Electrophoretic Mobility Shift Assay Analysis Research Articles

A Basic Leucine Zipper Transcription Factor, AsbZIP26, Positively Regulates the Synthesis of Alliin in Garlic (Allium sativum L.)

Garlic is a significant medicinal and culinary plant, with its primary bioactive compound being alliin. The bZIP transcription factor is pivotal in plant secondary metabolism and growth, yet its function in garlic remains unexplored. The previously identified key enzyme gene, AsFMO1 in garlic, is known to directly regulate alliin synthesis, however, the specific molecular mechanism is still unknown. In this research, the transcription factor AsbZIP26 was discovered in the leaves of purple-skinned garlic in PiZhou, demonstrating a positive regulatory role in alliin biosynthesis. Transcriptomic data indicated that mechanical damage stress highly induced the expression of the AsbZIP26 gene, a favorable condition for alliin synthesis. Subcellular localization revealed nuclear targeting of AsbZIP26. Employing yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA) analysis, and chromatin immunoprecipitation (ChIP), it was confirmed that AsbZIP26 specifically binds to the G-box region of the AsFMO1 promoter, directly activating its expression and playing a crucial function in garlic biosynthesis. Overexpression of AsbZIP26 in garlic callus tissues resulted in increased alliin levels. Overall, this study discovered a functional transcription factor AsbZIP26 in garlic, which can regulate the synthesis of alliin in garlic, filling the gap in the alliin synthesis pathway and providing new ideas for garlic breeding and selection for improving garlic varieties.

The Induction of Drug Uptake Transporter Organic Anion Transporting Polypeptide 1A2 by Radiation Is Mediated by the Nonreceptor Tyrosine Kinase v-YES-1 Yamaguchi Sarcoma Viral Oncogene Homolog 1.

Organic anion transporting polypeptides (OATP, gene symbol SLCO) are well-recognized key determinants for the absorption, distribution, and excretion of a wide spectrum of endogenous and exogenous compounds including many antineoplastic agents. It was therefore proposed as a potential drug target for cancer therapy. In our previous study, it was found that low-dose X-ray and carbon ion irradiation both upregulated the expression of OATP family member OATP1A2 and in turn, led to a more dramatic killing effect when cancer cells were cotreated with antitumor drugs such as methotrexate. In the present study, the underlying mechanism of the phenomenon was explored in breast cancer cell line MCF-7. It was found that the nonreceptor tyrosine kinase v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1 (YES-1) was temporally coordinated with the change of OATP1A2 after irradiation. The overexpression of YES-1 significantly increased OATP1A2 both at the mRNA and protein level. The signal transducer and activator of transcription 3 (STAT3) pathway is likely the downstream target of YES-1 because phosphorylation and nuclear accumulation of STAT3 were both enhanced after overexpressing YES-1 in MCF-7 cells. Further investigation revealed that there are two possible binding sites of STAT3 localized at the upstream sequence of SLCO1A2, the encoding gene of OATP1A2. Electrophoretic mobility shift assay and chromatin immunoprecipitation analysis suggested that these two sites bound to STAT3 specifically and the overexpression of YES-1 significantly increased the association of the transcription factor with the putative binding sites. Finally, inhibition or knockdown of YES-1 attenuated the induction effect of radiation on the expression of OATP1A2. SIGNIFICANCE STATEMENT: The present study found that the effect of X-rays on v-YES-1 Yamaguchi sarcoma viral oncogene homolog 1 (YES-1) and organic anion transporting polypeptides (OATP)1A2 was temporally coordinated. YES-1 phosphorylates and increases the nuclear accumulation of signal transducer and activator of transcription 3, which in turn binds to the upstream regulatory sequences of SLCO1A2, the coding gene for OATP1A2. Hence, inhibitors of YES-1 may suppress the radiation induction effect on OATP1A2.

Unraveling the role of PlARF2 in regulating deed formancy in Paeonia lactiflora.

