Nanko, S., Hirasawa, H., Kato, N., Nabika, T., Kobayashi, S., 2003. Brain-derived neurotrophic factor gene and schizophrenia: polymorphism screening and association analysis. Schizophr. Res. 62, 281–283.] and pls expand this too: Hong et al. (2003) to be significantly associated with schizophrenia. However, subsequently several studies obtained conflicting results. Methods We compared the allele/genotype frequencies of the C-270T and Val66Met polymorphisms and the haplotype frequencies at the two polymorphisms in a moderate independent patient–control sample from the Han Chinese population. Two systematic meta-analyses were performed to assess the collective evidence for association across studies for each of the two polymorphisms. Results No statistically significant differences were found in allele or genotype or haplotype frequencies between patient and normal control subjects for either of the two polymorphisms. On the other hand, the meta-analysis of all published population-based association studies showed statistically significant evidence for heterogeneity among each of the two polymorphisms. Stratification of the studies by ethnicity of the samples yielded no significant evidence for an association with the polymorphisms in the Caucasian population (for C-270T polymorphism: pooled OR Caucasian = 0.736, 95% CI = 0.476–1.139, p = 0.169; for Val66Met polymorphism: pooled OR Caucasian = 1.027, 95% CI = 0.796–1.325, p = 0.835), nor in the Asian population (for C-270T polymorphism: pooled OR Asian = 0.445, 95% CI = 0.144–1.373, p = 0.159; for Val66Met polymorphism: pooled OR Asian = 0.962, 95% CI = 0.820–1.128, p = 0.635). Conclusions Our population-based study and meta-analysis demonstrate that the BDNF C-270T and Val66Met polymorphisms do not play major roles in the susceptibility to schizophrenia in either Caucasian or Asian populations. But we can not rule out the possibility that other polymorphisms with the BDNF gene are involved in the pathophysiology of schizophrenia.

Studies of discordance in monozygotic twins have demonstrated that environmental effects play an important role in the pathogenesis of schizophrenia. DNA microarray analysis has revealed upregulation of the DRD2 gene in peripheral blood lymphocytes of schizophrenic patients. We hypothesized that this expression alteration could involve the DNA (CpG) methylation status that is implicated to the transcription status of the gene and in this study we used bisulfited sequence analysis to determine the DNA methylation status of a typical CpGs island within the 5'-regulatory region of DRD2 in peripheral blood lymphocytes from 48 discordant sib pairs suffering from schizophrenia. We found that the methylated cytosines occurred mainly in three clusters. No statistically significant difference in frequency of site-specific cytosine methylation modification of DRD2 between patients and normal controls was found nor did we find any significant association between sex, age on admission or age at onset of schizophrenia and methylated cytosines of DRD2. Our study did not support the hypothesis that site-specific cytosine methylation of DRD2 plays a role in the psychopathology of schizophrenia.

Aim: The huge interindividual difference of CYP3A4 expression may contribute to the variability of drug response. Post-transcriptional regulation of CYP3A4 remains elusive although transcriptional regulation has been studied much more clearly. microRNAs (miRNAs) were reported to be one of factors to regulate the expression of CYP3A4 previously. Materials & methods: Based on the in silico prediction of 3′-UTR-bindind site of microRNA-27a (miR-27a), the transcriptional and post-transcriptional regulation of miR-27a were investigated through luciferase reporter assay, real-time PCR and immunoblot. Results: The significantly decrease of CYP3A4 3′-UTR-luciferase activity in human embryonic kidney 293 and Hep3B cells was detected after transfected with plasmid that expressed miRNA-27a in luciferase reporter assay. Correlation study was conducted in human livers (n = 26) and significant correlation has been discovered between miRNA-27a and CYP3A4 mRNA and protein level. Conclusion: Together, these findings suggest that miR-27a might be involved in the regulation of CYP3A4 gene expression.

