Luc Reporter Research Articles

Bioactive Tape With BMP-2 Binding Peptides Captures Endogenous Growth Factors and Accelerates Healing After Anterior Cruciate Ligament Reconstruction

Background: The anterior cruciate ligament (ACL) has poor regenerative capacity, and an injury leads to loss of function, limiting quality of life and increasing the incidence of osteoarthritis. Surgical interventions can stabilize the joint and improve functional recovery. The delivery of growth factors (GFs) enhances the healing process; however, this is complex in its regulation, is high in costs, has side effects, and can only be accomplished with supraphysiological concentrations and thus is currently not clinically feasible. However, the immobilization of a patient’s endogenous GFs in biomaterials can overcome these problems. Purpose: To develop a method to capture endogenous bone morphogenetic protein–2 (BMP-2) and ultimately show enhanced ACL healing in vivo using this novel methodology. Study Design: Controlled laboratory study. Methods: BMP-2 binding peptides were synthetized, purified, and immobilized on polycaprolactone (PCL) films. The affinity between the peptide and human BMP-2 (hBMP-2) was confirmed with immunofluorescence and enzyme-linked immunosorbent assay. The C2C12 Luc reporter cell line was used to confirm the bioactivity of immobilized BMP-2. For in vivo experiments, the same functionalization technology was applied to the commercially available Polytape, and the functionalized tape was sutured together with the graft used for ACL reconstruction in rats. Each animal underwent reconstruction with either native Polytape (n = 3) or Polytape with BMP-2 binding peptides (n = 3). At 2 and 6 weeks after surgery, the graft was assessed by histology and micro–computed tomography. Results: The covalent immobilization of the peptide in PCL was successful, allowing the peptide to capture hBMP-2, which remained bioactive and led to the osteogenic differentiation of C2C12. In vivo experiments confirmed the potential of the Polytape functionalized with the BMP-2 binding peptide to capture endogenous BMP-2, leading to enhanced bone formation inside the femoral and tibial tunnels and ultimately improving the graft’s quality. Conclusion: The incorporation of BMP-2 binding peptides into materials used for ACL reconstruction can capture endogenous hBMP-2, which enhances the healing process inside the bone tunnels. Clinical Relevance: These results demonstrate the potential of using synthetic peptides to endow biomaterials with novel biological functions, namely to capture and immobilize endogenous GFs.

Synergistic effects of tetrandrine combined with ionizing radiation on a murine colorectal carcinoma‑bearing mouse model.

Tetrandrine (TET), a traditional Chinese clinical agent, has been used for the treatment of many diseases, including cancers. The purpose of the present study was to investigate the combined effects of TET and ionizing radiation (IR) on murine CT26 colorectal adenocarcinoma cells in vitro and in vivo. A CT26 cell line transfected with dual HSV-1 thymidine kinase and firefly luciferase (luc) reporter genes was used. The half-maximal inhibitory concentration (IC50) of TET in CT26/tk-luc cells was ~10 µM. An additive effect was observed after combination of both agents based on a colony formation assay. Apoptosis and cleaved caspase-3 levels were increased significantly in cells after combination treatment, as shown by flow cytometric analysis, DNA fragmentation and western blotting. However, tumor growth inhibition and therapeutic efficacy of TET combined with IR in vivo were identified to be synergistic, as monitored by tumor growth delay time, measured with a digital caliper. A significant inhibition of tumor growth was identified in the combination group compared with the radiation only group. Furthermore, non-invasive bioluminescent imaging (BLI) and gamma scintigraphy were also used to evaluate therapeutic efficacy. Both modalities revealed that the best tumor growth control was under combination treatment among all groups. The present study demonstrated that TET is not only beneficial for chemotherapy, but also has potential as a radiosensitizer for the treatment of cancer.

Inhibition of expression of BmNPV cg30 by bmo-miRNA-390 is a host response to baculovirus invasion.

