We used the fluorescence ubiquitination-based cell cycle indicator (FUCCI) to monitor cell cycle arrest after treatment of FUCCI-expressing HeLa cells (FUCCI-HeLa) with a traditional Chinese medicine (TCM) herbal mixture LQ, previously shown to have anti-tumor and anti-metastatic activity in mouse models. Paclitaxel was used as the positive control. In 2D monolayer culture, the untreated control had approximately 45% of the cells in S/G₂/M phase. In contrast, the LQ-treated cells (9 mg/ml) were mostly in the G₀/G₁ (>90%) after 72 hours. After treatment with paclitaxel (0.01 μm), for 72 hours, 95% of the cells were in S/G₂/M. In 2.5D Matrigel culture, the colonies in the untreated control group had 40% of the cells in S/G₂/M. LQ arrested the cells in G₀/G₁ after 72 hours. Paclitaxel arrested almost all the cells in S/G₂/M after 72 hours. In 3D Gelfoam culture, the untreated control culture had approximately 45% of cells in G₂/M. In contrast, the LQ-treated cells were mostly in G₀/G₁ phase (>80%) after 72 hours treatment. Paclitaxel resulted in 90% of the cells arrested in S/G₂/M after 72 hours. The present report suggests the non-toxic LQ has potential to maintain cancers in a quiescent state for long periods of time.

We established co-cultures of invasive or non-invasive NSCLC cell lines and various types of fibroblasts (FBs) to more precisely characterize the molecular mechanism of tumor-stroma crosstalk in lung cancer. The HGF-MET-ERK1/2-CREB-axis was shown to contribute to the onset of the invasive phenotype of Calu-1 with HGF being secreted by FBs. Differential expression analysis of the respective mono- and co-cultures revealed an upregulation of NFκB-related genes exclusively in co-cultures with Calu-1. Cytokine Array- and ELISA-based characterization of the "cytokine fingerprints" identified CSF2 (GM-CSF), CXCL1, CXCL6, VEGF, IL6, RANTES and IL8 as being specifically upregulated in various co-cultures. Whilst CXCL6 exhibited a strictly FB-type-specific induction profile regardless of the invasiveness of the tumor cell line, CSF2 was only induced in co-cultures of invasive cell lines regardless of the partnered FB type. These cultures revealed a clear link between the induction of CSF2 and the EMT signature of the cancer cell line. The canonical NFκB signaling in FBs, but not in tumor cells, was shown to be responsible for the induced and constitutive CSF2 expression. In addition to CSF2, cytokine IL6, IL8 and IL1B, and chemokine CXCL1 and CXCL6 transcripts were also shown to be increased in co-cultured FBs. In contrast, their induction was not strictly dependent on the invasiveness of the co-cultured tumor cell. In a multi-reporter assay, additional signaling pathways (AP-1, HIF1-α, KLF4, SP-1 and ELK-1) were found to be induced in FBs co-cultured with Calu-1. Most importantly, no difference was observed in the level of inducibility of these six signaling pathways with regard to the type of FBs used. Finally, upon tumor fibroblast interaction the massive induction of chemokines such as CXCL1 and CXCL6 in FBs might be responsible for increased recruitment of a monocytic cell line (THP-1) in a transwell assay.

Background. White adipose tissue browning may be a promising strategy to combat obesity. UCP1 is strongly induced in White adipose tissue with β3-adrenergic agonist treatment, but the causes of this increase have not been fully elucidated. This study aims to explore more miRNAs involved in the process of browning of visceral adipose tissue. Methods. Total of fourteen mice were randomly divided into control and study group. Study group mice were injected intraperitoneally with CL316243 once daily for seven days; meanwhile the control group were treated with 0.9% NaCl. After a 7-day period, the expression of genes involved in WAT browning and potential UCP1-targeting miRNAs in adipose tissues was analyzed by qPCR. Results. qPCR analysis revealed that UCP1, DIO2, CIDEA, and CPT1B in epididymal adipose tissue were overexpressed in CL316243 group. Furthermore, potential UCP1-targeting miR-9 and miR-338-3p in epididymal adipose tissue were significantly decreased in CL316243 group. Conclusion. This suggests that potential UCP1-targeting miR-9 and miR-338-3p may be involved in the browning of epididymal adipose tissue by regulating UCP1 gene expression. In this study, we demonstrated that this increase of UCP1 is due, at least in part, to the decreased expression of certain UCP1-targeting miRNAs in epididymal adipose tissue compared to control.