PlARF2 can positively regulate the seed dormancy in Paeonia lactiflora Pall. and bind the RY cis-element. Auxin, a significant phytohormone influencing seed dormancy, has been demonstrated to be regulated by auxin response factors (ARFs), key transcriptional modulators in the auxin signaling pathway. However, the role of this class of transcription factors (TFs) in perennials with complex seed dormancy mechanisms remains largely unexplored. Here, we cloned and characterized an ARF gene from Paeonia lactiflora, named PlARF2, which exhibited differential expression levels in the seeds during the process of seed dormancy release. The deduced amino acid sequence of PlARF2 had high homology with those of other plants and contained typical conserved Auxin_resp domain of the ARF family. Phylogenetic analysis revealed that PlARF2 was closely related to VvARF3 in Vitis vinifera. The subcellular localization and transcriptional activation assay showed that PlARF2 is a nuclear protein possessing transcriptional activation activity. The expression levels of dormancy-related genes in transgenic callus indicated that PlARF2 was positively correlated with the contents of PlABI3 and PlDOG1. The germination assay showed that PlARF2 promoted seed dormancy. Moreover, TF Centered Yeast one-hybrid assay (TF-Centered Y1H), electrophoretic mobility shift assay (EMSA) and dual-luciferase reporter assay analysis (Dual-Luciferase) provided evidence that PlARF2 can bind to the 'CATGCATG' motif. Collectively, our findings suggest that PlARF2, as TF, could be involved in the regulation of seed dormancy and may act as a repressor of germination.

Anti-carbamylated protein antibodies drive AEC II toward a profibrotic phenotype by interacting with carbamylated TLR5.

This study looked at the role of anti-carbamylated protein (anti-CarP) antibodies in contributing to lung fibrosis in CTD-associated interstitial lung disease (ILD) in an autoantigen-dependent manner. ELISA was used to test serum samples, including 89 from the CTD-ILD group and 170 from the non-CTD-ILD group, for anti-CarP levels. Male C57BL/6 mice were used for the pulmonary fibrosis model and anti-CarP treatment in vivo (n = 5) and patient serum-derived or commercialized anti-CarP was used for cell treatment. We identified the carbamylated membrane protein via immunofluorescence (IF) and co-immunoprecipitation followed by mass spectrometry (MS) analysis. Quantitative RT-PCR, IF and western blot were performed to explore the antigen-dependent role of anti-CarP. A native electrophoretic mobility shift assay and MS analysis were used to verify direct interaction and carbamylation sites. A significantly higher serum anti-CarP level was observed in CTD with ILD than without ILD. In vivo, intrapulmonary delivery of anti-CarP induces epithelial-mesenchymal transition (EMT) and microfibrotic foci. Carbamylation was enriched in type II alveolar epithelial cells (AEC II). A novel carbamylated membrane receptor, specifically recognized by anti-CarP, was identified as toll-like receptor 5 (TLR5). We found anti-CarP induces the nuclear translocation of NF-κB and downstream events, including EMT and expression of inflammatory cytokines in AEC II, which were reversed by TLR5 blocking or TLR5 knockdown. Moreover, up to 12 lysine carbamylation sites were found in TLR5 ectodomain, allowing the interaction of anti-CarP with carbamylated TLR5. Overall, we found anti-CarP drives aberrant AEC II activation by interacting with carbamylated TLR5 to promote ILD progression.

Antitoxin MqsA decreases antibiotic susceptibility through the global regulator AgtR in Pseudomonas fluorescens.

