NFAT is a cytoplasm-localized hyper-phosphorylated transcription factor that is activated through dephosphorylation by calcineurin, a Ca2+/calmodulin-dependent phosphatase. A non-palindromic NFAT-response element (RE) found in the IL2 promoter region has been commonly used for a Ca2+-response reporter gene system, but requirement of concomitant activation of AP-1 (Fos/Jun) often complicates the interpretation of obtained results. A new nanoluciferase (NanoLuc) reporter gene containing nine-tandem repeats of a pseudo-palindromic NFAT-RE located upstream of the IL8 promoter was designed to monitor Ca2+-induced transactivation activity of NFAT in human embryonic kidney (HEK) 293 cells by measuring luciferase activities of NanoLuc and co-expressed firefly luciferase for normalization. Ionomycin treatment enhanced the relative luciferase activity (RLA), which was suppressed by calcineurin inhibitors. HEK293 cells that stably express human STIM1 and Orai1, components of the store-operated calcium entry (SOCE) machinery, gave a much higher RLA by stimulation with thapsigargin, an inhibitor of sarcoplasmic/endoplamic reticulum Ca2+-ATPase (SERCA). HEK293 cells deficient in a penta-EF-hand Ca2+-binding protein ALG-2 showed a higher RLA value than the parental cells by stimulation with an acetylcholine receptor agonist carbachol. The novel reporter gene system is found to be useful for applications to cell signaling research to monitor biological endpoint effects of cellular Ca2+ mobilization.

Cell division cycle 6 (CDC6), an androgen receptor (AR) target gene, is implicated in regulating DNA replication and checkpoint mechanisms. CDC6 expression is increased during prostate cancer (PCa) progression and positively correlates with AR in PCa tissues. AR or CDC6 knockdown, together with AZD7762, a Chk1/2 inhibitor, results in decreased TopBP1-ATR-Chk1 signaling and markedly increased ataxia-telangiectasia-mutated (ATM) phosphorylation, a biomarker of DNA damage, and synergistically increases treatment efficacy. Combination treatment with the AR signaling inhibitor enzalutamide (ENZ) and the Chk1/2 inhibitor AZD7762 demonstrates synergy with regard to inhibition of AR-CDC6-ATR-Chk1 signaling, ATM phosphorylation induction, and apoptosis in VCaP (mutant p53) and LNCaP-C4-2b (wild-type p53) cells. CDC6 overexpression significantly reduced ENZ- and AZD7762-induced apoptosis. Additive or synergistic therapeutic activities are demonstrated in AR-positive animal xenograft models. These findings have important clinical implications, since they introduce a therapeutic strategy for AR-positive, metastatic, castration-resistant PCa, regardless of p53 status, through targeting AR-CDC6-ATR-Chk1 signaling.

Our previous studies have found that single nucleotide polymorphisms (SNPs) of tribbles homolog 3 (TRIB3) are related to the hypotensive effects of calcium-channel blockers (CCBs) and angiotensin-converting enzyme (ACE) inhibitors. In this study, we aimed at exploring and validating the effect of TRIB3 polymorphism on antihypertensive drugs responses.

AtHSP70 expression exhibits both stress and non-stress temperature response, however, the molecular mechanisms underlying these temperature signaling pathways remain elusive. Here we performed truncation and deletion assay to investigate the cis-elements within the promoter region of AtHSP70-4 (AT3G12580). And found the region between -1000 and -1100 bp from the translation initiation site (TIS) was indispensable for the non-stress temperature response of AtHSP70. Further deletion assay of several candidate motifs within this region suggested that one 'GCGCCAAA' sequence played the critical role. This motif was found as the reverse DNA-binding motif of cell cycle transcription factor E2F family. EMSA assay verified one number of Arabidopsis E2F family-E2F2 could bind to AtHSP70-4 promoter via this motif. These results indicated the temperature regulated expression of AtHSP70-4 may be mediated by cell cycle transcription factors and participate in plant acclimations to non-stress temperature changes.

The cucumber is one of the most important vegetables worldwide and is used as a research model for study of phloem transport, sex determination and temperature-photoperiod physiology. The shoot apex is the most important plant tissue in which the cell fate and organ meristems have been determined. In this study, a series of whole-genome small RNA, degradome and transcriptome analyses were performed on cucumber shoot apical tissues treated with high vs. low temperature and long vs. short photoperiod.