The transcription factor activator protein-1 (AP-1) proteins are implicated to play a major role in the regulation of numerous genes involved in the function and development of the immune system, cell differentiation, proliferation, apoptosis, etc. It can bind to promoter of its target genes in a sequence-specific manner to transactivate or repress them. In this study, the full-length cDNA of an AP-1 was identified from Venerupis philippinarum (denoted as VpAP-1) by EST analysis and RACE approaches. Phylogenetic analysis indicated that VpAP-1 had higher evolutional conservation to invertebrate than vertebrate counterparts and should be a new member of the AP-1 protein family. Spatial expression analysis found that VpAP-1 transcript was most abundantly expressed in the hemocytes and hepatopancreas, weakly expressed in the tissues of the gills, mantle and muscle. After Vibrio anguillarum challenge, the expression of VpAP-1 transcript in overall hemocyte population was up-regulated in the first 6h, and then decreased to 1.5-fold of the control group at 12h. As time progressed, a second peak of VpAP-1 expression was detected at 24h post-infection, which was 5-fold compared with that of the control group (P<0.01). After that, the expression level was sharply decreased and dropped to 0.5-fold of the control at 96h. The above results indicated that VpAP-1 was perhaps involved in the immune responses against microbe infection and might be contributed to the clearance of bacterial pathogens.

Our previous studies indicated that transcription factor Brn-4 is upregulated in the surgically denervated hippocampus in vivo, promoting neuronal differentiation of hippocampal neural stem cells (NSCs) in vitro. The molecules mediating Brn-4 upregulation in the denervated hippocampus remain unknown. In this study we examined the levels of insulin-like growth factor-1 (IGF-1) in hippocampus following denervation. Surgical denervation led to a significant increase in IGF-1 expression in vivo. We also report that IGF-1 treatment on NSCs in vitro led to a marked acceleration of Brn-4 expression and cell differentiation down neuronal pathways. The promotion effects were blocked by PI3K-specific inhibitor (LY294002), but not MAPK inhibitor (PD98059); levels of phospho-Akt were increased by IGF-1 treatment. In addition, inhibition of IGF-1 receptor (AG1024) and mTOR (rapamycin) both attenuated the increased expression of Brn-4 induced by IGF-1. Together, the results demonstrated that upregulation of IGF-1 induced by hippocampal denervation injury leads to activation of the PI3K/Akt signaling pathway, which in turn gives rise to upregulation of the Brn-4 and subsequent stem cell differentiation down neuronal pathways.

Chemotherapy has been widely used to treat cancer, however, the appearance of multiple drug resistance (MDR) in cancer patients is regarded as a major clinical obstacle to successful chemotherapy. MicroRNAs (miRNAs) are evolutionary conserved small RNAs that regulate gene expression at the post-transcriptional level and have been shown to regulate cell differentiation, development, proliferation and apoptosis. Nevertheless, the involvement of miRNAs and their roles in the development of MDR in liver cancer are not fully understood. Our study found that the expression of miR-27a was down-regulated in the multidrug-resistant hepatocellular carcinoma cell line BEL-7402/5-fluorouracil (BEL/5-FU) compared with its parental BEL-7402 cell line, while the MDR1/P-glycoprotein expression was elevated. Overexpression of miR-27a by transfecting with miR-27a mimics in the BEL/5-FU cells could reduce the MDR1/P-glycoprotein and β-catenin expressions, enhance the sensitivity of these cells to 5-fluorouracil and 5-fluorouracil-induced apoptosis. Moreover, up-regulation of miR-27a did not decrease the FZD7 mRNA level, but significantly reduce its protein expression in BEL/5-FU cells. It was also confirmed that reduction of FZD7 by RNA interference induced inhibitory effects on the expression of MDR1/P-glycoprotein and β-catenin, similar to miR-27a. Taken together, our findings suggest that miR-27a could function as a novel regulator to reverse MDR in hepatocellular carcinoma cells by inhibiting the FZD7/β-catenin pathway.