Type II toxin-antitoxin systems are highly prevalent in bacterial genomes and play crucial roles in the general stress response. Previously, we demonstrated that the type II antitoxin PfMqsA regulates biofilm formation through the global regulator AgtR in Pseudomonas fluorescens. Here, we found that both the C-terminal DNA-binding domain of PfMqsA and AgtR are involved in bacterial antibiotic susceptibility. Electrophoretic mobility shift assay (EMSA) analyses revealed that AgtR, rather than PfMqsA, binds to the intergenic region of emhABC-emhR, in which emhABC encodes an resistance-nodulation-cell division efflux pump and emhR encodes a repressor. Through quantitative real-time reverse-transcription PCR and EMSA analysis, we showed that AgtR directly activates the expression of the emhR by binding to the DNA motif [5´-CTAAGAAATATACTTAC-3´], leading to repression of the emhABC. Furthermore, we demonstrated that PfMqsA modulates the expression of EmhABC and EmhR. These findings enhance our understanding of the mechanism by which antitoxin PfMqsA contributes to antibiotic susceptibility.

Transcription factor OxyR regulates sulfane sulfur removal and L-cysteine biosynthesis in Corynebacterium glutamicum.

Sulfane sulfur, a collective term for hydrogen polysulfide and organic persulfide, often damages cells at high concentrations. Cells can regulate intracellular sulfane sulfur levels through specific mechanisms, but these mechanisms are unclear in Corynebacterium glutamicum. OxyR is a transcription factor capable of sensing oxidative stress and is also responsive to sulfane sulfur. In this study, we found that OxyR functioned directly in regulating sulfane sulfur in C. glutamicum. OxyR binds to the promoter of katA and nrdH and regulates its expression, as revealed via in vitro electrophoretic mobility shift assay analysis, real-time quantitative PCR, and reporting systems. Overexpression of katA and nrdH reduced intracellular sulfane sulfur levels by over 30% and 20% in C. glutamicum, respectively. RNA-sequencing analysis showed that the lack of OxyR downregulated the expression of sulfur assimilation pathway genes and/or sulfur transcription factors, which may reduce the rate of sulfur assimilation. In addition, OxyR also affected the biosynthesis of L-cysteine in C. glutamicum. OxyR overexpression strain Cg-2 accumulated 183 mg/L of L-cysteine, increased by approximately 30% compared with the control (142 mg/L). In summary, OxyR not only regulated sulfane sulfur levels by controlling the expression of katA and nrdH in C. glutamicum but also facilitated the sulfur assimilation and L-cysteine synthesis pathways, providing a potential target for constructing robust cell factories of sulfur-containing amino acids and their derivatives. IMPORTANCE C. glutamicum is an important industrial microorganism used to produce various amino acids. In the production of sulfur-containing amino acids, cells inevitably accumulate a large amount of sulfane sulfur. However, few studies have focused on sulfane sulfur removal in C. glutamicum. In this study, we not only revealed the regulatory mechanism of OxyR on intracellular sulfane sulfur removal but also explored the effects of OxyR on the sulfur assimilation and L-cysteine synthesis pathways in C. glutamicum. This is the first study on the removal of sulfane sulfur in C. glutamicum. These results contribute to the understanding of sulfur regulatory mechanisms and may aid in the future optimization of C. glutamicum for biosynthesis of sulfur-containing amino acids.

The transcription factor EMB1444-like affects tomato fruit ripening by regulating YELLOW-FRUITED TOMATO 1, a core component of ethylene signaling transduction.

The fleshy fruit of tomato (Solanum lycopersicum) are climacteric and, as such, ethylene plays a pivotal role in their ripening and quality traits. In this study, a basic helix-loop-helix transcription factor, EMB1444-like, was found to induce the expression of YELLOW-FRUITED TOMATO 1 (YFT1), which encodes the SlEIN2 protein, a key element in the ethylene signaling pathway. Yeast one-hybrid and EMSA analyses revealed that EMB1444-like binds to the E-box motif (CACTTG, -1295 bp to -1290 bp upstream of the ATG start codon) of the YFT1 promoter (pYFT1). Suppression of EMB1444-like expression in tomato lines (sledl) using RNAi reduced ethylene production by lowering the expression of 1-AMINOCYCLOPROPANE-1-CARBOXYLATE SYNTHASE 2/4 (ACS2/4) and ACC OXIDASE1 (ACO1) in a positive feedback loop. sledl tomato also showed differences in numerous quality traits related to fruit ripening, compared with the wild type, such as delayed chromoplast differentiation, a decrease in carotenoid accumulation, and delayed fruit ripening in an ethylene-independent manner, or at least upstream of ripening mediated by YFT1/SlEIN2. This study elucidates the regulatory framework of fruit ripening in tomato, providing information that may be used to breed tomato hybrid cultivars with an optimal balance of shelf-life, durability, and high quality.