Bombyx mori larvae exhibit in vivo defensive reactions immediately after invasion by a virus. One of these defense systems is to express appropriate microRNAs (miRNAs) to respond to the infection. A novel Bombyx mori-encoded miRNA, bmo-miR-390, was identified previously by high-throughput sequencing. Based on bioinformatic predictions, the Bombyx mori nuclear polyhedrosis virus cg30 gene (BmNPV-cg30) is one of the target genes of bmo-miR-390. In this study, expression vectors with an enhanced green fluorescence protein (EGFP) or a luciferase (luc) reporter gene together with bm-miR-390 or the cg30 3' UTR were constructed and used to co-transfect BmN cells. Using a dual luciferase reporter (DLR) assay, we found that bmo-miR-390 significantly downregulates the expression of BmNPV-cg30 (P < 0.05) in vitro. Moreover, artificially synthesized bmo-miR-390 mimics enhanced the regulatory effect of bmo-miR-390, while an inhibitor eliminated the inhibitory effect. These results show for the first time that bmo-miR-390 can effectively downregulate the expression of BmNPV-cg30 in BmNPV-infected BmN cells.

Agrobacterium-mediated and electroporation-mediated transformation of Chlamydomonas reinhardtii: a comparative study

BackgroundChlamydomonas reinhardtii is an unicellular green alga used for functional genomics studies and heterologous protein expression. A major hindrance in these studies is the low level and instability of expression of nuclear transgenes, due to their rearrangement and/or silencing over time.ResultsWe constructed dedicated vectors for Agrobacterium-mediated transformation carrying, within the T-DNA borders, the Paromomycin (Paro) selectable marker and an expression cassette containing the Luciferase (Luc) reporter gene. These vectors and newly developed co-cultivation methods were used to compare the efficiency, stability and insertion sites of Agrobacterium- versus electroporation-mediated transformation. The influence of different transformation methods, of the cell wall, of the virulence of different Agrobacterium strains, and of transgene orientation with respect to T-DNA borders were assessed. False positive transformants were more frequent in Agrobacterium-mediated transformation compared to electroporation, compensating for the slightly lower proportion of silenced transformants observed in Agrobacterium-mediated transformation than in electroporation. The proportion of silenced transformants remained stable after 20 cycles of subculture in selective medium. Next generation sequencing confirmed the nuclear insertion points, which occurred in exons or untraslated regions (UTRs) for 10 out of 10 Agrobacterium-mediated and 9 out of 13 of electroporation-mediated insertions. Electroporation also resulted in higher numbers of insertions at multiple loci.ConclusionsDue to its labor-intensive nature, Agrobacterium transformation of Chlamydomonas does not present significant advantages over electroporation, with the possible exception of its use in insertional mutagenesis, due to the higher proportion of within-gene, single-locus insertions. Our data indirectly support the hypothesis that rearrangement of transforming DNA occurs in the Chlamydomonas cell, rather than in the extracellular space as previously proposed.

Cell-specific resetting of mouse islet cellular clocks by glucagon, glucagon-like peptide 1 and somatostatin.

Molecular clocks, operative in pancreatic islet cells, represent an intrinsic mechanism regulating intracellular metabolism and hormone secretion. Glucagon, somatostatin and glucagon-like peptide 1 (GLP-1) are essential coordinators of islet physiology. Here, we assess the synchronizing capacity of glucagon, somatostatin and GLP-1 on pancreatic α- and β-cell circadian clocks. Triple transgenic mice, expressing a circadian PER2::luciferase (luc) reporter combined with α- and β-cell-specific fluorescent reporters, were employed. Isolated pancreatic islets and fluorescence-activated cell sorting-separated α- and β-cells were synchronized with glucagon, somatostatin analogue or GLP-1 mimetics, with subsequent real-time PER2::luc bioluminescence recording. Gene expression of Gcgr, Sstr2, Sstr3 and Glp1r in islet cells was assessed by RNA sequencing and RT-qPCR. Glucagon and GLP-1 mimetics (liraglutide and exenatide) induced high-amplitude rhythmic expression of the PER2::luc reporter in β-cells, but not in α-cells, while the somatostatin analogue octreotide generated a significant phase shift between α- and β-cells. Enrichment of Gcgr and Glp1r transcripts was detected in β-cells compared to their α-cell counterparts. The synchronizing effect of glucagon was dose-dependent and mediated by the adenylate cyclase signalling cascade, as it was diminished by adenylate cyclase inhibitor. We conclude that proglucagon-derived peptides and somatostatin exhibit receptor-mediated cell-specific synchronizing effects for mouse α- and β-cell oscillators. Differential islet cell clock modulation by glucagon and somatostatin may represent a physiological mechanism underlying paracrine regulation of rhythmic glucagon and insulin secretion. The reported here strong synchronizing properties of GLP-1 mimetics, widely used for treatment of type 2 diabetes, are of high clinical relevance.