Chromatin remodeling processes are among the most important regulatory mechanisms in controlling cell proliferation and regeneration. Drosophila intestinal stem cells (ISCs) exhibit self-renewal potentials, maintain tissue homeostasis, and serve as an excellent model for studying cell growth and regeneration. In this study, we show that Brahma (Brm) chromatin-remodeling complex is required for ISC proliferation and damage-induced midgut regeneration in a lineage-specific manner. ISCs and enteroblasts exhibit high levels of Brm proteins; and without Brm, ISC proliferation and differentiation are impaired. Importantly, the Brm complex participates in ISC proliferation induced by the Scalloped-Yorkie transcriptional complex and that the Hippo (Hpo) signaling pathway directly restricted ISC proliferation by regulating Brm protein levels by inducing caspase-dependent cleavage of Brm. The cleavage resistant form of Brm protein promoted ISC proliferation. Our findings highlighted the importance of Hpo signaling in regulating epigenetic components such as Brm to control downstream transcription and hence ISC proliferation. DOI:http://dx.doi.org/10.7554/eLife.00999.001.

Cancer therapy that specifically targets malignant cells with minimal or no toxicity to normal tissue has been a long-standing goal of cancer research. Rad51 expression is elevated in a wide range of cancers and Rad51 promoter has been used to transcriptionally target tumor cells, however, a large size of Rad51 promoter limits its application for gene therapy. To identify novel tumor-specific promoters, we examined expression levels of Rad51 paralogs, Rad51B, Rad51C, and Rad51D as well as Rad52 in a panel of normal and tumor cell lines. We found that Rad51C is significantly overexpressed in cancer cells. The expression was up-regulated by approximately 6-fold at the mRNA level and 9-fold at the protein level. Interestingly, the 2064 bp long Rad51C promoter fragment was approximately 300-fold higher in cancer cells than in normal cells. A construct containing Rad51C promoter driving diphtheria toxin A efficiently killed several types of cancer cells with very mild effect to normal cells. These results underscore the potential of targeting the homologous recombination pathway in cancer cells and provide a proof of principle that the Rad51C promoter fragment can be used to transcriptionally target cancer cells.

In the past few years, the bone field has witnessed great advances in genome-wide association studies (GWASs) of osteoporosis, with a number of promising genes identified. In particular, meta-analysis of GWASs, aimed at increasing the power of studies by combining the results from different study populations, have led to the identification of novel associations that would not otherwise have been identified in individual GWASs. Recently, the first whole genome sequencing study for osteoporosis and fractures was published, reporting a novel rare nonsense mutation. This review summarizes the important and representative findings published by December 2013. Comments are made on the notable findings and representative studies for their potential influence and implications on our present understanding of the genetics of osteoporosis. Potential limitations of GWASs and their meta-analyses are evaluated, with an emphasis on understanding the reasons for inconsistent results between different studies and clarification of misinterpretation of GWAS meta-analysis results. Implications and challenges of GWAS are also discussed, including the need for multi- and inter-disciplinary studies.

Recent large-scale transcriptome analyses have found large numbers of transcripts, including that of long non-coding RNAs (lncRNAs), which are aberrant in various diseases, especially cancers. However, it is not clear whether lncRNAs are involved specifically in renal cell carcinoma (RCC). We investigated the expression patterns of lncRNAs in five RCC tumor samples (T) relative to those of matched adjacent non-tumor tissues (N) via microarray.