Functional analysis of CTLA4 promoter variant and its possible implication in colorectal cancer immunotherapy.

Colorectal cancer (CRC) is a prevalent cancer, ranking as the third most common. Recent advances in our understanding of the molecular causes of this disease have highlighted the crucial role of tumor immune evasion in its initiation and progression. CTLA4, a receptor that acts as a negative regulator of T cell responses, plays a pivotal role in this process, and genetic variations in CTLA4 have been linked to CRC susceptibility, prognosis, and response to therapy. We conducted a case-control study involving 98 CRC patients and 424 controls. We genotyped the CTLA4 c.-319C > T variant (rs5742909) and performed an association analysis by comparing allele frequencies between the patients and controls. To assess the potential functional impact of this variant, we first performed an In Silico analysis of transcription factor binding sites using Genomatix. Finally, to validate our findings, we conducted a luciferase reporter gene assay using different cell lines and an electrophoretic mobility shift assay (EMSA). The case-control association analysis revealed a significant association between CTLA4 c.-319C > T and CRC susceptibility (p = 0.023; OR 1.89; 95% CI = 1.11-3.23). Genomatix analysis identified LEF1 and TCF7 transcription factors as specific binders to CTLA4 c.-319C. The reporter gene assay demonstrated notable differences in luciferase activity between the c.-319 C and T alleles in COS-7, HCT116, and Jurkat cell lines. EMSA analysis showed differences in TCF7 interaction with the CTLA4 C and T alleles. CTLA4 c.-319C > T is associated with CRC susceptibility. Based on our functional validation results, we proposed that CTLA4 c.-319C > T alters gene expression at the transcriptional level, triggering a stronger negative regulation of T-cells and immune tumoral evasion.

Overexpression of a novel small auxin-up RNA gene, OsSAUR11, enhances rice deep rootedness

BackgroundDeep rooting is an important factor affecting rice drought resistance. However, few genes have been identified to control this trait in rice. Previously, we identified several candidate genes by QTL mapping of the ratio of deep rooting and gene expression analysis in rice.ResultsIn the present work, we cloned one of these candidate genes, OsSAUR11, which encodes a small auxin-up RNA (SAUR) protein. Overexpression of OsSAUR11 significantly enhanced the ratio of deep rooting of transgenic rice, but knockout of this gene did not significantly affect deep rooting. The expression of OsSAUR11 in rice root was induced by auxin and drought, and OsSAUR11-GFP was localized both in the plasma membrane and cell nucleus. Through an electrophoretic mobility shift assay and gene expression analysis in transgenic rice, we found that the transcription factor OsbZIP62 can bind to the promoter of OsSAUR11 and promote its expression. A luciferase complementary test showed that OsSAUR11 interacts with the protein phosphatase OsPP36. Additionally, expression of several auxin synthesis and transport genes (e.g., OsYUC5 and OsPIN2) were down-regulated in OsSAUR11-overexpressing rice plants.ConclusionsThis study revealed a novel gene OsSAUR11 positively regulates deep rooting in rice, which provides an empirical basis for future improvement of rice root architecture and drought resistance.

The XylR/NtrC-type regulator CbsR positively regulates upstream pathway of chlorobenzene degradation in Pandoraea pnomenusa.

