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Abstract: Objective: The deregulation of E-cadherin is associated with Src/FAK signaling axis and histone deacetylase (HDAC)/EZH2 activity. However, the association between EZH2 and FAK and its clinical significance in endometrial carcinoma has not been reported. Methods: 202 archived cases of endometrial carcinoma (1996–2000) were reviewed and divided into two subtypes. TMAs were developed as per established procedures. EZH2, FAK, and pFAK immunohistochemical stains were performed and the expression was scored as negative (0), low (1) and high (2). Proper statistical analysis was used to assess the correlation between the expression profiles and the clinicopathological parameters and clinical outcome. Results: A total of 141 (69.8%) type-1 tumors and 61 (30.2%) type-2 tumors were identified. EZH2 overexpression was identified in 7.6% of type-1 tumors vs. 63% of type-2 tumors (p<0.001). FAK and pFAK overexpression was only seen in 24.8% and 1.7% of Type-1 tumors as compared to 72% and 58.8% of type-2 tumors, respectively (p<0.001). A positive correlation between the expression of EZH2, FAK, pFAK and PTEN (p<0.0001) was found. The overexpression of EZH2, FAK, and pFAK were significantly associated with high histologic grade, angiolymphatic invasion, lymph node metastasis, myometrial invasion and cervical involvement (p<0.01). Kaplan–Meier analysis demonstrates that the overexpression of EZH2 (p=0.0024), FAK and pFAK (p=0.0001) was significantly associated with decreased overall survival. Conclusion: The overexpression of EZH2, FAK and pFAK correlates with well established pathologic risk factors and may predict a more aggressive biologic behavior in endometrial carcinoma, transforming these proteins into potential therapeutic targets for treatment of endometrial cancer. [Copyright &y& Elsevier]

Abstract: Some of the most serious diseases are characterized by the presence of a specific secondary structure within DNA or RNA, often in the promoter or the coding region of the responsible gene, that enhances or disrupts expression of the protein. Structural elements that impact cellular function may also be formed in other genomic regions such as telomeres. Compounds that interact with such structural elements may be useful in diagnosis or treatment of patients. In this report, we present a FRET melting assay that allows testing of libraries of compounds against four different nucleic acid structures. Compounds are tested to determine whether they stabilize preformed secondary structures (i.e., whether they cause an increase in melting temperature (Tm )). This property is described by the ΔTm parameter, which is the difference between the Tm of the compound-stabilized structure and the Tm of the unbound structure. Model oligonucleotides are labeled with FAM as a fluorescent donor and TAMRA as an acceptor. The intensity of FAM fluorescence is recorded as a function of temperature. Melting temperatures are determined by the FRET method in 96-well plates; this assay could easily be converted into 384-well format. [Copyright &y& Elsevier]

Abstract: Selenoprotein K (SelK), an endoplasmic reticulum (ER) resident protein, its biological function has been less-well studied. To investigate the role of SelK in the ER stress response, effects of SelK gene silence and ER stress agents on expression of SelK and cell apoptosis in HepG2 cells were studied. The results showed that SelK was regulated by ER stress agents, Tunicamycin (Tm) and β-Mercaptoethanol (β-ME), in HepG2 cells. Moreover, the SelK gene silence by RNA interference could significantly aggravate HepG2 cell death and apoptosis induced by the ER stress agents. These results suggest that SelK is an ER stress-regulated protein and plays an important role in protecting HepG2 cells from ER stress agent-induced apoptosis. [ABSTRACT FROM AUTHOR]

Abstract: In the present study, cDNA encoding a heat shock protein 60 (LvHSP60) gene in Litopenaeus vannamei was cloned using a combination of homology and rapid amplification of cDNA end (RACE) methods. The full length of the LvHSP60 cDNA was found to be 2379bp, with a 1737bp open reading frame. The translated amino acid sequence consisted of 579 residues with a calculated molecular mass of 60.8kD and an isoelectronic point (pI) of 5.97. Comparison of the deduced amino acid sequence showed that it has high identity (85–89%) with HSP60/chaperonins from insects and mammals. Quantitative real-time PCR and Western blot analysis were carried out to investigate the expression patterns and distribution profiles of LvHSP60 before and after stimulation with the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Vibrio alginolyticus. LvHSP60 mRNA was found to be both constitutive and inducible, and was highly expressed in haemocytes and almost all tissues examined, including muscle, stomach, heart, hepatopancreas and gill tissue, but it was less strongly expressed in the intestine. The expression analysis revealed that LvHSP60 was significantly up-regulated in the gills, hepatopancreas and haemocytes after bacterial challenge. Transcription of LvHSP70 was also induced in haemocytes and the hepatopancreas after different bacteria injection. Subsequent flow cytometry analysis showed that the concentration of Ca2+ ions increased significantly within bacteria-challenged haemocytes by 1.5h after injection. The results indicate that LvHSP60 and LvHSP70 may play important roles in mediating the immune responses of L. vannamei to bacterial challenge, and that the Ca2+ signalling transduction pathway may be involved in the initiation of the shrimp’s immune responses in early stages of infection. [Copyright &y& Elsevier]

