Firefly Luciferase Research Articles

Development of bioluminescent Group B streptococcal strains for longitudinal infection studies.

Group B Streptococcus (GBS) remains the leading bacterial cause of invasive neonatal disease, resulting in substantial morbidity and mortality. New therapeutic approaches beyond antibacterial treatment to prevent neonatal disease outcomes are urgent. One significant limitation in studying GBS disease and progression is the lack of non-invasive technologies for longitudinal studies. Here, we develop and compare three bioluminescent GBS strains for in vivo pathogenic analysis. Bioluminescence is based on the luxABCDE operon on a replicative vector (luxGBS-CC17), and the red-shifted firefly luciferase on a replicative vector (fflucGBS-CC17) or integrated in the genome (glucGBS-CC17). We show that luxGBS-CC17 is suitable for in vitro analysis but does not produce a significant bioluminescent signal in infected pups. In contrast, the fflucGBS-CC17 results in a strong bioluminescent signal proportional to the organ colonisation level. However, the stability of the replicative vector depends on the route of infection, especially when pups acquire the bacteria from infected vaginal mucosa. Stable chromosomal integration of luciferase in glucGBS-CC17 leads to significant bioluminescence in both haematological and vertical infection models associated with high systemic colonisation. These strains will allow the preclinical evaluation of treatment efficacy against GBS invasive disease using whole-mouse bioluminescence imaging.

Phage LysSA163-CBD mediated specific recognition coupled with ATP bioluminescence for the sensitive detection of viable Staphylococcus aureus in food matrices

BackgroundStaphylococcus aureus is a significant foodborne pathogen, commonly detected in milk and meat products. Conventional detection methods have limited sensitivity and specificity, which are time-consuming and susceptible to background interference from complex samples, and cannot effectively distinguish live and dead bacteria. ResultsHerein, we developed a novel adenosine triphosphate (ATP) bioluminescence method coupled with magnetic separation, which is based on phage-encoded endolysin LysSA163-CBD (CBD 163) for rapid and specific detection of viable Staphylococcus aureus. The expressed protein (CBD 163) was derived from the phage LSA2301 and was successfully expressed in Escherichia coli BL21 following an induction period of 4 h at 37 °C, with a molecular weight approximating 40 kDa. The optimal conditions for CBD-magnetic beads (cMBs) to capture S. aureus cells were determined to be 100 μL/mL cMBs at 25 °C for 30 min. The viable S. aureus cells were disrupted by the Cetyl trimethyl ammonium bromide (CTAB) to release intracellular ATP. Then, the ATP reacted with the firefly luciferase and D-Luciferin-based bioluminescence (BL) reagents solution to generate intensive BL signal. The CBD-magnetic separation-ATP bioluminescence (cMS-BL) assay was able to quickly detect viable S. aureus via ATP bioluminescence in 45 min, with a detection range from 5 × 103 to 5 × 107 CFU/mL and limit of detection (LOD) of 190 CFU/mL. Additionally, the cMS-BL method exhibited high specificity and anti-interference ability, which has been successfully applied to quantify S. aureus cells in crayfish-tail, chicken, and skim milk. Significance and noveltyThese results demonstrate the potential of CBD 163 as a versatile and robust biorecognition element for rapid and specific detection of viable S. aureus in food matrices. The proposed phage-derived bacteria-binding proteins-based protocol for BL detection shows various advantages, including high sensitivity, simple operation, and the capability to distinguish live bacteria, providing a strategy for designing high-quality biorecognition element toward foodborne pathogens.

Near-infrared in vivo imaging system for dynamic visualization of lung-colonizing bacteria in mouse pneumonia.

Conventional animal models of infectious diseases have traditionally relied upon average assessments involving numerous individuals, meaning they do not directly reflect changes in the pathology of an individual. Moreover, in recent years, ethical concerns have resulted in the demand to reduce the number of animals used in such models. Although in vivo imaging offers an effective approach for longitudinally evaluating the pathogenesis of infectious diseases in individual animals, a standardized method has not yet been established. To our knowledge, this study is the first to develop a highly versatile in vivo pulmonary bacterial quantification system utilizing near-infrared luminescence, plasmid-mediated expression of firefly luciferase in bacteria, and a scientific complementary metal-oxide semiconductor camera. Our research holds promise as a useful tool for assessing the efficacy of therapeutic drugs and pathogenesis of infectious diseases.