Pandoraea pnomenusa MCB032 completely degrades chlorobenzene, whose metabolic pathway is encoded by cbs and clc gene clusters. The putative regulatory factors ClcR and CbsR are predicted to regulate the cbs and clc gene clusters. This research aims to understand the function of ClcR and CbsR. RT-PCR analyses demonstrated that the cbsFAaAbAcAdB operon that encodes catabolic pathways for the degradation of chlorobenzene to chlorocatechol is located on an operon. Moreover, the clcABCDE operon is involved in the 3-chlorocatechol pathway. Gene knockout and transcriptional analysis showed that the transcription of the cbsFAaAbAcAdB operon is positively regulated by CbsR, whereas the clcABCDE operon is activated by ClcR. Primer extension analysis was used to locate the transcription start sites of the cbsFAaAbAcAdB and cbsR operons. Electrophoretic mobility shift assay analyses showed that CbsR is bound to the sites in the promoter regions of cbsFAaAbAcAdB and cbsR operons. The XylR/NtrC-type regulator CbsR positively regulates the transcription of the cbsFAaAbAcAdB operon encoding the upstream pathway of chlorobenzene catabolism, while the LysR-type regulator ClcR activates the clcABCDE operon encoding the downstream pathway.

Identification and characterization of the Doublesex gene and its mRNA isoforms in the brine shrimp Artemia franciscana.

Doublesex (DSX) proteins are members of the Doublesex/mab-3-related (DMRT) protein family and play crucial roles in sex determination and differentiation among the animal kingdom. In the present study, we identified two Doublesex (Dsx)-like mRNA isoforms in the brine shrimp Artemia franciscana (Kellogg 1906), which are generated by the combination of alternative promoters, alternative splicing and alternative polyadenylation. The two transcripts exhibited sex-biased enrichment, which we termed AfrDsxM and AfrDsxF. They share a common region which encodes an identical N-terminal DNA-binding (DM) domain. RT-qPCR analyses showed that AfrDsxM is dominantly expressed in male Artemia while AfrDsxF is specifically expressed in females. Expression levels of both isoforms increased along with the developmental stages of their respective sexes. RNA interference with dsRNA showed that the knockdown of AfrDsxM in male larvae led to the appearance of female traits including an ovary-like structure in the original male reproductive system and an elevated expression of vitellogenin. However, silencing of AfrDsxF induced no clear phenotypic change in female Artemia. These results indicated that the male AfrDSXM may act as inhibiting regulator upon the default female developmental mode in Artemia. Furthermore, electrophoretic mobility shift assay analyses revealed that the unique DM domain of AfrDSXs can specifically bind to promoter segments of potential downstream target genes like AfrVtg. These data show that AfrDSXs play crucial roles in regulating sexual development in Artemia, and further provide insight into the evolution of sex determination/differentiation in sexual organisms.

Electrophoretic Mobility Shift Assay (EMSA) and Microscale Thermophoresis (MST) Methods to Measure Interactions Between tRNAs and Their Modifying Enzymes.

The Elongator complex is a unique tRNA acetyltransferase; it was initially annotated as a protein acetyltransferase, but in-depth biochemical analyses revealed its genuine function as a tRNA modifier. The substrate recognition and binding of the Elongator is mainly mediated by its catalytic Elp3 subunit. In this chapter, we describe protocols to generate fluorescently labeled RNAs and outline the principles underlying electrophoretic mobility shift assays (EMSA) and microscale thermophoresis (MST). These two methods allow qualitative and quantitative examinations of the binding affinity of various tRNAs toward the homologs of Elp3 from various organisms. The rather qualitative results from EMSA analyses can be nicely complemented by MST measurements allowing precise determination of the dissociation constant (KD). We also provide detailed notes for users to mitigate potential ambiguities and technical pitfalls during the procedures.

Inhibitory Effects of Mismatch Binding Molecules on the Repair Reaction of Uracil-Containing DNA.