PEGylated dendrimer-entrapped gold nanoparticles with low immunogenicity for targeted gene delivery.

The high efficiency and specificity of gene therapy are mainly ascribed to the excellent transfection ability of the gene carrier. Non-viral polymer vectors have attracted extensive attention because of their low cytotoxicity and outstanding genetic loading capacity compared with viral vectors. For safe and efficient transfection of nuclear acids, here we report a novel gene delivery system, dendrimer-entrapped gold nanoparticles modified with a folate-conjugated poly (ethylene glycol) (Au DENPs-PEG-FA), possessing superior gene transfection efficiency than that of partially hydrophilic methoxy poly(ethylene glycol) (mPEG)-modified dendrimer-entrapped gold nanoparticles (Au DENPs-mPEG). The prepared Au DENPs-PEG-FA were well characterized, and our data revealed that the vector showed good cytocompatibility. Additionally, the quantification of inflammatory cytokines detected by qRT-PCR showed that the vectors displayed low innate immune response. The efficiency of nucleic acid (encoding enhanced green fluorescent protein (EGFP) and luciferase (Luc) reporter) transfection evaluated via flow cytometry and confocal microscopic imaging suggested that the Au DENPs-PEG-FA were able to transfect nucleic acid into HeLa cells with enhanced transfection efficiency. Furthermore, the existence of FA rendered the Au DENPs with excellent targeting performance via FA receptor-ligand binding interaction. The designed Au DENPs-PEG-FA with low immunogenicity and enhanced gene transfection efficiency may hold a great promise to be a superior vector for gene therapy.

Long-chain polyunsaturated fatty acids and extensively hydrolyzed casein-induced browning in a Ucp-1 reporter mouse model of obesity.

Browning in adipose tissues, which can be affected by diet, may mitigate the detrimental effects of adiposity and improve longer-term metabolic health. Here, browning-inducing effects of long-chain polyunsaturated fatty acids, e.g., arachidonic acid (ARA)/docosahexaenoic acid (DHA) and extensively hydrolyzed casein (eHC) were investigated in uncoupling protein 1 (Ucp-1) reporter mice. To address the overall functionality, their potential role in supporting a healthy metabolic profile under obesogenic dietary challenges later in life was evaluated. At weaning Ucp1+/LUC reporter mice were fed a control low fat diet (LFD) with or without ARA + DHA, eHC or eHC + ARA + DHA for 8 weeks until week 12 after which interventions continued for another 12 weeks under a high-fat diet (HFD) challenge. Serology (metabolic responses and inflammation) and in vivo and ex vivo luciferase activity were determined; in the meantime browning-related proteins UCP-1 and the genes peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α), PR domain containing 16 (PRDM16) and Ucp-1 were examined. ARA + DHA, eHC or their combination reduced body weight gain and adipose tissue weight compared to the HFD mice. The interventions induced Ucp-1 expression in adipose tissues prior to and during the HFD exposure. Ucp-1 induction was accompanied by higher PGC1a and PRDM16 expression. Glucose tolerance and insulin sensitivity were improved coinciding with lower serum cholesterol, triglycerides, free fatty acids, insulin, leptin, resistin, fibroblast growth factor 21, alanine aminotransferase, aspartate aminotransferase and higher adiponectin than the HFD group. HFD-associated increased systemic (IL-1β and TNF-α) and adipose tissue inflammation (F4/80, IL-1β, TNF-α, IL-6) was reduced. Studies in a Ucp-1 reporter mouse model revealed that early intervention with ARA/DHA and eHC improves metabolic flexibility and attenuates obesity during HFD challenge later in life. Increased browning is suggested as, at least, part of the underlying mechanism.