Chromatin insulators or boundary elements protect genes from regulatory activities from neighboring genes or chromatin domains. In the Drosophila Abdominal-B (Abd-B) locus, the deletion of such elements, such as Frontabdominal-7 (Fab-7) or Fab-8 led to dominant gain of function phenotypes, presumably due to the loss of chromatin barriers. Homologous chromosomes are paired in Drosophila, creating a number of pairing dependent phenomena including transvection, and whether transvection may affect the function of Polycomb response elements (PREs) and thus contribute to the phenotypes are not known. Here, we studied the chromatin barrier activity of Fab-8 and how it is affected by the zygosity of the transgene, and found that Fab-8 is able to block the silencing effect of the Ubx PRE on the DsRed reporter gene in a CTCF binding sites dependent manner. However, the blocking also depends on the zygosity of the transgene in that the barrier activity is present when the transgene is homozygous, but absent when the transgene is heterozygous. To analyze this effect, we performed chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR) experiments on homozygous transgenic embryos, and found that H3K27me3 and H3K9me3 marks are restricted by Fab-8, but they spread beyond Fab-8 into the DsRed gene when the two CTCF binding sites within Fab-8 were mutated. Consistent with this, the mutation reduced H3K4me3 and RNA Pol II binding to the DsRed gene, and consequently, DsRed expression. Importantly, in heterozygous embryos, Fab-8 is unable to prevent the spread of H3K27me3 and H3K9me3 marks from crossing Fab-8 into DsRed, suggesting an insulator bypass. These results suggest that in the Abd-B locus, deletion of the insulator in one copy of the chromosome could lead to the loss of insulator activity on the homologous chromosome, and in other loci where chromosomal deletion created hemizygous regions of the genome, the chromatin barrier could be compromised. This study highlights a role of homologous chromosome pairing in the regulation of gene expression in the Drosophila genome.

WRKY transcription factors are critical for plant growth, development, and adaptation to stress. This paper focuses on the expression characteristic to abiotic stress and phytohormones of OsWRKY24, OsWRKY53, and OsWRKY70. Three OsWRKY TFs contained two conserved domains and there were multiple cis-elements in response to adversity stress and hormone signaling in their promoters. Real-time PCR analysis revealed their widespread expression in normal tissues during seedling and heading stages. Under various stresses such as darkness, low temperature, salt, and drought, or treatment with hormones like ABA, SA, MeJA, and GA, transcript levels of these genes had changed significantly in wild-type seedlings. The expression level of OsWRKY24 was upregulated by darkness, cold, SA, and MeJA but downregulated by salt, drought, ABA, and GA treatments. The transcripts of OsWRKY53 were induced by darkness, low-temperature, salt, drought, ABA, and JA, while inhibited by SA and GA. In addition, OsWRKY70 expression level was elevated under darkness, low-temperature, SA, and JA but suppressed with salt, drought, ABA, and GA. These findings provide valuable insights into the regulatory mechanisms by which WRKY TFs adapt to stress via plant-hormone signaling.

LncRNA GAS5 plays a tumor suppressive role in a variety of human cancers and promises to be a novel diagnostic biomarker, therapy target, as well as prognostic biomarker. However, the role of GAS5 in nasopharyngeal carcinoma (NPC) remains elusive. The objective of the present study was to evaluate the effect of single nucleotide polymorphisms (SNPs) in GAS5 on treatment efficacy and toxicity in NPC patients receiving chemoradiotherapy. Three potentially functional SNPs of GAS5 were genotyped in 267 NPC patients and validated in another 238 NPC patients treated with chemoradiotherapy from southern China. Multivariate logistic regression analyses and stratification analyses were used to estimate the association of candidate SNPs and chemoradiotherapy efficacy and toxic reactions. Our results showed that rs2067079 kept a consistent association with severe myelosuppression and severe neutropenia in discovery set (OR=2.403, P=0.009; OR=2.454, P=0.015; respectively), validation set (OR=3.653, P=0.027; OR=4.767, P=0.016; respectively), and combined dataset (OR=1.880, P=0.007; OR=2.079, P=0.005; respectively). rs2067079 CT genotype carriers presented an even more remarkable increased risk of severe myelosuppression (OR=3.878, P=0.003) and severe neutropenia (OR=3.794, P=0.009) in subgroups taking paclitaxel+platinum as concurrent chemoradiotherapy regimen. Besides, we found a gene-does effect of rs6790, with the incidence rate of severe myelosuppression decreased from 23.56% to 17.21% to 10% and the incidence rate of severe neutropenia decreased from 30.4% to 20.9% to 17.1% for rs6790 GG vs GA vs AA genotype carriers. Our results indicate the potential role of lncRNA GAS5 polymorphisms rs2067079 and rs6790 as predictive biomarkers for chemoradiotherapy induced toxic reactions in NPC patients.