The stable R-loop formed during transcription induces enzyme-mediated deamination of cytosine, and the uracil in the DNA produced activates the base excision repair (BER) pathway. DNA cleavage involved in the BER pathway is thought to be one of the possible causes of trinucleotide repeat instability. Here, we performed an in vitro assay to investigate the effect of a DNA-binding small molecule, naphthyridine carbamate dimer (NCD), on BER enzyme reactions. The gel electrophoretic mobility shift assay (EMSA) and thermal melting analysis revealed the binding of NCD to a 5'-XGG-3'/5'-XGG-3' triad (X = C or U or apurinic/apyrimidinic site), which is a mimic of a BER enzyme substrate. Polyacrylamide gel electrophoresis (PAGE) of the reaction products of these substrates with hSMUG1 and APE1 enzymes in the presence of NCD showed that NCD interfered with the repair reaction in the 5'-XGG-3'/5'-XGG-3' triad. These findings would broaden the potential of small molecules in modulating trinucleotide repeat instability.

Novel mycoplasma nucleomodulin MbovP475 decreased cell viability by regulating expression of CRYAB and MCF2L2

ABSTRACT Nucleomodulins are secreted bacterial proteins whose molecular targets are located in host cell nuclei. They are gaining attention as critical virulence factors that either modify the epigenetics of host cells or directly regulate host gene expression. Mycoplasma bovis is a major veterinary pathogen that secretes several potential virulence factors. The aim of this study was to determine whether any of their secreted proteins might function as nucleomodulins. After an initial in silico screening, the nuclear localization of the secreted putative lipoprotein MbovP475 of M. bovis was demonstrated in bovine macrophage cell line (BoMac) experimentally infected with M. bovis. Through combined application of ChIP-seq, Electrophoretic mobility shift assay (EMSA) and surface plasmon resonance (SPR) analysis, MbovP475 was determined to bind the promoter regions of the cell cycle central regulatory genes CRYAB and MCF2L2. MbovP475 has similar secondary structures with the transcription activator-like effectors (TALEs). Screening of various mutants affecting the potential DNA binding sites indicated that the residues 242NI243 within MbovP475 loop region of the helix-loop-helix domain were essential to its DNA binding activity, thereby contributing to decrease in BoMac cell viability. In conclusion, this is the first report to confirm M. bovis secretes a conserved TALE-like nucleomodulin that binds the promoters of CRYAB and MCF2L2 genes, and subsequently down-regulates their expression and decreases BoMac cell viability. Therefore, this study offers a new understanding of mycoplasma pathogenesis.

LuxR-Type SCO6993 Negatively Regulates Antibiotic Production at the Transcriptional Stage by Binding to Promoters of Pathway-Specific Regulatory Genes in Streptomyces coelicolor.

SCO6993 (606 amino acids) in Streptomyces coelicolor belongs to the large ATP-binding regulators of the LuxR family regulators having one DNA-binding motif. Our previous findings predicted that SCO6993 may suppress the production of pigmented antibiotics, actinorhodin, and undecylprodigiosin, in S. coelicolor, resulting in the characterization of its properties at the molecular level. SCO6993-disruptant, S. coelicolor ΔSCO6993 produced excess pigments in R2YE plates as early as the third day of culture and showed 9.0-fold and 1.8-fold increased production of actinorhodin and undecylprodigiosin in R2YE broth, respectively, compared with that by the wild strain and S. coelicolor ΔSCO6993/SCO6993+. Real-time polymerase chain reaction analysis showed that the transcription of actA and actII-ORF4 in the actinorhodin biosynthetic gene cluster and that of redD and redQ in the undecylprodigiosin biosynthetic gene cluster were significantly increased by SCO6993-disruptant. Electrophoretic mobility shift assay and DNase footprinting analysis confirmed that SCO6993 protein could bind only to the promoters of pathway-specific transcriptional activator genes, actII-ORF4 and redD, and a specific palindromic sequence is essential for SCO6993 binding. Moreover, SCO6993 bound to two palindromic sequences on its promoter region. These results indicate that SCO6993 suppresses the expression of other biosynthetic genes in the cluster by repressing the transcription of actII-ORF4 and redD and consequently negatively regulating antibiotic production.

