Firefly Luciferase Gene Research Articles

Bioluminescence Imaging to Study Recombinant Orthopoxvirus Infection in Animal Models.

Bioluminescent images of viral replication in live animals (in vivo) reveal disease dynamics and effects of medical countermeasures over time. After selecting an appropriate orthopoxvirus animal model for the study, a recombinant virus with the firefly luciferase gene inserted in the genome is used to infect the animals. On the day of bioluminescent imaging, the substrate, D-luciferin, is prepared; animals are sedated and injected with the substrate and IVIS imager is utilized; various bioluminescent images are acquired; then animals recover and are able to continue in the study. Ex vivo imaging can also be completed after animals are euthanized at experimental endpoint. This approach allows real-time imaging of viral kinetics within an animal, and analysis of images can provide an additional quantitative measure throughout the study. Bioluminescent imaging not only provides scientific benefits but also benefits to animal welfare. For these reasons, bioluminescent imaging should be considered for any in vivo orthopoxvirus study.

Evolution and post-transcriptional regulation insights of m6A writers, erasers, and readers in plant epitranscriptome.

As a dynamic and reversible post-transcriptional marker, N6-methyladenosine (m6A) plays an important role in the regulation of biological functions, which are mediated by m6A pathway components including writers (MT-A70, FIP37, VIR and HAKAI family), erasers (ALKBH family) and readers (YTH family). There is an urgent need for a comprehensive analysis of m6A pathway components across species at evolutionary levels. In this study, we identified 4062 m6A pathway components from 154 plant species including green algae, utilizing large-scale phylogenetic to explore their origin and evolution. We discovered that the copy number of writers was conserved among different plant lineages, with notable expansions in the ALKBH and YTH families. Synteny network analysis revealed conserved genomic contexts and lineage-specific transpositions. Furthermore, we used Direct RNA Sequencing (DRS) to reveal the Poly(A) length (PAL) and m6A ratio profiles in six angiosperms species, with a particular focus on the m6A pathway components. The ECT1/2-Poeaece4 sub-branches (YTH family) with unique genomic contexts exhibited significantly higher expression level than genes of other ECT1/2 poeaece sub-branches (ECT1/2-Poeaece1-3), accompanied by lower m6A modification and PAL. Besides, conserved m6A sites distributed in CDS and 3'UTR were detected in the ECT1/2-Poaceae4, and the dual-luciferase assay further demonstrated that these conserved m6A sites in the 3'UTR negatively regulated the expression of Firefly luciferase (LUC) gene. Finally, we developed transcription factor regulatory networks for m6A pathway components, using yeast one-hybrid assay demonstrated that PheBPC1 could interact with the PheECT1/2-5 promoter. Overall, this study presents a comprehensive evolutionary and functional analysis of m6A pathway components and their modifications in plants, providing a valuable resource for future functional analysis in this field.

In vivo bioluminescence imaging revealed the change of the time window of BDNF expression in the brain elicited by a single bout of exercise following repeated exercise

Exercise increases the expression of brain-derived neurotrophic factor (BDNF) in the brain and contributes to cognitive and sensorimotor functions. This study aimed to elucidate how repeated exercise modifies BDNF expression elicited by a single bout of exercise in the brain using in vivo bioluminescence imaging (BLI). Bdnf-luciferase (Luc) mice with the firefly luciferase gene inserted at the translation start point of the Bdnf gene were used for BLI to monitor changes in BDNF expression in the brain. The treadmill exercise at a speed of 10 m/s for 60 min was repeated 5 days a week for 4 weeks. BLI in individual subjects was repeated four times: before the exercise intervention, on the first exercise day, and 14 and 28 days after the start of the intervention. Each BLI was performed after a single bout of exercise and monitored for 8 h after exercise. Repetitive BLI showed that the exercise regimen enhanced BDNF expression in the brain, specifically at 4–8 h after a single bout of exercise. Repeated exercise for 2 weeks accelerated the start of enhancement after a single bout of exercise, but not after 4 weeks of repeated exercise. This study showed that repeated exercise modulated the time window of exercise-enhanced BDNF expression, suggesting that repeated exercise could change the sensitivity of gene expression to a single bout of exercise. These findings can be attributed to the advantages of in vivo BLI, which allowed us to precisely measure the time course of BDNF expression after repeated exercise in individual subjects.