Resistance mechanisms to immune checkpoint blockade therapy (ICBT) limit its response duration and magnitude. Paradoxically, Interferon γ (IFNγ), a key cytokine for cellular immunity, can promote ICBT resistance. Using syngeneic mouse tumour models, we confirm that chronic IFNγ exposure confers resistance to immunotherapy targeting PD-1 (α-PD-1) in immunocompetent female mice. We observe upregulation of poly-ADP ribosyl polymerase 14 (PARP14) in chronic IFNγ-treated cancer cell models, in patient melanoma with elevated IFNG expression, and in melanoma cell cultures from ICBT-progressing lesions characterised by elevated IFNγ signalling. Effector T cell infiltration is enhanced in tumours derived from cells pre-treated with IFNγ in immunocompetent female mice when PARP14 is pharmacologically inhibited or knocked down, while the presence of regulatory T cells is decreased, leading to restoration of α-PD-1 sensitivity. Finally, we determine that tumours which spontaneously relapse in immunocompetent female mice following α-PD-1 therapy upregulate IFNγ signalling and can also be re-sensitised upon receiving PARP14 inhibitor treatment, establishing PARP14 as an actionable target to reverse IFNγ-driven ICBT resistance.

Avian leukosis virus (ALV) is detrimental to poultry health and causes substantial economic losses from mortality and decreased performance. Because tumorigenesis is a complex mechanism, the regulatory architecture of the immune system is likely to include the added dimensions of modulation by miRNAs and long-noncoding RNA (lncRNA). To characterize the response to ALV challenge, we developed a novel methodology that combines four datasets: mRNA expression and the associated regulatory factors of miRNA and lncRNA, and ALV gene expression. Specific Pathogen-Free (SPF) layer chickens were infected with ALV-J or maintained as non-injected controls. Spleen samples were collected at 40 days post injection (dpi), and sequenced. There were 864 genes, 7 miRNAs and 17 lncRNAs differentially expressed between infected and non-infected birds. The combined analysis of the 4 RNA expression datasets revealed that ALV infection is detected by pattern-recognition receptors (TLR9 and TLR3) leading to a type-I IFN mediated innate immune response that is modulated by IRF7 and IRF1. Co-expression network analysis of mRNA with miRNA, lncRNA and virus genes identified key elements within the complex networks utilized during ALV response. The integration of information from the host transcriptomic, epigenetic and virus response also has the potential to provide deeper insights into other host-pathogen interactions.

Background: Nonalcoholic fatty liver disease (NAFLD) is a serious threat to human health worldwide, with a high genetic susceptibility. Rs2302685, a functional germline variant of LRP6, has been recently found to associate with NAFLD risk. This study was aimed to clarify the underlying mechanism associated with rs2302685 risk and its impact on pharmacotherapy in treatment of NAFLD. Methods: Venous blood samples were collected from NAFLD and non-NAFLD patients for SNP genotyping by using mass spectrometry. The Lrp6-floxdel mouse (Lrp6(+/-)) was generated to model the partial function associated with human rs2302685. The liver injury and therapeutic effects of silibinin were compared between Lrp6(+/-) and Lrp6(+/+) mice received a methionine-choline deficient (MCD) diet or normal diet. The effect of Lrp6 functional alteration on Wnt/β-catenin-Cyp2e1 signaling activities was evaluated by a series of cellular and molecular assays. Results: The T allele of LRP6 rs2302685 was confirmed to associate with a higher risk of NAFLD in human subjects. The carriers of rs2302685 had reduced level of AST and ALT as compared with the noncarriers. The Lrp6(+/-) mice exhibited a less severe liver injury induced by MCD but a reduced response to the treatment of silibinin in comparison to the Lrp6(+/+) mice, suggesting Lrp6 as a target of silibinin. Wnt/β-catenin-Cyp2e1 signaling together with ROS generation could be exacerbated by the overexpression of Lrp6, while decreased in response to Lrp6 siRNA or silibinin treatment under NAFLD modeling. Conclusions: The Lrp6 function affects individual susceptibility to NAFLD and the therapeutic effect of silibinin through the Wnt/β-catenin-Cyp2e1 signaling pathway. The present work has provided an underlying mechanism for human individual susceptibility to NAFLD associated with Lrp6 polymorphisms as well as a rationale for the effective use of silibinin in NAFLD patients.