Transcriptional repression of MaRBOHs by MaHsf26 is associated with heat shock-alleviated chilling injury in banana fruit

Both heat shock factors (Hsfs) and reactive oxygen species (ROS) are widely perceived to be involved in chilling stress response. However, the particular mechanism of Hsfs mediated-ROS metabolism related to chilling stress remains largely unknown, especially during chilling injury development of postharvest economically cold-sensitive fruit such as banana. In this study, we found that heat shock treatment (HST) could effectively alleviate postharvest banana chilling injury and reduce ROS accumulation during storage. The expression levels of several respiratory burst oxidase homolog (RBOH) genes, encoding ROS-producing enzymes, increased consistently with the progression, whereas were inhibited upon HST. Significantly, we identified an Hsfs MaHsf26, which exhibited a opposite expression pattern as that of MaRBOHs, as the putative binding protein of MaRBOHB-like1, MaRBOHE-like and MaRBOHF-like promoters using yeast one-hybrid (Y1H) screening analysis. Further electrophoretic mobility shift assay (EMSA) and dual luciferase reporter (DLR) analysis displayed MaHsf26 suppressed the transcriptions of MaRBOHB-like1, MaRBOHE-like and MaRBOHF-like through binding to the heat shock elements (HSE) in their promoters, respectively. In general, our results suggested that MaHsf26 plays as a transcriptional repressor of ROS generation by suppressing the transcriptions of MaRBOHs and thereby involving in the HST-mitigated chilling injury in postharvest banana fruit.

PPAR gamma2: The main isoform of PPARγ that positively regulates the expression of the chicken Plin1 gene

Perilipin1 (PLIN1) is a major phosphorylated protein that specifically coats the surface of neutral lipid droplets (LDs) in adipocytes and plays a crucial role in regulating the accumulation and hydrolysis of triacylglycerol (TG). Mammalian studies have shown that Plin1 gene transcription is mainly regulated by peroxisome proliferator-activated receptor-gamma (PPARγ), the master regulator of adipogenesis. However, the regulatory mechanism of the chicken Plin1 (cPlin1) gene is poorly understood. The present study aimed to investigate whether Plin1 is regulated by PPARγ in chickens and identify its exact molecular mechanism. Reporter gene and expression assays showed that PPARγ2, but not PPARγ1, activated (P<0.01) the cPlin1 gene promoter. An electrophoretic mobility shift assay and mutational analysis revealed that PPARγ2 bound to a special site in the cPlin1 gene promoter to enhance its expression. In summary, our results show that PPARγ promotes the expression of the cPlin1 gene and that PPARγ2 is the main regulatory isoform.

Cotton miR319b-Targeted TCP4-Like Enhances Plant Defense Against Verticillium dahliae by Activating GhICS1 Transcription Expression

Teosinte branched1/Cincinnata/proliferating cell factor (TCP) transcription factors play important roles in plant growth and defense. However, the molecular mechanisms of TCPs participating in plant defense remain unclear. Here, we characterized a cotton TCP4-like fine-tuned by miR319b, which could interact with NON-EXPRESSER OF PATHOGEN-RELATED GENES 1 (NPR1) to directly activate isochorismate synthase 1 (ICS1) expression, facilitating plant resistance against Verticillium dahliae. mRNA degradome data and GUS-fused assay showed that GhTCP4-like mRNA was directedly cleaved by ghr-miR319b. Knockdown of ghr-miR319b increased plant resistance to V. dahliae, whereas silencing GhTCP4-like increased plant susceptibility by the virus-induced gene silencing (VIGS) method, suggesting that GhTCP4-like is a positive regulator of plant defense. According to the electrophoretic mobility shift assay and GUS reporter analysis, GhTCP4-like could transcriptionally activate GhICS1 expression, resulting in increased salicylic acid (SA) accumulation. Yeast two-hybrid and luciferase complementation image analyses demonstrated that GhTCP4-like interacts with GhNPR1, which can promote GhTCP4-like transcriptional activation in GhICS1 expression according to the GUS reporter assay. Together, these results revealed that GhTCP4-like interacts with GhNPR1 to promote GhICS1 expression through fine-tuning of ghr-miR319b, leading to SA accumulation, which is percepted by NPR1 to increase plant defense against V. dahliae. Therefore, GhTCP4-like participates in a positive feedback regulation loop of SA biosynthesis via NPR1, increasing plant defenses against fungal infection.