Indeterminate thyroid nodules form a heterogenous group of lesions that constitute 5% to 30% of thyroid cytology diagnoses. We introduce a triple immunostaining protocol for subtyping. Galectin-3, HBME-1, and p27 triple immunostaining, performed on destained cytology slides and formalin-fixed paraffin-embedded tissue, was developed and applied to 51 patients retrospectively with preoperative cytologic diagnoses of follicular lesion of undetermined significance (n=40), atypia of undetermined significance (n=6), and suspicious for follicular neoplasm (n=5). The malignant rate in this series was 43.1% (22/51). A hierarchal evaluation algorithm was generated based on digital image quantitation of triple-stained histologic sections, and applied to both cytology and histology specimens. Fifty of 51 cytology cases have triple staining validated by internal controls. In cytology specimens, the individual sensitivities and specificities of p27, Galectin3, and HBME1 for cancer with 95% confidence interval are: 86.2% (0.674, 0.955)/66.7% (0.431, 0.845); 77.3% (0.542, 0.913)/72.4% (0.525, 0.866); and 72.7% (0.496, 0.884)/93.1% (0.758, 0.988), respectively. Sensitivity is increased to 95.5% (0.751, 0.998), but specificity is decreased to 69.0% (0.490, 0.840), if Galectin3 and HBME1 are both used in combination as markers for malignancy. However, the level of specificity is increased to 86.2% (0.674, 0.955) and sensitivity remains high 100% (0.808, 1) if in addition, using the Galectin3/HBME1:p27 ratio (ratio ≥2 indicating malignancy) for 2 or 3 markers positive cases. Thus, the triple staining method on cytology slides and histology sections shows a similar sensitivity/specificity/positive predictive value/negative predictive value of 100.0%/86.2%/84.0%/100.0% and 95.5%/86.2%/84.0%/96.2%, respectively (P=0.92). Overall, p27 is the most frequent single positive marker (19/50, 38% in cytology), consistent with benign nature of most indeterminate thyroid nodules. Galectin-3 and HBME-1 colocalization (positive in the same cell) was demonstrated in thyroid cancer in 45.5% (10/22) of histology sections, but in none of the normal thyroid tissues and benign thyroid lesions. This supports the notion that synchronous activation of Galectin-3 and HBME-1 occurs in thyroid malignancy and is highly specific for malignancy. We have demonstrated the performance and pattern of triple immunostaining for subtyping indeterminate thyroid nodules. Further studies and validation in different larger populations are warranted.

Most of the natural variation in flowering time in Arabidopsis thaliana can be attributed to allelic variation at the gene FRIGIDA (FRI, AT4G00650), which activates expression of the floral repressor FLOWERING LOCUS C (FLC, AT5G10140). Usually, late-flowering accessions carry functional FRI alleles (FRI-wt), whereas early flowering accessions contain non-functional alleles. The two most frequent alleles found in early flowering accessions are the ones present in the commonly used lab strains Columbia (FRI-Col) and Landsberg erecta (FRI-Ler), which contain a premature stop codon and a deletion of the start codon respectively.

Neddylation promotes the process of ubiquitination, which plays a critical role in the degradation of numerous proteins, including cell cycle and apoptosis regulators. In our previous study, an increase in neddylation was identified in melanoma cell lines. In the present study, the upregulation of neddylation was detected in melanoma tissues which confirmed the results of our previous study on melanoma cell lines. To explore the mechanism by which the process of neddylation was increased, the enzymes that regulate the process were investigated. These neddylation-related regulatory enzymes are potential targets for melanoma therapy. Downregulation of UBA3, a subunit of the E1 enzyme, by RNA interference caused cell cycle arrest at G0/G1 in the M14 cell line. In addition, cyclin D expression declined, whereas p27, p21 and bax expression increased. These findings suggest that interfering with the neddylation pathway may decrease the proliferation of melanoma through the modulation of cell cycle regulators and apoptosis promoters.

Although ample evidence suggests that emotion and response inhibition are interrelated at the behavioral and neural levels, neural substrates of response inhibition to negative facial information remain unclear. Thus we used event-related potential (ERP) methods to explore the effects of explicit and implicit facial expression processing in response inhibition.