The Secondary Bile acid, Lithocholic Acid, Inhibits Cl− Secretion Across Colonic Epithelial Cells in Vitro

Lithocholic acid (LCA) is a secondary bile acid formed in the colon by bacterial metabolism of chenodeoxycholic acid. Our previous studies have shown the bile acid to be protective under conditions of colonic inflammation. Here, we sought to determine if LCA might also play a role in regulating fluid and electrolyte transport. T84 cell monolayers were mounted in Ussing chambers for measurements of Cl− secretion, the primary driving force for fluid secretion into the colon. qRT-PCR and Western blotting were used to analyze mRNA and protein expression. Results were expressed as mean ± SEM and data were analyzed by one way ANOVA and Tukey’s post hoc test or by mixed-effects analysis and Dunnett's post hoc test. To assess the effects of LCA on CFTR promoter activity, we used a luciferase promoter/reporter system, where HEK293 cells were transfected with a plasmid containing the firefly luciferase gene under control of the CFTR promoter. Co-transfections were carried out with 2 additional plasmids expressing either the nuclear bile acid receptor, FXR, or its dimerization partner, RXR. Pretreatment of T84 cell monolayers with LCA inhibited subsequent Cl− secretory responses to the cAMP-dependent agonist, forskolin (FSK; 10 μM), in a concentration (1 – 10 μM) and time-dependent (3 - 24 hrs) manner. Maximal effects of LCA were observed at a concentration of 10 μM after treatment for 24 hrs, when responses to FSK were reduced to 50.9 ± 8.5% of those in controls (n = 6; p < 0.01). LCA (10 μM; 24 hrs) also inhibited responses to the Ca2+-dependent secretagogues, thapsigargin (2 μM) and histamine (100 μM), by 59.4 ± 2.4% (n = 4; p < 0.001) and 52.2 ± 1.9% (n = 5; p < 0.001), respectively. In further experiments, using nystatin-permeabilized T84 monolayers to isolate apical Cl− conductances, LCA (10 μM; 24 hrs) reduced FSK-stimulated responses to 72.7 ± 6.6% (n = 17; p < 0.001) of those in control cells. Analysis of CFTR expression, the primary exit pathway for Cl− in colonic epithelial cells, revealed that LCA treatment reduced mRNA and protein expression of the channel to 0.65 ± 0.05 fold (n = 7; p < 0.01) and 0.43 ± 0.06 fold (n = 6; p < 0.001), respectively. In CFTR promoter assays, LCA (10 μM) reduced CFTR promoter activity to 0.7 ± 0.02 fold of that in control cells (n = 5; p < 0.01), with co-expression of FXR being required for this effect to be observed. Finally, while LCA activated both FXR and the vitamin D receptor (VDR) in T84 cells, its effects in downregulating CFTR expression and Cl− conductances were mimicked by the FXR agonist, GW4064, but not by the VDR agonist, calcitriol. In conclusion, LCA, at physiologically relevant concentrations, inhibits Cl− secretion across colonic epithelial cells, likely through a mechanism involving FXR activation and inhibition of CFTR expression. These data add to the growing pool of knowledge regarding important regulatory actions of LCA in the colon and highlight its potential role as a target for the treatment of intestinal disorders. This work was supported by a grant from Science Foundation Ireland. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Engineered Luminescent Oncolytic Vaccinia Virus Activation of Photodynamic-Immune Combination Therapy for Colorectal Cancer.