Acetohydroxyacid synthase (AHAS), also called acetolactate synthase, is a key enzyme involved in the first step of the biosynthesis of the branched-chain amino acids valine, isoleucine and leucine. Acetohydroxyacid synthase-inhibiting herbicides (AHAS herbicides) are five chemical families of herbicides that inhibit AHAS enzymes, including imidazolinones (IMI), sulfonylureas (SU), pyrimidinylthiobenzoates, triazolinones and triazolopyrimidines. Five AHAS genes have been identified in rapeseed, but little information is available regarding the role of miRNAs in response to AHAS herbicides. In this study, an AHAS herbicides tolerant genotype and a sensitive genotype were used for miRNA comparative analysis. A total of 20 small RNA libraries were obtained of these two genotypes at three time points (0h, 24 h and 48 h) after spraying SU and IMI herbicides with two replicates. We identified 940 conserved miRNAs and 1515 novel candidate miRNAs in Brassica napus using high-throughput sequencing methods combined with computing analysis. A total of 3284 genes were predicted to be targets of these miRNAs, and their functions were shown using GO, KOG and KEGG annotations. The differentiation expression results of miRNAs showed almost twice as many differentiated miRNAs were found in tolerant genotype M342 (309 miRNAs) after SU herbicide application than in sensitive genotype N131 (164 miRNAs). In additiond 177 and 296 miRNAs defined as differentiated in sensitive genotype and tolerant genotype in response to SU herbicides. The miR398 family was observed to be associated with AHAS herbicide tolerance because their expression increased in the tolerant genotype but decreased in the sensitive genotype. Moreover, 50 novel miRNAs from 39 precursors were predicted. There were 8 conserved miRNAs, 4 novel miRNAs and 3 target genes were validated by quantitative real-time PCR experiment. This study not only provides novel insights into the miRNA content of AHAS herbicides tolerant rapeseed in response to AHAS herbicides, but also demonstrates that miRNAs may be involved in AHAS herbicides tolerance.

Mitogen-activated protein kinase (MAPK) cascades have important functions in plant growth, development, and response to various stresses. The MAPKK and MAPKKK gene families in tomato have never been systematically analyzed. In this study, we performed a genome-wide analysis of the MAPKK and MAPKKK gene families in tomato and identified 5 MAPKK genes and 89 MAPKKK genes. Phylogenetic analyses of the MAPKK and MAPKKK gene families showed that all the MAPKK genes formed four groups (groups A, B, C, and D), whereas all the MAPKKK genes were classified into three subfamilies, namely, MEKK, RAF, and ZIK. Evolutionary analysis showed that whole genome or chromosomal segment duplications were the main factors responsible for the expansion of the MAPKK and MAPKKK gene families in tomato. Quantitative real-time RT-PCR analysis showed that the majority of MAPKK and MAPKKK genes were expressed in all tested organs with considerable differences in transcript levels indicating that they might be constitutively expressed. However, the expression level of most of these genes changed significantly under heat, cold, drought, salt, and Pseudomonas syringae treatment. Furthermore, their expression levels exhibited significant changes in response to salicylic acid and indole-3-acetic acid treatment, implying that these genes might have important roles in the plant hormone network. Our comparative analysis of the MAPKK and MAPKKK families would improve our understanding of the evolution and functional characterization of MAPK cascades in tomato.

A hydrophilic interaction liquid chromatography (HILIC)-ultra high-pressure liquid chromatography (UHPLC) coupled with tandem mass spectrometry (MS/MS) method was developed and applied to profile metabolite changes in human Huh-7 cells exposed to the potent aryl hydrocarbon receptor (AHR) ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Comparisons of sensitivity (limit of detection as low as 0.01 µM) and reproducibility (84% of compounds had an interday relative standard deviation (RSD) less than 10.0%; 83% of compounds had an intraday RSD less than 15.0%) were assessed for all the metabolites. The exposure of Huh-7 cells to the hepatotoxic carcinogen TCDD at low doses (1 nM and 10 nM for 4 h and 24 h, respectively) was reflected by the disturbance of amino acid metabolism, energy metabolism (glycolysis, TCA cycle), and nucleic acid metabolism. TCDD caused a significant decrease in amino acids such as serine, alanine, and proline while promoting an increase in arginine levels with 24 h treatment. Energy metabolism intermediates such as phosphoenolpyruvate and acetyl-CoA and nucleosides such as UMP, XMP, and CMP were also markedly decreased. These results support the application of HILIC-UHPLC-MS/MS for robust and reliable analysis of the cellular response to environmentally relevant toxicants at lower doses.