Characteristics of heart failure with preserved ejection fraction (HFPEF) have not yet been fully understood. The objectives of this pilot study are to detect protein expression profile in the sera of HFPEF patients, and to identify potential biomarkers for the disease. Five hundred and seven proteins were detected in the sera of healthy volunteers and patients with either HFPEF or hypertension using antibody microarrays (three in each group). The results showed that the serum concentrations of 17 proteins (e.g. angiogenin, activin A and artemin) differed considerably between HFPEF and non-HFPEF patients (hypertensive patients and healthy controls), while a protein expression pattern distinct from that in non-HFPEF patients was associated with HFPEF patients. The up-regulation of angiogenin in both HFPEF patients with LVEF ≥50% (P = 0.004) and a subset of HFPEF patients with LVEF = 41-49% (P < 0.001) was further validated in 16 HFPEF patients and 16 healthy controls. Meanwhile, angiogenin distinguished HFPEF patients from controls with a mean area under the receiver operating characteristic curve of 0.88 (P < 0.001) and a diagnostic cut-off point of 426 ng/ml. Moreover, the angiogenin levels in HFPEF patients were positively correlated with Lg(N-terminal pro-B-type natriuretic peptide, NT-proBNP) (P < 0.001). In addition, high angiogenin level (≥426 ng/ml) was a predictor of all-cause death within a short-term follow-up duration, but not in the longer term of 36 months. This pilot study indicates that the aforementioned 17 potential biomarkers, such as angiogenin, may hold great promise for both diagnosis and prognosis assessment of HFPEF.

Melatonin is an indolamine synthesized in the pineal gland that has a wide range of physiological functions, and it has been under clinical investigation for expanded applications. Increasing evidence demonstrates that melatonin can ameliorate cadmium-induced hepatotoxicity. However, the potentially protective effects of melatonin against cadmium-induced hepatotoxicity and the underlying mechanisms of this protection remain unclear. This study investigates the protective effects of melatonin pretreatment on cadmium-induced hepatotoxicity and elucidates the potential mechanism of melatonin-mediated protection. We exposed HepG2 cells to different concentrations of cadmium chloride (2.5, 5, and 10 μM) for 12 h. We found that Cd stimulated cytotoxicity, disrupted the mitochondrial membrane potential, increased reactive oxygen species production, and decreased mitochondrial mass and mitochondrial DNA content. Consistent with this finding, Cd exposure was associated with decreased Sirtuin 1 (SIRT1) protein expression and activity, thus promoted acetylation of PGC-1 alpha, a key enzyme involved in mitochondrial biogenesis and function, although Cd did not disrupt the interaction between SIRT1 and PGC-1 alpha. However, all cadmium-induced mitochondrial oxidative injuries were efficiently attenuated by melatonin pretreatment. Moreover, Sirtinol and SIRT1 siRNA each blocked the melatonin-mediated elevation in mitochondrial function by inhibiting SIRT1/ PGC-1 alpha signaling. Luzindole, a melatonin receptor antagonist, was found to partially block the ability of melatonin to promote SIRT1/ PGC-1 alpha signaling. In summary, our results indicate that SIRT1 plays an essential role in the ability of moderate melatonin to stimulate PGC-1 alpha and improve mitochondrial biogenesis and function at least partially through melatonin receptors in cadmium-induced hepatotoxicity.

In many regions, excessive fluoride and excessive iodide coexist in groundwater, which may lead to biphasic hazards to human thyroid. To explore fluoride-induced thyroid cytotoxicity and the mechanism underlying the effects of excessive iodide on fluoride-induced cytotoxicity, a thyroid cell line (Nthy-ori 3-1) was exposed to excessive fluoride and/or excessive iodide. Cell viability, lactate dehydrogenase (LDH) leakage, reactive oxygen species (ROS) formation, apoptosis, and the expression levels of inositol-requiring enzyme 1 (IRE1) pathway-related molecules were detected. Fluoride and/or iodide decreased cell viability and increased LDH leakage and apoptosis. ROS, the expression levels of glucose-regulated protein 78 (GRP78), IRE1, C/EBP homologous protein (CHOP), and spliced X-box-binding protein-1 (sXBP-1) were enhanced by fluoride or the combination of the two elements. Collectively, excessive fluoride and excessive iodide have detrimental influences on human thyroid cells. Furthermore, an antagonistic interaction between fluoride and excessive iodide exists, and cytotoxicity may be related to IRE1 pathway-induced apoptosis.