Oncolytic virus therapy is currently regarded as a promising approach in cancer immunotherapy. It has greater therapeutic advantages for colorectal cancer that is prone to distant metastasis. However, the therapeutic efficacy and clinical application of viral agents alone for colorectal cancer remain suboptimal. In this study, an engineered oncolytic vaccinia virus (OVV-Luc) that expresses the firefly luciferase gene is developed and loaded Chlorin e6 (Ce6) onto the virus surface through covalent coupling, resulting in OVV-Luc@Ce6 (OV@C). The OV@C infiltrates tumor tissue and induces endogenous luminescence through substrate catalysis, resulting in the production of reactive oxygen species. This unique system eliminates the need for an external light source, making it suitable for photodynamic therapy (PDT) in deep tissues. Moreover, this synergistic effect between PDT and viral immunotherapy enhances dendritic cell maturation, macrophage polarization, and reversal of the immunosuppressive microenvironment. This synergistic effect has the potential to convert a "cold" into a "hot" tumor, it offers valuable insights for clinical translation and application.

Abstract C052: Ketogenic diet enhanced nutrient metabolic pathways but did not inhibit tumor growth in an obesity-associated PDAC mouse model

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has a dismal 5-year survival rate due to ineffective treatments. Obesity is a recognized risk factor for PDAC. Thus, developing effective weight management strategies is essential to decrease obesity, and potentially mitigate PDAC risk. The high-fat, low-carbohydrate ketogenic diet (KD) promotes weight loss and is a promising therapeutic strategy in some cancer preclinical models by altering tumor metabolism. Purpose: The KD will disrupt cancer metabolism, promote weight loss, and delay tumor growth in obesity-associated PDAC. Methods: C57BL/6 mice were fed an obesogenic high-fat diet (HFD) for 15 weeks to induce obesity, after which were randomized to a control diet (CD), a KD, or continued in the HFD for 7 weeks. After, mice were orthotopically injected with mouse KRas G12D/Trp53−/−/PDX-1-CRE (mKPC) cells containing the firefly luciferase gene. Tumor growth was monitored using luminescence through an In Vivo Imaging System. Body weight, glucose tolerance tests, and ketone measurements were performed before, during, and at the study end. Tumors metabolomics were analyzed by Liquid Chromatography Mass Spectrometry. Results: Mice gained weight after 15 weeks of an obesogenic diet. Mice that were switched to a KD or CD lost weight compared to mice that remained on the HFD following diet intervention. Mice fed a KD maintained increased ketone concentrations and increased glucose tolerance after tumors. However, none of the dietary interventions decreased tumor growth. Tumors in mice fed a KD showed significant enrichment in metabolic processes, such as, vitamin B6 and vitamin B5 metabolism, the citrate cycle, and arginine biosynthesis compared to mice that remained on a HFD. Conclusions: Although the KD induced weight loss and altered tumor metabolism, it was insufficient to decrease tumor growth in an obesogenic PDAC mouse model. Our data identified vitamin B6 and vitamin B5 metabolism, the citrate cycle, and arginine biosynthesis as important compensatory metabolic processes induced by the KD. Understanding the availability and functions of these metabolic processes could identify mechanisms of diet interventions for obesity-associated PDAC to decrease obesity, and potentially mitigate PDAC growth. Citation Format: Ericka Velez-Bonet, Kristyn Gumpper-Fedus, Fouad Choueiry, Zhu Jiangjiang, Zobeida Cruz-Monserrate. Ketogenic diet enhanced nutrient metabolic pathways but did not inhibit tumor growth in an obesity-associated PDAC mouse model [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C052.

Human Single Domain Antibody-Based CAR-T Cells Targeting BAFF-R Demonstrate Promising Preclinical Activity in B Cell Malignancies