Grass carp (Ctenopharyngodon idella) is the largest economic fish in freshwater culture in China, which is predisposed to infectious diseases under high temperature. Under the background of global warming, the industrialization of the Pearl River Delta region has led to aggravated thermal pollution, which has increasingly serious impacts on the aquatic ecological environment. This will result in more frequent exposure of grass carp to overheated water temperatures. Previous studies have only identified the regulatory genes of fish that respond to pathogens or temperature stress, but the transcriptional response to both is unknown. In this study, the histopathological analysis showed heat stress exacerbated spleen damage induced by Aeromonas hydrophila. The transcriptional responses of the spleens from A. hydrophila lipopolysaccharide (LPS) - injected grass carp undergoing heat stress and at normal temperatures for 6, 24, and 72 h were investigated by mRNA and microRNA sequencing. We identified 28, 20, and 141 differentially expressed (DE) miRNAs and 126, 383, and 4841 DE mRNAs between the two groups after 6, 24, and 72 h, respectively. There were 67 DE genes mainly involved in the cytochrome P450 pathway, antioxidant defense, inflammatory response, pathogen recognition pathway, antigen processing and presentation, and the ubiquitin-proteasome system. There were 5 DE miRNAs involved in regulating apoptosis and inflammation. We further verified 17 DE mRNAs and 5 DE miRNAs using quantitative real-time PCR. Based on miRNAs and mRNAs analysis, continuous heat stress will affect the antibacterial responses of grass carp spleens, resulting in aggravation of spleen injury. Together, these results provide data for further understanding of the decreased tolerance of fish to pathogen infection in persistent high-temperature environments. • Continuous heat stress exacerbates spleen damage induced by Aeromonas hydrophila. • Continuous heat stress impaired the antioxidant and immune functions of grass carp. • HSPs are induced to persist after co-treatment with LPS and heat stress. • MicroRNAs mediate apoptosis and inflammation in the damaged spleen of grass carp. [ABSTRACT FROM AUTHOR]

Many regulators controlling arterial identity are well described; however, transcription factors that promote vein identity and vascular patterning have remained largely unknown. We previously identified the transcription factors Islet2 (Isl2) and Nr2f1b required for specification of the vein and tip cell identity mediated by notch pathway in zebrafish. However, the interaction between Isl2 and Nr2f1b is not known. In this study, we report that Nr2f2 plays minor roles on vein and intersegmental vessels (ISV) growth and dissect the genetic interactions among the three transcription factors Isl2, Nr2f1b, and Nr2f2 using a combinatorial knockdown strategy. The double knockdown of isl2/nr2f1b , isl2/nr2f2 , and nr2f1b/nr2f2 showed the enhanced defects in vasculature including less completed ISV, reduced veins, and ISV cells. We further tested the genetic relationship among these three transcription factors. We found isl2 can regulate the expression of nr2f1b and nr2f2 , suggesting a model where Isl2 functions upstream of Nr2f1b and Nr2f2. We hypothsized that Isl2 and Nr2f1b can function together through cis -regulatory binding motifs. In-vitro luciferase assay results, we showed that Isl2 and Nr2f1b can cooperatively enhance gene expression. Moreover, co-immunoprecipitation results indicated that Isl2 and Nr2f1b interact physically. Together, we showed that the interaction of the Nr2f1b and Nr2f2 transcription factors in combination with the Islet2 play coordinated roles in the vascular development of zebrafish. • The interaction of the Nr2f1b and Nr2f2 transcription factors in combination with the Islet2 play coordinated roles in the vascular development of zebrafish. • Physical interaction between Isl2 and Nr2f1b by using genetic and biochemical approaches. • Isl2 and Nr2f1b can cooperatively enhance gene expression via cis -regulatory element of target genes. [ABSTRACT FROM AUTHOR]

Abstract: Potato microtuber produced in vitro provides a model system to investigate photoperiod-dependent tuberization. However, the genes associated with potato tuberization remain to be elucidated. The present research involved three potato clones with distinct tuberization response to changes of photoperiod. Digital Gene Expression (DGE) Tag Profiling analysis of the short-day-sensitive clone identified 2218 genes that were regulated by day length. Both GO and KEGG pathway analysis provided insights into predominant biological processes and pathways, and enabled the selection of 56 genes associated with circadian rhythmicity, signal transduction, and development. Quantitative transcriptional analysis in the selected clones revealed 5 genes potentially associated with photoperiodic tuberization, which were predicted to encode a DOF protein, a blue light receptor, a lectin, a syntaxin-like protein, and a protein with unknown function. Our results strongly suggest that potato tuberization may be largely controlled by the homologs of genes shown to regulate flowering time in other plants. [Copyright &y& Elsevier]

Abstract: In this study the gene encoding HSP70 was isolated from Phascoloma esculenta by homologous cloning and rapid amplification of cDNA ends (RACE). The full-length of cDNA (2520 bp) consists of a 5′-terminal untranslated region (UTR) (125 bp), a 3′-terminal UTR (421 bp) with a canonical polyadenylation signal sequence (AATAAA), a poly (A) tail, and an open reading frame (ORF) (1974 bp). The predicted molecular mass and isoelectric point for HSP70 is 71.6 kDa and 5.15, respectively. BLAST analysis showed that P. esculenta HSP70 gene shared high similarity. Classical HSP signature motifs, ATP/GTP-Binding Site Motif A, Bipartite Nuclear Targeting Sequence, the cytosolic HSP70 could be expressed in Escherichia coli BL21. After purification, the recombinant pET-HSP70 protein was used to produce the polyclonal antibody in mice and the specificity of the antibody was confirmed by Western blot analysis. Fluorescent real-time quantitative PCR analysis showed that expression of Hsp70 in sipuncula was increased significantly after exposure to 10 mM Zn for12 h, Cd for 24 h, Cu for 48 h, and was exposure to 37 °C for 24 h sea water. [Copyright &y& Elsevier]