Metastasis is the leading cause of cancer-related death in almost all types of cancers, including colorectal cancer (CRC). Metastasis is a complex, multistep, dynamic biological event, and epithelial-mesenchymal transition (EMT) is a critical process during the cascade. Ajuba family proteins are LIM domain-containing proteins and are reported to be transcription repressors regulating different kinds of physiological processes. However, the expression and pathological roles of Ajuba family proteins in tumors, especial in tumor metastasis, remain poorly studied. Here, we found that JUB, but not the other Ajuba family proteins, was highly upregulated in clinical specimens and CRC cell lines. Ectopic expression of JUB induced EMT and enhanced motility and invasiveness in CRC, and vice versa. Mechanistic study revealed that JUB induces EMT via Snail and JUB is also required for Snail-induced EMT. The expression of JUB shows an inverse correlation with E-cadherin expression in clinical specimens. Taken together, these findings revealed that the LIM protein JUB serves as a tumor-promoting gene in CRC by promoting EMT, a critical process of metastasis. Thus, the LIM protein JUB may provide a novel target for therapy of metastatic CRC.

The most predominant HIV-1 strains in China's current epidemic is the Circulating Recombinant Form 07_BC (CRF07_BC). CRF07_BC is mainly considered as a CCR5-tropic (R5) virus, since CXCR4-tropic (X4) viruses have thus far not been found in this subtype, and the molecular determinants of coreceptor switching remain unknown. To investigate the mechanisms underlying coreceptor requirement in CRF07_BC viruses, we characterized a panel of pNL4-3-based chimeric viruses with mutated V3 loop regions derived from an HIV-1 CRF07_BC infectious clone pXJDC13. Among 17 chimeric viruses, seven were dual-tropic and induced syncytium formation in MT-2 cells. Two amino acid insertions between positions 13 and 14, as well as arginine substitution at position 11 or 16 (IG insertion and P16R mutation or MG insertion and S11R mutation), conferred the chimeric viruses CXCR4-tropic features, which were same as subtype C X4 viruses. Next, to construct CRF07_BC X4 variants, mutated V3 loops were cloned into the CRF07_BC infectious clone pXJDC13. These V3 loops, which in the pNL4-3 backbone conferred chimeric viruses with CXCR4-using ability, abrogated infectivity completely in the CRF07_BC pXJDC13 genetic background. Similarly, IG insertion or MG insertion and S11R mutation dramatically diminished or completely abolished viral infectivity in other envelopes of subtype C or CRF07_BC. These results suggest that the effects of IG insertion and P16R mutation or MG insertion and S11R mutation on CXCR4 usage are context dependent, and additional mutations elsewhere in the envelope are needed to compensate for these fitness-reducing alterations.

Hepatocellular carcinoma (HCC) is one of the most frequently diagnosed cancers worldwide. However, the treatment of patients with HCC is particularly challenging. Long non-coding RNA maternally expressed gene 3 (MEG3) has been identified as a potential suppressor of several types of tumors, but the delivery of long RNA remains problematic, limiting its applications. In the present study, we designed a novel delivery system based on MS2 virus-like particles (VLPs) crosslinked with GE11 polypeptide. This vector was found to be fast, effective and safe for the targeted delivery of lncRNA MEG3 RNA to the epidermal growth factor receptor (EGFR)-positive HCC cell lines without the activation of EGFR downstream pathways, and significantly attenuated both in vitro and in vivo tumor cell growth. Our study also revealed that the targeted delivery was mainly dependent on clathrin-mediated endocytosis and MEG3 RNA suppresses tumor growth mainly via increasing the expression of p53 and its downstream gene GDF15, but decreasing the expression of MDM2. Thus, this vector is promising as a novel delivery system and may facilitate a new approach to lncRNA based cancer therapy.