Background: B-cell activating factor-receptor (BAFF-R) is a potential B-cell specific target, highly expressed in B cell malignancies and regulates B cell proliferation and survival. Single-chain antibody-based CAR-T cells targeting BAFF-R (PMB101) has demonstrated antitumor effects against human B cell malignancies and can overcome CD19 antigen loss (Qin et al. Sci Transl Med. 2019;11(511)). However, CARs built on antigen-specific single chain antibody variable fragment (scFv) may have some immunological risks. Recently, single variable domain of heavy chain antibodies are becoming an alternative to scFv to construct CARs. To improve the function of BAFF-R CAR-T cells, we designed CARs with only a fully human heavy-chain variable domain. Methods: A fully human antibody phage display library (IMARS, IASO Biotherapeutics) was used to select anti-BAFF-R clones using optimal protein/cell alternate panning. The enzyme-linked immunosorbent assay (ELISA) and flow cytometry were applied to evaluate the specificity of clones. The screened clones were grafted into a second-generation CAR with CD8α hinge, 4-1BB costimulatory and CD3ζ intracellular signaling domains to generate CAR-T cells. PMB101 and CAR-T cells constructed with VAY736 were used as positive controls. Non-transduced T cells from the same donor were used as a negative control. Results: Four candidate clones including clone 1, 5, 77 and 80 were finally screened by in vitro functional evaluation, among which clone 5, 77 and 80 were variable domain heavy chain antibodies. The expression levels of the degranulation marker CD107a on CAR-T cells constructed with the four candidate clones were significantly upregulated after coculture with BAFF-R positive tumor cells, while BAFF-R negative tumor cells could not effectively activate CAR-T cells (data not shown). The four clones exhibited similar cytotoxicity in CAR-T cells compared with the PMB101 and VAY736 CAR-T cells using the luciferase-based cytotoxicity assay (data not shown). Xenografts were established in NPG mice following intravenous injection of 1 x 10 6 jeko-1 cells expressing the firefly luciferase gene on day - 5, and a single dose of 4 x 10 6 CAR T cells were infused IV on day 0. Compared to the other groups, no progressive tumor growth was discovered in mice treated with clone 5 CAR-T cells until day 49. Clone 5 CAR-T cells showed excellent in vivo antitumor activity to PMB101 and conferred long-term survival (data not shown). Conclusion: We developed a novel BAFF-R CAR-T cell product structured on single-domain antibody. This product demonstrated promising preclinical activity, and it may provide an alternative choice for patients with B cell malignancies.

Quantification of Xylem-Specific Thermospermine-Dependent Translation of SACL Transcripts with Dual Luciferase Reporter System.

Thermospermine (Tspm) is a polyamine found to play a crucial role in xylem development in Arabidopsis thaliana. Tspm promotes the translation of the SACL genes by counteracting the activity of a cis element in their 5'-leader region that suppresses the translation of the main ORF. Here we describe a method to test the Tspm-dependent translational regulation of the 5'-leader of the SACL mRNAs in Nicotiana benthamiana leaves and A. thaliana mesophyll protoplasts with a dual luciferase assay. The dual luciferase reporter system is used to assess gene expression and is based on the detection of the Firefly luciferase luminescence driven by a specific promoter. However, it can also be used to evaluate the cis elements found in 5'-leader that influence the translation of the main ORF in a transcript. We have used a modified version of the pGreenII 0800 LUC plasmid carrying a double 35S promoter, followed by a poly-linker sequence in phase with the Firefly luciferase gene (pGreen2x35SLUC) where the full 5'-leader sequence of SACL3 was cloned. This construct was used for Agrobacterium tumefaciens infiltration of N. benthamiana leaves and for transfection of A. thaliana mesophyll protoplasts, followed by mock or Tspm treatments. The resulting translation of the Firefly luciferase in these organisms and conditions was then tested by measuring luminescence with the dual luciferase assay and a luminometer. These experiments have allowed us to quantify the positive effect of Tspm in the translation of SACL3 transcripts.