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.

Characterization of MSCs expressing islet neogenesis associated protein (INGAP): INGAP secretion and cell survival in vitro and in vivo

Mesenchymal stem/stromal cells (MSCs) are emerging as a new therapy for diabetes. Here we investigate the properties of MSCs engineered to express Islet Neogenesis Associated Protein (INGAP) previously shown to reverse diabetes in animal models and evaluate their potential for anti-diabetic applications in mice. Mouse bone marrow-derived MSCs retrovirally transduced to co-express INGAP, Firefly Luciferase and EGFP (INGAP-MSCs), were characterized in vitro and implanted intraperitoneally (IP) into non-diabetic and diabetic C57BL/6 mice (Streptozotocin model) and tracked by live bioluminescence imaging (BLI). Distribution and survival of IP injected INGAP-MSCs differed between diabetic and non-diabetic mice, with a rapid clearance of cells in the latter, and a stronger retention (up to 4 weeks) in diabetic mice concurring with homing towards the pancreas. Interestingly, INGAP-MSCs inhibited the progression of hyperglycemia starting at day 3 and lasting for the entire 6 weeks of the study. Pursuing greater retention, we investigated the survival of INGAP-MSCs in hydrogel matrices. When mixed with Matrigel™ and injected subcutaneously into non-diabetic mice, INGAP-MSCs remained in the implant up to 16 weeks. In vitro tests in three matrices (Matrigel™, Type I Collagen and VitroGel®-MSC) demonstrated that INGAP-MSCs survive and secrete INGAP, with best results at the density of 1–2 x 106 cells/mL. However, all matrices induced spontaneous adipogenic differentiation of INGAP-MSCs in vitro and in vivo, which requires further investigation of its potential impact on MSC therapeutic properties. In summary, based on their ability to stop the rise in hyperglycemia in STZ-treated mice, INGAP-MSCs are a promising therapeutic tool against diabetes but require further research to improve cell delivery and survival.

High-throughput sensitive screening of small molecule modulators of microexon alternative splicing using dual Nano and Firefly luciferase reporters

Disruption of alternative splicing frequently causes or contributes to human diseases and disorders. Consequently, there is a need for efficient and sensitive reporter assays capable of screening chemical libraries for compounds with efficacy in modulating important splicing events. Here, we describe a screening workflow employing dual Nano and Firefly luciferase alternative splicing reporters that affords efficient, sensitive, and linear detection of small molecule responses. Applying this system to a screen of ~95,000 small molecules identified compounds that stimulate or repress the splicing of neuronal microexons, a class of alternative exons often disrupted in autism and activated in neuroendocrine cancers. One of these compounds rescues the splicing of several analyzed microexons in the cerebral cortex of an autism mouse model haploinsufficient for Srrm4, a major activator of brain microexons. We thus describe a broadly applicable high-throughput screening system for identifying candidate splicing therapeutics, and a resource of small molecule modulators of microexons with potential for further development in correcting aberrant splicing patterns linked to human disorders and disease.

A single dose of VEGF-A circular RNA sustains in situ long-term expression of protein to accelerate diabetic wound healing