MIR137 , which encodes microRNA-137 (miR-137), and several of its target genes exhibit genome-wide significant associations with schizophrenia. In a previous study, we analyzed the SNPs in a group of predicted MIR137 target genes and detected genome-wide significant association of schizophrenia with rs2944829 in the CALN1 gene. However, no experimental evidence for CALN1 and MIR137 interaction has yet been reported. In this study, we first computationally analyzed the putative miR-137 target site on CALN1 and predicted that miR-137 binds CALN1 at nucleotide (nt) position 236–242 in the 3′UTR. Then we assayed gene expression by transfecting miR-137 mimics into HEK293 and SH-SY5Y cell lines. Quantitative real-time RT-PCR results showed that the expression level of CALN1 significantly decreased in cells co-transfected with miR-137 mimics compared to cells transfected with the blank control ( P = .0046 in HEK293 cell lines, P = .038 in SH-SY5Y cells lines). Finally, we co-transfected different combinations of miRNA mimics and either wild type CALN1 3′UTR or mutant 3′UTR reporters into HEK293 and SH-SY5Y cell lines and assessed the specificity of miRNA binding using a luciferase reporter assay. The transfection of miR-137 mimics corresponded with a considerable reduction of luciferase activity on vectors carrying the target fragment ( P = 1.17 × 10 −5 , 68% reduction in HEK293 cell line, and P = 5.09 × 10 −6 , 32% reduction in SH-SY5Y cell line). This inhibition was impaired by site-directed mutagenesis of the miR-137 target fragment. Our results provide strong evidence that CALN1 is a target of miR-137.

Background: Epidemiological studies have identified prenatal exposure to famine as a risk factor for schizophrenia, and animal models of prenatal malnutrition display structural and functional brain abnormalities implicated in schizophrenia. Methods: The offspring of the RLP50 rat, a recently developed animal model of prenatal famine malnutrition exposure, was used to investigate the changes of gene expression and epigenetic modifications in the brain regions. Microarray gene expression analysis was carried out in the prefrontal cortex and the hippocampus from 8 RLP50 offspring rats and 8 controls. MBD-seq was used to test the changes in DNA methylation in hippocampus depending on prenatal malnutrition exposure. Results: In the prefrontal cortex, offspring of RLP50 exhibit differences in neurotransmitters and olfactory-associated gene expression. In the hippocampus, the differentially-expressed genes are related to synaptic function and transcription regulation. DNA methylome profiling of the hippocampus also shows widespread but systematic epigenetic changes; in most cases (87%) this involves hypermethylation. Remarkably, genes encoded for the plasma membrane are significantly enriched for changes in both gene expression and DNA methylome profiling screens ( p = 2.37×10–9 and 5.36×10–9, respectively). Interestingly, Mecp2 and Slc2a1 , two genes associated with cognitive impairment, show significant down-regulation, and Slc2a1 is hypermethylated in the hippocampus of the RLP50 offspring. Conclusions: Collectively, our results indicate that prenatal exposure to malnutrition leads to the reprogramming of postnatal brain gene expression and that the epigenetic modifications contribute to the reprogramming. The process may impair learning and memory ability and result in higher susceptibility to schizophrenia.

Recent clinical trials have provided evidence that pioglitazone reduces cardiovascular events in patients with type 2 diabetes. However, the underlying mechanisms are not well understood. Because it has been well established that disruption of atherosclerotic plaques is a key event involved in acute myocardial infarction, we hypothesized that pioglitazone reduces cardiovascular events by stabilizing atherosclerotic lesions. In this study, we used an animal model to test our hypothesis. Low-density lipoprotein receptor-deficient (LDLR-/-) male mice were first fed a high-fat diet for 4 months to induce the formation of aortic atherosclerotic plaques and then treated with pioglitazone for the next 3 months. Analysis of atherosclerotic plaques at the end of the study showed that treatment with pioglitazone at 20 mg/kg/day reduced the progression of atherosclerotic plaques as compared to untreated mice. Furthermore, gene array analysis, quantitative real-time polymerase chain reaction, and immunohistochemical analysis showed that pioglitazone inhibited high-fat diet-induced upregulation of matrix metalloproteinase (MMP) expression. Finally, Sirius red staining showed that atherosclerotic lesions in mice receiving pioglitazone had higher collagen contents than those in untreated mice. This study demonstrated for the first time that administration of pioglitazone in LDLR-/- mice inhibited lesion progression and MMP expression in established atherosclerotic plaques and thus delineated a potential mechanism by which pioglitazone reduces cardiovascular events in patients with type 2 diabetes.

The ability to regulate endogenous gene expression is critical in biological research. Existing technologies, such as RNA interference, zinc-finger regulators, transcription-activator-like effectors, and CRISPR-mediated regulation, though proved to be competent in significantly altering expression levels, do not provide a quantitative adjustment of regulation effect. As a solution to this problem, we place CRISPR-mediated interference under the control of blue light: while dCas9 protein is constitutively expressed, guide RNA transcription is regulated by YF1-FixJ-PFixK2, a blue light responding system. With a computer-controlled luminous device, the quantitative relationship between target gene expression and light intensity has been determined. As the light intensifies, the expression level of target gene gradually ascends. This remarkable property enables sensor-CRISPRi to accurately interrogate cellular activities.