ADAMTSL3 - a potential direct target of miR-144-3p in chondrocyte cell

The miR-144-3p was discovered to upregulate in osteoarthritis. However, the function of this molecule is still poorly understood. Identification of microRNA target genes could shed a light on miRNA functions. In this project, we aim to identify a direct target of miR-144-3p. Potential targets of miR-144-3p were predicted by bioinformatic algorithms. Expression of ADAMTSL3 in miRNA-144-3p gain-and- loss- of function experiment was quantified by Realtime-PCR. Next, the 3’UTR of ADAMTSL3 containing miR-144-3p-binding sites was subcloned in the 3’;UTR of the Firefly luciferase gene on the pmiRGlo. To test the complementary binding of miR-144-3p to ADAMTSL3 mRNA, we employed the luciferase assay using pmiRGlo-ADAMTSL3 vector or pmiRGlo-ADAMTSL3 mutant in which miR-144-3p binding sites in the 3’UTR was mutated. Results showed that miRNA-144-3p directly inhibits the expression of ADAMTSL3 through its ability to pair at “seed” sites. This study provides evidence for the interaction of miRNA-144-3p and its target, ADAMTSL3 mRNA, in chondrocytes.

Masitinib Inhibits Hepatitis A Virus Replication.

The hepatitis A virus (HAV) infection causes acute hepatitis. HAV also induces acute liver failure or acute-on-chronic liver failure; however, no potent anti-HAV drugs are currently available in clinical situations. For anti-HAV drug screening, more convenient and useful models that mimic HAV replication are needed. In the present study, we established HuhT7-HAV/Luc cells, which are HuhT7 cells stably expressing the HAV HM175-18f genotype IB subgenomic replicon RNA harboring the firefly luciferase gene. This system was made by using a PiggyBac-based gene transfer system that introduces nonviral transposon DNA into mammalian cells. Then, we investigated whether 1134 US Food and Drug Administration (FDA)-approved drugs exhibited in vitro anti-HAV activity. We further demonstrated that treatment with tyrosine kinase inhibitor masitinib significantly reduced both HAV HM175-18f genotype IB replication and HAV HA11-1299 genotype IIIA replication. Masitinib also significantly inhibited HAV HM175 internal ribosomal entry-site (IRES) activity. In conclusion, HuhT7-HAV/Luc cells are adequate for anti-HAV drug screening, and masitinib may be useful for the treatment of severe HAV infection.

Systematic identification of endogenous strong constitutive promoters from the diazotrophic rhizosphere bacterium Pseudomonas stutzeri DSM4166 to improve its nitrogenase activity

BackgroundBiological nitrogen fixation converting atmospheric dinitrogen to ammonia is an important way to provide nitrogen for plants. Pseudomonas stutzeri DSM4166 is a diazotrophic Gram-negative bacterium isolated from the rhizosphere of cereal Sorghum nutans. Endogenous constitutive promoters are important for engineering of the nitrogen fixation pathway, however, they have not been systematically characterized in DSM4166.ResultsTwenty-six candidate promoters were identified from DSM4166 by RNA-seq analysis. These 26 promoters were cloned and characterized using the firefly luciferase gene. The strengths of nineteen promoters varied from 100 to 959% of the strength of the gentamicin resistance gene promoter. The strongest P12445 promoter was used to overexpress the biological nitrogen fixation pathway-specific positive regulator gene nifA. The transcription level of nitrogen fixation genes in DSM4166 were significantly increased and the nitrogenase activity was enhanced by 4.1 folds determined by the acetylene reduction method. The nifA overexpressed strain produced 359.1 µM of extracellular ammonium which was 25.6 times higher than that produced by the wild-type strain.ConclusionsThe endogenous strong constitutive promoters identified in this study will facilitate development of DSM4166 as a microbial cell factory for nitrogen fixation and production of other useful compounds.

Development and optimization of a cell screening system for farnesoid X receptor agonist

This study aims to develop an improved cell screening system for farnesoid X receptor (FXR) agonists based on a dual luciferase reporter gene system. FXR response element (FXRE) fragments from FXR target genes were cloned and inserted into upstream of firefly luciferase (Luc) gene in the plasmid pGL4-luc2P-Hygro. In combination with the internal reference plasmid containing renilla luciferase, a dual luciferase reporter gene system was developed and used for high throughput screening of FXR agonists. After studying the effects of over-expression of RXR, mouse or human FXR, various FXRE fragments, and different ratio of FXR plasmid amount to reporter gene plasmid, induction efficiency of the screening system was optimized by the known FXR agonist GW4064, and Z factor for the system reached 0.83 under optimized conditions. In summary, an improved cell screening system based on double luciferase reporter gene detection system was developed to facilitate the discovery of FXR agonists, where a new enhanced FXRE element was formed by a superposition of multiple FXRE fragments from FXR target genes, instead of a superposition of traditional IR-1 (inverted repeats-1) fragments.