Diabetic foot ulcer (DFU), which is characterised by damage to minute blood vessels or capillaries around wounds, is one of the most serious and dreaded complications of diabetes. It is challenging to repair chronic non-healing DFU wounds. Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis and promotes wound healing in DFU. However, it is difficult to sustainably deliver VEGF to the wound site owing to its poor stability and easy degradation. To overcome this challenge, lipid nanoparticles (LNP) encapsulating circular RNA (circRNA) encoding VEGF-A have been developed to continuously generate and release VEGF-A and accelerate diabetic wound healing. First, VEGF-A circRNA was synthesized using group I intron autocatalysis strategy and confirmed by enzyme digestion, polymerase chain reaction, and sequencing assay. VEGF-A circRNA was encapsulated in ionizable lipid U-105-derived LNP (U-LNP) using microfluidic technology to fabricate U-LNP/VEGF-A circRNA. For comparison, a commercially ionizable lipid ALC-0315-derived LNP (A-LNP) encapsulating circRNA (A-LNP/circRNA) was used. Dynamic light scattering and transmission electron microscopy characterization indicated that U-LNP/circRNA had spherical structure with an average diameter of 108.5 nm, a polydispersity index of 0.22, and a zeta potential of -3.31 mV. The messenger RNA (mRNA) encapsulation efficiency (EE%) of U-LNP was 87.12%. In vitro transfection data confirmed better stability and long-term VEGF-A expression of circRNA compared with linear mRNA. Assessment of cytotoxicity and innate immunity further revealed that U-LNP/circRNA was biocompatible and induced a weak congenital immune response. Cell scratch and angiogenesis tests demonstrated the bioactivity of U-LNP/VEGF-A circRNA owing to its VEGF-A expression. In situ bioluminescence imaging of firefly luciferase (F-Luc) probe and ELISA demonstrated that circRNA had long-term and strong expression of VEGF-A in the first week, and a gradual decrease in the next week at the wound site and surrounding areas. Finally, a diabetic mouse model was used to validate the healing effect of U-LNP/VEGF-A circRNA formulation. The results showed that a single dose of U-LNP/VEGF-A circRNA administered by dripping resulted in almost complete wound recovery on day 12, which was significantly superior to that of U-LNP/VEGF-A linear mRNA, and it also outperformed recombinant human vascular endothelial growth factor (rhVEGF) injection and A-LNP/circRNA dripping. Histological analysis confirmed the healing efficiency and low toxicity of U-LNP/VEGF-A circRNA formulation. Together, VEGF-A circRNA delivered by U-105-derived LNP showed good performance in wound healing, which was ascribed to the long-term expression and continuous release of VEGF-A, and has potential applications for the treatment of diabetic foot ulcer wounds.

P-268 Unique Argonaute isoforms from mammalian oocytes: unveiling their true purpose

Abstract Study question How does the mouse oocyte-specific AGO2 isoform (AGO2ooc) affect RNA interference (RNAi) and microRNA (miRNA) pathways? Summary answer Mouse AGO2ooc presents reduced RNAi-like endonucleolytic cleavage of perfectly complementary targets. Functional analysis of its impact on fertility and oocyte transcriptome control is in progress. What is known already Argonaute proteins play a pivotal role in effector complexes dedicated to miRNA and RNAi pathways, where small RNAs mediate sequence-specific repression of target RNAs. While all mammalian AGO proteins (AGO1 to AGO4) can function in the miRNA pathway, inhibiting target translation, AGO2 uniquely engages in the RNAi pathway, directing the endonucleolytic cleavage of targets perfectly complementary to small RNAs. In mammalian somatic cells, miRNAs are part of a crucial posttranscriptional regulation network, while RNAi is mostly non-functional. Mouse oocytes, however, deviate from this norm: miRNA regulation proves dispensable, while the abrogation of RNAi results in meiotic spindle defects and sterility. Study design, size, duration Experimental study. Participants/materials, setting, methods We pooled 25 fully-grown GV oocytes in triplicates from non-stimulated 10-12 weeks-old C57BI/6NCrl females for RNA isolation and sequencing. To assess RNAi-like and miRNA-like activity, we transfected Ago2ooc/ooc clones and WT cells with NanoLuc luciferase reporters bearing one perfect complementary (1xP, RNAi-like), four bulged (4xB, miRNA-like) or four mutated (4xM, control) sites for Let-7a and miR-30c. A firefly luciferase reporter without any site was co-transfected for normalization purposes. Main results and the role of chance RNA sequencing revealed an alternative exon-1 in mouse oocyte AGO2, producing an isoform (AGO2ooc) with different amino acid composition in the N-terminus. Interestingly, the somatic version of this N-terminus presents amino acid residues known to be post-translationally modified. These residues are absent in AGO2ooc. To functionally analyze the oocyte-specific N-terminus, we genetically engineered NIH3T3 fibroblasts to express the oocyte alternative exon-1 (Ago2ooc/ooc). Notably, AGO2ooc-expressing cells presented reduced RNAi-like (2-5 fold) but indistinguishable miRNA-like activity when compared to WT cells in reporter assays for Let-7a and miR-30c. The lower RNAi-like activity presented by AGO2ooc-expressing cells was further confirmed in an independent RNAi assay employing the expression of dsRNA hairpins. Both isoforms showed similar abundance in fibroblasts and similar cleavage activity in vitro. Next, we wondered whether AGO2ooc also displays reduced RNAi activity in vivo. To answer that, we generated mouse models whose oocytes would express the somatic AGO2 isoform instead of the AGO2ooc, or N-terminal variants with mutations in putative key amino acid residues. These models will be further utilized to assess fertility and oocyte transcriptome. Limitations, reasons for caution We must investigate whether AGO2ooc also displays differential RNAi activity in mouse oocytes and is relevant for oocyte competence, using the developed mouse models. Wider implications of the findings Mouse oocytes are known to exceptionally operate RNAi. Remarkably, AGO2, the effector nuclease associated with RNAi-like target cleavage has a different N-terminus in oocytes, suggesting that this factor could have adapted to this atypical regulation. We hypothesize that AGO2ooc controls RNAi repression, gene expression and oocyte competence. Trial registration number not applicable