Expression of miR-145-5p During Chondrogenesis of Mesenchymal Stem Cells

Assessing the quality of tissue engineered (TE) cartilage has historically been performed by endpoint measurements including marker gene expression. Until the adoption of promoter-driven reporter constructs capable of quantitative and real time non-destructive expression analysis, temporal gene expression assessments along a timeline could not be performed on TE constructs. We further exploit this technique to utilize microRNA (miRNA or miR) through the use of firefly luciferase reporter (Luc) containing a 3' UTR perfect complementary target sequence to the mature miR-145-5p. We report the development and testing of a firefly luciferase (Luc) reporter responsive to miR-145-5p for longitudinal tracking of miR-145-5p expression throughout MSC chondrogenic differentiation. Plasmid reporter vectors containing a miR-145-5p responsive reporter (Luc reporter with a perfect complementary target sequence to the mature miR-145-5p sequence in the 3'UTR), a Luc reporter driven by a truncated Sox9 (one of the targets of miR-145-5p) promoter, or the Luc backbone (control) vector without a specific miRNA target were transfected into MSCs by electroporation. Transfected MSCs were mixed with untransfected MSC to generate chondrogenic pellets. Pellets were imaged by bioluminescent imaging (BLI) and harvested along a preset time line. The imaging signals from miR-145-5p responsive reporter and Sox9 promoter-driven reporter showed correlated time-courses (measured by BLI and normalized to Luc-control reporter; Spearman r=0.93, p=0.0002) during MSC chondrogenic differentiation. Expression analysis by qRT-PCR suggests an inverse relationship between miR-145-5p and Sox9 gene expression during MSC chondrogenic differentiation. Non-destructive cell-pellet imaging is capable of supplementing histological analyses to characterize TE cartilage. The miR-145-5p responsive reporter is relatively simple to construct and generates a consistent imaging signal responsive to miR-145-5p during MSC chondrogenesis in parallel to certain molecular and cellular events.

Hyperoxia-mediated transcriptional activation of cytochrome P4501A1 (CYP1A1) and decreased susceptibility to oxygen-mediated lung injury in newborn mice

Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. In this study, we tested the hypothesis that newborn transgenic mice carrying the human CYP1A1-Luc promoter will display transcriptional activation of the human CYP1A1 promoter in vivo upon exposure to hyperoxia, and that these mice will be less susceptible to hyperoxic lung injury and alveolar simplification than similarly exposed wild type (WT) mice. Newborn WT (CD-1) or transgenic mice carrying a 13.2 kb human CYP1A1 promoter and the luciferase (Luc) reporter gene (CYP1A1-luc) were maintained in room air or exposed to hyperoxia (85% O2) for 7–14 days. Hyperoxia exposure of CYP1A1-Luc mice for 7 and 14 days resulted in 4- and 30-fold increases, respectively, in hepatic Luc (CYP1A1) expression, compared to room air controls. In lung, hyperoxia caused a 2-fold induction of reporter Luc at 7 days, but the induction declined after 14 days. The newborn CYP1A1-Luc mice were less susceptible to lung injury and alveolar simplification than similarly exposed wild type (WT) CD-1 mice. Also, the CYP1A1-Luc mice showed increased levels of hepatic and pulmonary CYP1A1 expression and hepatic CYP1A2 activity after hyperoxia exposure. Hyperoxia also increased NADP(H) quinone reductase (NQO1) pulmonary gene expression in both CD-1 and CYP1A1-Luc mice at both time points, but this was more pronounced in the latter at 14 days. Our results support the hypothesis that hyperoxia activates the human CYP1A1 promoter in newborn mice, and that increased endogenous expression of CYP1A1 and NADP(H) quinone reductase (NQO1) contributes to the decreased susceptibilities to hyperoxic lung injury in the transgenic animals. This is the first report providing evidence of hyperoxia-mediated transcriptional activation of the human CYP1A1 promoter in newborn mice, and this in conjunction with decreased lung injury, suggests that these phenomena have important implications for BPD.