Alfaxalone anaesthesia increases brain derived neurotrophic factor levels and preserves postoperative cognition by activating pregnane-X receptors: an in vitro study and a double blind randomised controlled trial

BackgroundAlfaxalone is a fast acting intravenous anaesthetic with high therapeutic index. It is an analogue of the naturally-occurring neurosteroid allopregnanolone responsible for maintenance of cognition and neuroprotection by activation of brain pregnane X receptors and consequent increased production of mature brain-derived neurotrophic factor (m-BDNF). Two studies are reported here: an in vitro study investigated whether alfaxalone activates human pregnane X receptors (h-PXR) as effectively as allopregnanolone; and a clinical study that measured postoperative changes in serum m-BDNF and cognition in patients after alfaxalone anaesthesia compared with propofol and sevoflurane.MethodsIn vitro Activation of h-PXR by allopregnanolone and alfaxalone solutions (206 - 50,000 nM) was measured using human embryonic kidney cells expressing h-PXR hybridised and linked to the firefly luciferase gene. Light emission by luciferase stimulated by each ligand binding with h-PXR was measured.Clinical A double blind prospective randomised study of patients undergoing hip arthroplasty anaesthetised with alfaxalone TIVA (n = 8) or propofol TIVA (n = 3) or propofol plus sevoflurane inhalational anaesthesia (n = 4). The doses of anaesthetics were titrated to the same depth of anaesthesia (BIS 40-60). Subjects’ cognitive performance was assessed using the Grooved Pegboard Test, Digit Symbol Substitution Test (DSST) and Mini Mental State examination (MMSE) for 7 days postoperatively. Serum m-BDNF concentrations were measured for 7 postoperative days.ResultsIn vitro Allopregnanolone and alfaxalone both activated h-PXR, alfaxalone being more efficacious than allopregnanolone: 50,000 nM, p = 0.0019; 16,700 nM, p = 0.0472; 5600 nM, p = 0.0031.Clinical Alfaxalone treated subjects scored better than propofol and sevoflurane anaesthetised patients in the cognition tests: (MMSE p = 0.0251; Grooved Pegboard test dominant hand pre v post anaesthesia scores p = 0.8438 for alfaxalone and p = 0.0156 for propofol and propofol/sevoflurane combined). The higher cognition scores were accompanied by higher serum m-BDNF levels in the alfaxalone anaesthetised patients (p < 0.0001).ConclusionsThese results suggest that sedation and anaesthesia induced by the synthetic neuroactive steroid alfaxalone may be accompanied by effects normally caused by physiological actions of allopregnanolone at PXR, namely, increased secretion of m-BDNF and consequent neuroprotection and preservation of cognition.Trial registrationThe clinical trial was registered on 17/01/2018 with the Australian New Zealand Clinical Trials Registry: registration number ACTRN12618000064202 [Universal Trial Number U1111-1198-0412].

Expression of miRNA-Targeted and Not-Targeted Reporter Genes Shows Mutual Influence and Intercellular Specificity