Development and validation of Mayaro virus with luciferase reporter genes as a tool for antiviral assays

Arboviruses are etiological agents in an extensive group of emerging diseases with great clinical relevance in Brazil, due to the wide distribution of their vectors and the favorable environmental conditions. Among them, the Mayaro virus (MAYV) has drawn attention since its emergence as the etiologic agent of Mayaro fever, a highly debilitating disease. To study viral replication and identify new drug candidates, traditional antiviral assays based on viral antigens and/or plaque assays have been demonstrating low throughput, making it difficult to carry out larger-scale assays. Therefore, we developed and characterized two DNA-launched infectious clones reporter viruses based on the MAYV strain BeAr 20290 containing the reporter genes of firefly luciferase (FLuc) and nanoluciferase (NLuc), designated as MAYV-firefly and MAYV-nanoluc, respectively. The viruses replicated efficiently with similar properties to the parental wild-type MAYV, and luminescence expression levels reflected viral replication. Reporter genes were also preserved during passage in cell culture, remaining stably expressed for one round of passage for MAYV-firefly and three rounds for MAYV-nanoluc. Employing the infectious clone, we described the effect of Rimantadine, an FDA-approved Alzheimer's drug, as a repurposing agent for MAYV but with a broad-spectrum activity against Zika virus infection. Additionally, we validated MAYV-nanoluc as a tool for antiviral drug screening using the compound EIDD-2749 (4′-Fluorouridine), which acts as an inhibitor of alphavirus RNA-dependent RNA polymerase.

Development of Traceable Mouse Models of Advanced and Metastatic Bladder Cancer.

Bladder cancer (BC) is the fourth most common cancer in men, with a poor patient prognosis for advanced disease. The poor survival of patients with muscle-invasive bladder cancer (MIBC) and metastatic status emphasizes the urgent need to develop new therapies. Lacking in the field of BC is the availability of relevant advanced BC mouse models, especially metastatic ones, that accurately recapitulate the complexities of human pathology to test and study new therapeutic strategies. Addressing this need, we developed a traceable mouse model of BC that expresses tumor-associated antigens within the context of advanced muscle-invasive BC. This novel system was achieved through the deletion of the tp53 and pten genes, alongside the incorporation of the fusion construct of Firefly luciferase (Luc) and the SIYRYYGL (SIY) T-cell antigen. We validate that the presence of the transgene did not impact on the development of the tumors while allowing us to measure tumor progression by bioluminescence. We show that the transgene did not influence the composition of the immune tumor microenvironment. More importantly, we report that this model was unresponsive to anti-PD-1 treatment, as in the majority of patients with BC. We also develop a new model based on the orthotopic injection of BC clonal cell lines derived from our first model. We demonstrate that this new model invades the muscle layer and has a metastasis development rate of 83%. The advantage of this model is that we can visualize tumor growth and metastasis development in vivo. These mouse models' characteristics, displaying many similarities with the human pathology, provide a valuable tool for tracking tumor progression, metastasis spread in vivo, and treatment resistance, as well as exploring fundamental and translational aspects of BC biology. This work contributes to the improvement in the landscape of mouse models of advanced BC for testing new therapeutic strategies.