Characterization of a new pathway that activates lumisterol in vivo to biologically active hydroxylumisterols

Using LC/qTOF-MS we detected lumisterol, 20-hydroxylumisterol, 22-hydroxylumisterol, 24-hydroxylumisterol, 20,22-dihydroxylumisterol, pregnalumisterol, 17-hydroxypregnalumisterol and 17,20-dihydroxypregnalumisterol in human serum and epidermis, and the porcine adrenal gland. The hydroxylumisterols inhibited proliferation of human skin cells in a cell type-dependent fashion with predominant effects on epidermal keratinocytes. They also inhibited melanoma proliferation in both monolayer and soft agar. 20-Hydroxylumisterol stimulated the expression of several genes, including those associated with keratinocyte differentiation and antioxidative responses, while inhibiting the expression of others including RORA and RORC. Molecular modeling and studies on VDRE-transcriptional activity excludes action through the genomic site of the VDR. However, their favorable interactions with the A-pocket in conjunction with VDR translocation studies suggest they may act on this non-genomic VDR site. Inhibition of RORα and RORγ transactivation activities in a Tet-on CHO cell reporter system, RORα co-activator assays and inhibition of (RORE)-LUC reporter activity in skin cells, in conjunction with molecular modeling, identified RORα and RORγ as excellent receptor candidates for the hydroxylumisterols. Thus, we have discovered a new biologically relevant, lumisterogenic pathway, the metabolites of which display biological activity. This opens a new area of endocrine research on the effects of the hydroxylumisterols on different pathways in different cells and the mechanisms involved.

Gerontoxanthone B from Maclura cochinchinensis var. gerontogea exhibits anti-inflammatory potential as an aryl hydrocarbon receptor agonist

The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcriptional factor belonging to the basic helix-loop-helix-Per-Ahr/Arnt-Sim family. In this study, we evaluated the AhR agonistic activities of 12 xanthones isolated from the roots of M. cochinchinensis var. gerontogea using HepG2 cells transfected with pX4TK–Luc reporter plasmids. Gerontoxanthone B (GXB) showed the most potent activity at a concentration of 10μM, and the activity was inhibited by AhR antagonists such as GNF-351. GXB also increased cytochrome P450 1A1 mRNA and protein levels in HepG2 cells. Similar to the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin, however, GXB suppressed the IL-1β-induced mRNA level of SAA1, an acute-phase response gene that is up-regulated AhR-dependently but XRE-independently. Thus, GXB shows XRE-dependent transcriptional activity and XRE-independent activity involving AhR.

Site-specific phosphorylation of casein kinase 1 δ (CK1δ) regulates its activity towards the circadian regulator PER2.

Circadian rhythms are intrinsic ~24 hour cycles that regulate diverse aspects of physiology, and in turn are regulated by interactions with the external environment. Casein kinase 1 delta (CK1δ, CSNK1D) is a key regulator of the clock, phosphorylating both stabilizing and destabilizing sites on the PER2 protein, in a mechanism known as the phosphoswitch. CK1δ can itself be regulated by phosphorylation on its regulatory domain, but the specific sites involved, and the role this plays in control of circadian rhythms as well as other CK1-dependent processes is not well understood. Using a sensitized PER2::LUC reporter assay, we identified a specific phosphorylation site, T347, on CK1δ, that regulates CK1δ activity towards PER2. A mutant CK1δ T347A was more active in promoting PER2 degradation. This CK1δ regulatory site is phosphorylated in cells in trans by dinaciclib- and staurosporine-sensitive kinases, consistent with their potential regulation by cyclin dependent and other proline-directed kinases. The regulation of CK1δ by site-specific phosphorylation via the cell cycle and other signaling pathways provides a mechanism to couple external stimuli to regulation of CK1δ-dependent pathways including the circadian clock.