BrABF3 promotes flowering through the direct activation of CONSTANS transcription in pak choi.

Drought stress triggers the accumulation of the phytohormone abscisic acid (ABA), which in turn activates the expression of the floral integrator gene CONSTANS (CO), accelerating flowering. However, the molecular mechanism of ABA-induced CO activation remains elusive. Here, we conducted a yeast one-hybrid assay using the CO promoter from Brassica campestris (syn. Brassica rapa) ssp. chinensis (pak choi) to screen the ABA-induced pak choi library and identified the transcription activator ABF3 (BrABF3). BrABF3, the expression of which was induced by ABA in pak choi, directly bound to the CO promoter from both pak choi and Arabidopsis. The BrABF3 promoter is specifically active in the Arabidopsis leaf vascular tissue, where CO is mainly expressed. Impaired BrABF3 expression in pak choi decreased BrCO expression levels and delayed flowering, whereas ectopic expression of BrABF3 in Arabidopsis increased CO expression and induced earlier flowering under the long-day conditions. Electrophoretic mobility shift assay analysis showed that BrABF3 was enriched at the canonical ABA-responsive element-ABRE binding factor (ABRE-ABF) binding motifs of the BrCO promoter. The direct binding of BrABF3 to the ABRE elements of CO was further confirmed by chromatin immunoprecipitation quantitative PCR. In addition, the induction of BrCO transcription by BrABF3 could be repressed by BrCDF1 in the morning. Thus, our results suggest that ABA could accelerate the floral transition by directly activating BrCO transcription through BrABF3 in pak choi.

Tauroursodeoxycholic Acid Inhibits Nuclear Factor Kappa B Signaling in Gastric Epithelial Cells and Ameliorates Gastric Mucosal Damage in Mice.

Previous studies have reported the protective effects of tauroursodeoxycholic acid (TUDCA) on gastric epithelial cells in some animal models, but the precise mechanisms are unclear. This study examined the effects of TUDCA on NF-κB signaling in gastric epithelial cells. Moreover, the protective effects of TUDCA in experimental gastritis models induced by ethanol and NSAID were evaluated and compared with ursodeoxycholic acid (UDCA). After a pretreatment with TUDCA or UDCA, human gastric epithelial MKN-45 cells were stimulated with tumor necrosis factor (TNF)-α to activate NF-κB signaling. A real-time PCR (RT-PCR) for human interleukin (IL)-1 mRNA was performed. An electrophoretic mobility shift assay (EMSA) and immunoblot analyses were carried out. In murine models, after a pretreatment with TUDCA or UDCA, ethanol and indomethacin were administered via oral gavage. Macroscopic and microscopic assessments were performed to evaluate the preventive effects of TUDCA and UDCA on murine gastritis. A pretreatment with TUDCA downregulated the IL-1α mRNA levels in MKN-45 cells stimulated with TNF-α, as assessed by RT-PCR. As determined using EMSA, a pretreatment with TUDCA reduced the TNF-α-induced NF-κB DNA binding activity. A pretreatment with TUDCA inhibited IκBα phosphorylation induced by TNF-α, as assessed by immunoblot analysis. TUDCA attenuated the ethanol-induced and NSAID-induced gastritis in murine models, as determined macroscopically and microscopically. TUDCA inhibited NF-κB signaling in gastric epithelial cells and ameliorated ethanol- and NSAID-induced gastritis in murine models. These results support the potential of TUDCA for the prevention of gastritis in humans.