The regulation of translation by RNA-induced silencing complexes (RISCs) composed of Argonaute proteins and micro-RNAs is well established; however, the mechanisms underlying specific cellular responses to miRNAs and how specific complexes arise are not completely clear. To explore these questions, we performed experiments with Renilla and firefly luciferase reporter genes transfected in a psiCHECK-2 plasmid into human HCT116 or Me45 cells, where only the Renilla gene contained sequences targeted by microRNAs (miRNAs) in the 3'UTR. The effects of targeting were miRNA-specific; miRNA-21-5p caused strong inhibition of translation, whereas miRNA-24-3p or Let-7 family caused no change or an increase in reporter Renilla luciferase synthesis. The mRNA-protein complexes formed by transcripts regulated by different miRNAs differed from each other and were different in different cell types, as shown by sucrose gradient centrifugation. Unexpectedly, the presence of miRNA targets on Renilla transcripts also affected the expression of the co-transfected but non-targeted firefly luciferase gene in both cell types. Renilla and firefly transcripts were found in the same sucrose gradient fractions and specific anti-miRNA oligoribonucleotides, which influenced the expression of the Renilla gene, and also influenced that of firefly gene. These results suggest that, in addition to targeted transcripts, miRNAs may also modulate the expression of non-targeted transcripts, and using the latter to normalize the results may cause bias. We discuss some hypothetical mechanisms which could explain the observed miRNA-induced effects.

Bioluminescence imaging of mouse monocyte chemoattractant protein-1 expression in inflammatory processes.

Monocyte chemoattractant protein-1 (MCP-1) plays a crucial role in various inflammatory diseases. To reveal the impact of MCP-1 during diseases and to develop anti-inflammatory agents, we establish a transgenic mouse line. The firefly luciferase gene is incorporated into the mouse genome and driven by the endogenous MCP-1 promoter. A bioluminescence photographing system is applied to monitor luciferase levels in live mice during inflammation, including lipopolysaccharide-induced sepsis, concanavalin A-induced T cell-dependent liver injury, CCl 4-induced acute hepatitis, and liver fibrosis. The results demonstrate that the luciferase signal induced in inflammatory processes is correlated with endogenous MCP-1 expression in mice. Furthermore, the expressions of MCP-1 and the luciferase gene are dramatically inhibited by administration of the anti-inflammatory drug dexamethasone in a septicemia model. Our results suggest that the transgenic MCP-1-Luc mouse is a useful model to study MCP-1 expression in inflammation and disease and to evaluate the efficiency of anti-inflammatory drugs in vivo.

A pan-tumor-siRNA aptamer chimera to block nonsense-mediated mRNA decay inflames and suppresses tumor progression

Immune-checkpoint blockade (ICB) therapy has changed the clinical outcome of many types of aggressive tumors, but there still remain many cancer patients that do not respond to these treatments. There is an unmet need to develop a feasible clinical therapeutic platform to increase the rate of response to ICB. Here we use a previously described clinically tested aptamer (AS1411) conjugated with SMG1 RNAi (AS1411-SMG1 aptamer-linked siRNA chimeras [AsiCs]) to inhibit the nonsense-mediated RNA decay pathway inducing tumor inflammation and improving response to ICB. The aptamer AS1411 shows binding to numerous mouse and human tumor cell lines tested. AS1411 induces tumor cytotoxicity in long incubation times, which allows for the use of the aptamer as a carrier to target the RNAi inhibition to the tumor. The AS1411-SMG1 AsiCs induce a strong antitumor response in local and systemic treatment in different types of tumors. Finally, AS1411-SMG1 AsiCs are well tolerated with no detected side effects.

Establishment of a visualized mouse orthotopic model of nasopharyngeal carcinoma.

e18048 Background: Nasopharyngeal carcinoma, one of the most common head and neck tumors, is particularly prevalent in Southeast Asia and is characterized by high rates of metastasis and recurrence. Although mouse orthotopic tumor models are commonly employed in studies investigating the mechanisms underlying tumor development and progression, as well as preclinical treatment, currently no such model exists for nasopharyngeal carcinoma. The aim of the current study is to, therefore, establish an orthotopic murine model for nasopharyngeal carcinoma. Methods: To this end, human nasopharyngeal carcinoma C666-1-luc cells, stably expressing the firefly luciferase gene, were injected subcutaneously into the right axilla of BALB/C nude mice. Four weeks later, the resulting subcutaneous tumors were cut into small blocks and grafted into the nasopharynx of immunodeficient BALB/C nude mice to induce tumor formation. Tumor growth was monitored by bioluminescence imaging and small animal magnetic resonance imaging. The histological and immunological antigen expression associated with orthotopic nasopharyngeal carcinoma were analyzed by tissue section analysis and immunohistochemistry (IHC). Results: The tumor formation rate was over 90%. Fluorescence signal detection, micro-magnetic resonance imaging and hematoxylin and eosin staining revealed the successful growth of tumors in the nasopharynx of nude mice. Moreover, IHC analysis detected positive CK, CK5/6, P40 and P63 expression in the mouse orthotopic tumors, which is consistent with the reported antigen expression in the nasopharyngeal tumors of patients. Conclusions: Hence, we successfully established a mouse orthotopic model of nasopharyngeal carcinoma that is highly reproducible, and simple in operation, with low mortality and high tumor formation rates. Moreover, this model facilitates real-time monitoring of tumor growth via in vivo imaging technology, thus, providing a platform for researching nasopharyngeal carcinoma that is more conducive to preclinical research.