Low-Dose Dose-Response for In Vitro Nrf2-ARE Activation in Human HepG2 Cells.

Kelch-like ECH-associated protein 1 (Keap1), nuclear factor erythroid 2-like factor 2-related factor 2 (Nrf2), and the antioxidant response element (ARE) are interacting components of a master regulatory signaling pathway that coordinates redox homeostasis, cytoprotective responses, and shifts in stem cell state. This study reexamined detailed dose–response (DR) data reported for in vitro Nrf2-ARE activation in human hepatoblastoma HepG2 cell lines containing either a ARE-bla or ARE-luc reporter at 12 different concentrations of each of 15 chemicals. The normalized study data were combined among chemicals exhibiting a positive response, yielding n = 531 (179) DR data for 9 (7) chemicals using the ARE-bla (ARE-luc) assay. Three-parameter linear/kth-power regression fits obtained to each combined set of ARE-bla- or ARE-luc-assay response data provided good fits (R 2 = .99 or .91, respectively, P fit > .99) that each incorporate a highly significant negative initial linear slope (P = 4 × 10−5 or .00025) and an overall J-shaped DR pattern. Results from this reanalysis of high-resolution ARE response data support the hypothesis that nonlinear ARE-mediated adaptive cellular responses to oxidative stress are governed by an ultrasensitive molecular switch.

Imaging of Tumor-Associated Macrophages in a Transgenic Mouse Model of Orthotopic Ovarian Cancer.

Tumor-associated macrophages (TAMs) are often associated with a poor prognosis in cancer. To gain a better understanding of cellular recruitment and dynamics of TAM biology during cancer progression, we established a novel transgenic mouse model for in vivo imaging of luciferase-expressing macrophages. B6.129P2-Lyz2tm1(cre)Ifo/J mice, which express Cre recombinase under the control of the lysozyme M promoter (LysM) were crossed to Cre-lox Luc reporter mice (RLG), to produce LysM-LG mice whose macrophages express luciferase. Cell-type-specific luciferase expression in these mice was verified by flow cytometry, and via in vivo bioluminescence imaging under conditions where macrophages were either stimulated with lipopolysaccharide or depleted with clodronate liposomes. The distribution of activated macrophages was longitudinally imaged in two immunocompetent LysM-LG mouse models with either B16 melanoma or ID8 ovarian cancer cells. In vivo imaging of LysM-LG mice showed luciferase activity was generated by macrophages. Clodronate liposome-mediated depletion of macrophages lowered overall bioluminescence while lipopolysaccharide injection increased macrophage bioluminescence in both the B16 and ID8 models. Tracking macrophages weekly in tumor-bearing animals after intraperitoneal (i.p.) or intraovarian (i.o.) injection resulted in distinct, dynamic patterns of macrophage activity. Animals with metastatic ovarian cancer after i.p. injection exhibited significantly higher peritoneal macrophage activity compared to animals after i.o. injection. The LysM-LG model allows tracking of macrophage recruitment and activation during disease initiation and progression in a noninvasive manner. This model provides a tool to visualize and monitor the benefit of pharmacological interventions targeting macrophages in preclinical models.

DNA methylation of the Fthl17 5'-upstream region regulates differential Fthl17 expression in lung cancer cells and germline stem cells.