The mitogen-activated protein kinase (MAPK) cascade consists of three types of reversibly phosphorylated kinases, namely, MAPK, MAPK kinase (MAPKK/MEK), and MAPK kinase kinase (MAPKKK/MEKK), playing important roles in plant growth, development, and defense response. The MAPK cascade genes have been investigated in detail in model plants, including Arabidopsis, rice, and tomato, but poorly characterized in cucumber (Cucumis sativus L.), a major popular vegetable in Cucurbitaceae crops, which is highly susceptible to environmental stress and pathogen attack.

Neoadjuvant PD-1 blockade may be efficacious in some individuals with high-risk, resectable oral cavity head and neck cancer. To explore correlates of response patterns to neoadjuvant nivolumab treatment and post-surgical recurrences, we analyzed longitudinal tumor and blood samples in a cohort of 12 individuals displaying 33% responsiveness. Pretreatment tumor-based detection of FLT4 mutations and PTEN signature enrichment favors response, and high tumor mutational burden improves recurrence-free survival. In contrast, preexisting and/or acquired mutations (in CDKN2A, YAP1, or JAK2) correlate with innate resistance and/or tumor recurrence. Immunologically, tumor response after therapy entails T cell receptor repertoire diversification in peripheral blood and intratumoral expansion of preexisting T cell clones. A high ratio of regulatory T to T helper 17 cells in pretreatment blood predicts low T cell receptor repertoire diversity in pretreatment blood, a low cytolytic T cell signature in pretreatment tumors, and innate resistance. Our study provides a molecular framework to advance neoadjuvant anti-PD-1 therapy for individuals with resectable head and neck cancer.

Prevention or amelioration of declining β cell mass is a potential strategy to cure diabetes. Here, we report the pathways utilized by β cells to robustly replicate in response to acute insulin resistance induced by S961, a pharmacological insulin receptor antagonist. Interestingly, pathways that include CENP-A and the transcription factor E2F1 that are independent of insulin signaling and its substrates appeared to mediate S961-induced β cell multiplication. Consistently, pharmacological inhibition of E2F1 blocks β-cell proliferation in S961-injected mice. Serum from S961-treated mice recapitulates replication of β cells in mouse and human islets in an E2F1-dependent manner. Co-culture of islets with adipocytes isolated from S961-treated mice enables β cells to duplicate, while E2F1 inhibition limits their growth even in the presence of adipocytes. These data suggest insulin resistance-induced proliferative signals from adipocytes activate E2F1, a potential therapeutic target, to promote β cell compensation.

Podocyte plays an important role in maintaining the integrity and function of the glomerular filtration barrier. Various studies reported that forkhead transcription factor (Fox) O1 played a key role in anti-oxidative signaling. This study aimed to investigate the role of Stat1 in high glucose (HG) -induced podocyte injury.

Salt stress is one of the most representative abiotic stresses that severely affect plant growth and development. MicroRNAs (miRNAs) are well known for their significant involvement in plant responses to abiotic stresses. Although miRNAs implicated in salt stress response have been widely reported in numerous plant species, their regulatory roles in the adaptive response to salt stress in radish (Raphanus sativus L.), an important root vegetable crop worldwide, remain largely unknown.

Basic leucine zipper (bZIP) transcription factors play crucial roles in various abiotic stress responses as well as anthocyanin accumulation. Anthocyanins are most abundant in colorful skin radish, which exhibit strong antioxidant activity that offers benefits for human health. Here, a total of 135 bZIP-encoding genes were identified from radish genome. Synteny analysis showed that 104 radish and 63 Arabidopsis bZIP genes were orthologous. Transcriptome analysis revealed that 10 RsbZIP genes exhibited high-expression levels in radish taproot (RPKM>10). Specifically, RsbZIP010 exhibited down-regulated expression under Cd, Cr and Pb stresses, whereas RsbZIP031 and RsbZIP059 showed significant down-regulation under heat and salt stresses, respectively. RT-qPCR analysis indicated that RsbZIP011 and RsbZIP102 were significantly up-regulated in the tissues of radish with high anthocyanin contents. Furthermore, the promoter sequences of 39 anthocyanin-related genes were found to contain G-box or ACE-box elements that could be recognized by bZIP family members. Taken together, several RsbZIPs might be served as critical regulators in radish taproot under Cd, Cr, Pb, heat and salt stresses. RsbZIP011 and RsbZIP102 were the potential participants in anthocyanin biosynthesis pathway of radish. These results facilitate further investigation on functional characterization of bZIP genes in response to abiotic stress and anthocyanin biosynthesis in radish.