Hypericin nanoparticles for self-illuminated photodynamic cytotoxicity based on bioluminescence resonance energy transfer

The purpose of this study was to investigate the self-sensitization of photosensitizer without an external light source to produce a photodynamic therapy (PDT) effect based on the principle of bioluminescence resonance energy transfer (PDT-BRET). First, we demonstrated that HeLa cells could efficiently express firefly luciferase (FLase) after the firefly luciferase gene was transfected with the FLase-gene plasmid (FLase-GP), and proved that FLase could act on the substrate firefly D-luciferin (FLuc) to produce photons. The generated photons activate the photosensitizer hypericin (Hyp) and induce cytotoxicity. Then, we successfully prepared carboxymethyl chitosan-modified poly(lactic-co-glycolic acid) nanoparticles (CPNPs) loaded with FLuc (FLuc-CPNPs) and with loaded Hyp (Hyp-CPNPs). Their physicochemical and pharmaceutical characteristics indicated that they were an excellent drug delivery system. Characterization of the biological effects showed that they could be located in the mitochondrial, had higher ROS generation and stronger cytotoxicity. In vivo results also showed that PDT-BRET was as effective as classic PDT. PDT-BRET and the related drug delivery system are expected to become a new platform for anticancer therapy.

Bioluminescent Monitoring of Circadian Rhythms in Isolated Mesophyll Cells of Arabidopsis at Single-Cell Level.

A bioluminescent monitoring system is used to detect the circadian rhythms of individual plant cells. Transgenic Arabidopsis carrying the firefly luciferase (FLuc) gene driven by a circadian-regulated promoter is used as the material for protoplast isolation. The bioluminescence of these protoplasts in the culture medium is separately captured using a highly sensitive camera system. The time-series data of the bioluminescent imaging reveals the circadian rhythms of these isolated cells, enabling the native properties of the cellular circadian clocks to become elucidated.

Biodistribution and Non-linear Gene Expression of mRNA LNPs Affected by Delivery Route and Particle Size.

PurposeLipid nanoparticles (LNPs) are widely utilized as means to deliver mRNA molecules. However, metric connections between biodistribution and pharmacokinetics (PK) of the nanoparticle carrier and transgene expression dynamics remain largely unknown.MethodsLNPs containing mRNAs encoding the firefly luciferase gene were prepared with varying sizes. Biodistributions of injected LNPs in mice were measured by fluorescence bioimaging or liquid chromatography with tandem mass spectrometry. In addition, luciferase expression levels were determined by bioluminescence imaging and enzyme activity assays.ResultsSome intramuscularly injected LNPs were found circulating in the system, resulting in accumulation in the liver and spleen, especially when the LNP sizes were relatively small. Bigger LNPs were more likely to remain at the injection site. Transgene expression in the liver was found most prominent compared with other organs and tissues.ConclusionsBiomolecules such as mRNAs encapsulated in locally injected LNPs can reach other organs and tissues via systemic circulation. Gene expression levels are affected by the LNP biodistribution and pharmacokinetics (PK), which are further influenced by the particle size and injection route. As transfection efficiency varies in different organs, the LNP exposure and mRNA expression are not linearly correlated.