The Ferritin heavy polypeptide-like 17 (Fthl17) gene is a member of the cancer/testis antigen gene family, and is preferentially expressed in cancer cells and in testis. Although DNA methylation has been linked to the regulation of human FTHL17 gene expression, detailed epigenetic regulation of its expression has not been investigated. To address this, we assessed the epigenetic regulation of murine Fthl17 gene expression in cancer cells and germ cells. Fthl17 was more highly expressed in testis, a murine lung cancer cell line, KLN205, and in germline stem cells (GSCs) than in normal lung tissues. Furthermore, the Fthl17 expression level in GSCs was significantly higher than in KLN205 cells. We performed bisulfite-sequencing and luciferase (luc) reporter assays to examine the role of DNA methylation of the Fthl17 promoter in the regulation of Fthl17 expression. In KLN205 cells, testis, and GSCs, the Fthl17 5’-upstream region was hypo-methylated compared with normal lung tissues. Luc reporter assays indicated that hypo-methylation of the -0.6 kb to 0 kb region upstream from the transcription start site (TSS) was involved in the up-regulation of Fthl17 expression in KLN205 cells and GSCs. Because the -0.6 kb to -0.3 kb or the -0.3 kb to 0 kb region were relatively more hypo-methylated in KLN205 cells and in GSCs, respectively, compared with other regions between -0.6 kb to 0 kb, those regions may contribute to Fthl17 up-regulation in each cell type. Following treatment with 5-Azacytidine, the -0.3 kb to 0 kb region became hypo-methylated, and Fthl17 expression was up-regulated in KLN205 cells to a level comparable to that in GSCs. Together, the results suggest that hypo-methylation of different but adjacent regions immediately upstream of the Fthl17 gene contribute to differential expression levels in lung cancer cells and GSCs, and hypo-methylation of the TSS-proximal region may be critical for high level expression.

An incoherent feed-forward loop mediates robustness and tunability in a plant immune network.

Immune signaling networks must be tunable to alleviate fitness costs associated with immunity and, at the same time, robust against pathogen interferences. How these properties mechanistically emerge in plant immune signaling networks is poorly understood. Here, we discovered a molecular mechanism by which the model plant species Arabidopsis thaliana achieves robust and tunable immunity triggered by the microbe-associated molecular pattern, flg22. Salicylic acid (SA) is a major plant immune signal molecule. Another signal molecule jasmonate (JA) induced expression of a gene essential for SA accumulation, EDS5 Paradoxically, JA inhibited expression of PAD4, a positive regulator of EDS5 expression. This incoherent type-4 feed-forward loop (I4-FFL) enabled JA to mitigate SA accumulation in the intact network but to support it under perturbation of PAD4, thereby minimizing the negative impact of SA on fitness as well as conferring robust SA-mediated immunity. We also present evidence for evolutionary conservation of these gene regulations in the family Brassicaceae Our results highlight an I4-FFL that simultaneously provides the immune network with robustness and tunability in A.thaliana and possibly in its relatives.

Molecular and functional evaluation of a novel HIF inhibitor, benzopyranyl 1,2,3-triazole compound.

Hypoxia occurs in a variety of pathological events, including the formation of solid tumors. Hypoxia-inducible factor (HIF)-1α is stabilized under hypoxic conditions and is a key molecule in tumor growth and angiogenesis. Seeking to develop novel cancer therapeutics, we investigated small molecules from our in-house chemical libraries to target HIF-1α. We employed a dual-luciferase assay that uses a luciferase (Luc) reporter vector harboring five copies of hypoxia-responsive element (HRE) in the promoter. Under hypoxic conditions that increased Luc reporter activity by four-fold, we screened 144 different compounds, nine of which showed 30–50% inhibition of hypoxia-induced Luc reporter activity. Among these, “Compound 12, a benzopyranyl 1,2,3-triazole” was the most efficient at inhibiting the expression of HIF-1α under hypoxic conditions, reducing its expression by 80%. Under hypoxic conditions, the half maximal IC50 of the compound was 24 nM in HEK-293 human embryonic kidney cells, and 2 nM in A549 human lung carcinoma cells. Under hypoxic conditions, Compound 12 increased hydroxylated HIF-1α levels and HIF-1α ubiquitination, and also dose-dependently decreased HIF-1α target gene expression as well as vascular endothelial growth factor (VEGF) secretion. Furthermore, this compound inhibited VEGF-induced in vitro angiogenesis in human umbilical vein endothelial cells (HUVECs), and in vivo, it inhibited chick chorioallantoic membrane angiogenesis. In allogaft assays, cotreatment with Compound 12 and gefitinib significantly inhibited tumor growth and angiogenesis. Compound 12 can be a novel inhibitor of HIF-1α by accelerating its degradation, and shows much potential as an anti-cancer agent through its ability to suppress tumor growth and angiogenesis.