MicroRNAs are a family of small, non-coding RNAs that regulate gene expression in a sequence-specific manner. The two founding members of the microRNA family were originally identified in Caenorhabditis elegans as genes that were required for the timed regulation of developmental events. Since then, hundreds of microRNAs have been identified in almost all metazoan genomes, including worms, flies, plants and mammals. MicroRNAs have diverse expression patterns and might regulate various developmental and physiological processes. Their discovery adds a new dimension to our understanding of complex gene regulatory networks.

Objective— It has been shown that plasma level of C-reactive protein (CRP) is an independent predictor for acute coronary syndromes and is associated with plaque weakening. However, the underlying mechanisms are not well understood. In this study, we investigated the effect of CRP on the expression of matrix metalloproteinase-1 (MMP-1) that has been implicated in plaque vulnerability by human U937 histiocytes and monocyte-derived macrophages. Methods and Results— Enzyme-linked immunosorbent assay of MMP-1 in conditioned medium showed that treatment of U937 cells with 100 μg/mL of CRP for 24 hour led to a 3- to 5-fold increase in MMP-1 secretion. CRP also markedly stimulated MMP-1 release from human monocyte-derived macrophages. In contrast, CRP had no effect on tissue inhibitor of metalloproteinase-1 (TIMP-1) secretion. Northern blot showed that CRP upregulated MMP-1 mRNA expression. Collagenase activity assay showed that CRP increased collagen-degrading activity in cell-conditioned medium. Furthermore, results showed that the stimulation of MMP-1 secretion by CRP was inhibited by anti-CD32, but not by anti-CD64 antibody, and by mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) inhibitor PD98059. Finally, Western blot showed that CRP stimulated phosphorylation of extracellular signal-regulated kinase. Conclusions— This study demonstrates that CRP stimulates MMP-1 expression by U937 cells through FcγRII and extracellular signal-regulated kinase pathway. These findings suggest that CRP may promote matrix degradation and thus contribute to plaque vulnerability.

Macroautophagy (hereafter termed autophagy) is a highly evolutionarily conserved pathway that degrades intracellular components such as damaged organelles in lysosome. Autophagy occurs at low basal levels in virtually all types of cells, which is required for the maintenance of cellular homeostasis. Beclin 1 protein, encoded by the beclin 1 gene, plays a central role in the regulation of autophagy. Beclin 1 primarily functions as a scaffolding protein assembling Beclin 1 interactome to regulate Class III PI3K/VPS34 activity, which in turn, tightly controls autophagy at multiple stages. In addition to autophagy, Beclin 1 participates in the regulation of other biological processes such as endocytosis, apoptosis and phagocytosis. Fine-tuning of Beclin 1 protein levels, intracellular localization and the assembly of its interactome is pivotal for the proper execution of these biological functions. Deregulation of Beclin 1 contributes to the pathogenesis of a variety of human diseases. In this review, we summarize biology of Beclin 1 and its role in human pathology, with an emphasis on heart disease.

CYP3A4 metabolizes more than 50% of the drugs on the market. The large inter-individual differences of CYP3A4 expression may contribute to the variability of human drug responses. Post-transcriptional regulation of CYP3A4 is poorly understood, whereas transcriptional regulation has been studied much more thoroughly. In this study, we used multiple software programs to predict miRNAs that might bind to CYP3A4 and identified 112 potentially functional miRNAs. Then a luciferase reporter system was used to assess the effect of the overexpression of each potentially functional miRNA in HEK 293T cells. Fourteen miRNAs that significantly decreased reporter activity were measured in human liver samples (N = 27) as candidate miRNAs. To establish a more effective way to analyze in vivo data for miRNA candidates, the relationship between functional miRNA and target mRNA was modeled mathematically. Taking advantage of this model, we found that hsa-miR-577, hsa-miR-1, hsa-miR-532-3p and hsa-miR-627 could significantly downregulate the translation efficiency of CYP3A4 mRNA in liver. This study used in silico, in vitro and in vivo methods to progressively screen functional miRNAs for CYP3A4 and to enhance our understanding of molecular events underlying the large inter-individual differences of CYP3A4 expression in human populations.