The In Vitro Effect of Acidic-Pepsin on Nuclear Factor KappaB Activation and Its Related Oncogenic Effect on Normal Human Hypopharyngeal Cells.

BackgroundExtra-esophageal carcinogenesis has been widely discussed in relation to the chronic effects of laryngopharyngeal reflux and most prominently with pepsin historically central to this discussion. With refluxate known to include gastric (pepsin) and duodenal (bile) fluids, we recently demonstrated the mechanistic role of NF-κB in mediating the preneoplastic effects of acidic-bile. However, the role of pepsin in promoting hypopharyngeal premalignant events remains historically unclear. Here, we investigate the in vitro effect of acidic-pepsin on the NF-κB oncogenic pathway to better define its potential role in hypopharyngeal neoplasia.MethodsHuman hypopharyngeal primary cells (HHPC) and keratinocytes (HHK) were repetitively exposed to physiologic pepsin concentrations (0.1 mg/ml) at pH 4.0, 5.0 and 7.0. Cellular localization of phospho-NF-κB and bcl-2 was determined using immunofluorescence and western blotting. NF-κB transcriptional activity was tested by luc reporter and qPCR. Analysis of DNA content of pepsin treated HHK and HHPC was performed using Fluorescence-activated-cell sorting assay. To explore a possible dose related effect, pepsin concentration was reduced from 0.1 to 0.05 and 0.01 mg/ml.ResultsAt physiologic concentration, acidic-pepsin (0.1 mg/ml at pH 4.0) is lethal to most normal hypopharyngeal cells. However, in surviving cells, no NF-κB transcriptional activity is noted. Acidic-pepsin fails to activate the NF-κB or bcl-2, TNF-α, EGFR, STAT3, and wnt5α but increases the Tp53 mRNAs, in both HHPC and HHK. Weakly acidic-pepsin (pH 5.0) and neutral-pepsin (pH 7.0) induce mild activation of NF-κB with increase in TNF-α mRNAs, without oncogenic transcriptional activity. Lower concentrations of pepsin at varying pH do not produce NF-κB activity or transcriptional activation of the analyzed genes.ConclusionOur findings in vitro do not support the role of acidic-pepsin in NF-κB related hypopharyngeal carcinogenesis.

In-depth Characterization of Firefly Luciferase as a Reporter of Circadian Gene Expression in Mammalian Cells.

Firefly luciferase (Fluc) is frequently used to report circadian gene expression rhythms in mammalian cells and tissues. During longitudinal assays it is generally assumed that enzymatic substrates are in saturating excess, such that total bioluminescence is directly proportional to Fluc protein level. To test this assumption, we compared the enzyme kinetics of purified luciferase with its activity in mammalian cells. We found that Fluc activity in solution has a lower Michaelis constant (Km) for luciferin, lower temperature dependence, and lower catalytic half-life than Fluc in cells. In consequence, extracellular luciferin concentration significantly affects the apparent circadian amplitude and phase of the widely used PER2::LUC reporter in cultured fibroblasts, but not in SCN, and we suggest that this arises from differences in plasma membrane luciferin transporter activity. We found that at very high concentrations (>1 mM), luciferin lengthens circadian period, in both fibroblasts and organotypic SCN slices. We conclude that the amplitude and phase of circadian gene expression inferred from bioluminescence recordings should be treated with some caution, and we suggest that optimal luciferin concentration should be determined empirically for each luciferase reporter and cell type.

Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase.

Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a cytotoxic gene. In the present study we developed a new cancer specific binary expression system activated by the Integrase (Int) of the lambdoid phage HK022. We demonstrate the validity of this system by the specific expression of a luciferase (luc) reporter in human embryonic kidney 293T (HEK293T) cells and in a lung cancer mouse model. Due to the absence viral vectors and of cytotoxicity the Int based binary system offers advantages over previously described counterparts and may therefore be developed into a safer cancer cell killing system.