High-content analysis of proteostasis capacity in cellular models of amyotrophic lateral sclerosis (ALS)

Disrupted proteome homeostasis (proteostasis) in amyotrophic lateral sclerosis (ALS) has been a major focus of research in the past two decades. However, the proteostasis processes that become disturbed in ALS are not fully understood. Obtaining more detailed knowledge of proteostasis disruption in association with different ALS-causing mutations will improve our understanding of ALS pathophysiology and may identify novel therapeutic targets and strategies for ALS patients. Here we describe the development and use of a novel high-content analysis (HCA) assay to investigate proteostasis disturbances caused by the expression of several ALS-causing gene variants. This assay involves the use of conformationally-destabilised mutants of firefly luciferase (Fluc) to examine protein folding/re-folding capacity in NSC-34 cells expressing ALS-associated mutations in the genes encoding superoxide dismutase-1 (SOD1A4V) and cyclin F (CCNFS621G). We demonstrate that these Fluc isoforms can be used in high-throughput format to report on reductions in the activity of the chaperone network that result from the expression of SOD1A4V, providing multiplexed information at single-cell resolution. In addition to SOD1A4V and CCNFS621G, NSC-34 models of ALS-associated TDP-43, FUS, UBQLN2, OPTN, VCP and VAPB mutants were generated that could be screened using this assay in future work. For ALS-associated mutant proteins that do cause reductions in protein quality control capacity, such as SOD1A4V, this assay has potential to be applied in drug screening studies to identify candidate compounds that can ameliorate this deficiency.

Temperature-dependent intracellular crystallization of firefly luciferase in mammalian cells is suppressed by D-luciferin and stabilizing inhibitors

Firefly luciferase (Fluc) from Photinus pyralis is one of the most widely used reporter proteins in biomedical research. Despite its widespread use, Fluc's protein phase transition behaviors and phase separation characteristics have not received much attention. Current research uncovers Fluc's intrinsic property to phase separate in mammalian cells upon a simple cell culture temperature change. Specifically, Fluc spontaneously produced needle-shaped crystal-like inclusion bodies upon temperature shift to the hypothermic temperatures ranging from 25 °C to 31 °C. The crystal-like inclusion bodies were not associated with or surrounded by membranous organelles and were likely built from the cytosolic pool of Fluc. Furthermore, the crystal-like inclusion formation was suppressed when cells were cultured in the presence of D-luciferin and its synthetic analog, as well as the benzothiazole family of so-called stabilizing inhibitors. These two classes of compounds inhibited intracellular Fluc crystallization by different modes of action as they had contrasting effects on steady-state luciferase protein accumulation levels. This study suggests that, under substrate insufficient conditions, the excess Fluc phase separates into a crystal-like state that can modulate intracellular soluble enzyme availability and protein turnover rate.

Establishment of a Cleavage-Based Single-Plasmid Dual-Luciferase Surrogate Reporter for the Cleavage Efficiency Evaluation of CRISPR-Cas12a Systems and Its Primary Application.

CRISPR-Cas technology is a widely utilized gene-editing tool that involves gRNA-guided sequence recognition and Cas nuclease-mediated cleavage. The design and evaluation of gRNA are essential for enhancing CRISPR/Cas editing efficiency. Various assays such as single-strand annealing, in vitro cleavage, and T7 endonuclease I (T7EI) are commonly used to assess gRNA-mediated Cas protein cleavage activity. In this study, a firefly luciferase and Renilla luciferase co-expressed and a cleavage-based single-plasmid dual-luciferase surrogate reporter was built to evaluate the gRNA-mediated Cas12a cleavage efficiency. The cleavage activities of CRISPR-Cas12a can be quantitatively determined by the recovery degree of firefly luciferase activity. The cleavage efficiency of CRISPR-Cas12a can be quantitatively measured by the recovery of firefly luciferase activity. By using this system, the cleavage efficiency of CRISPR-Cas12a on hepatitis B virus (HBV)/D expression plasmid was evaluated, revealing a negative correlation between gRNA cleavage efficiency and HBV gene expression measured using an enzyme-linked immunosorbent assay. This simple, efficient, and quantifiable system only requires the dual-luciferase vector and CRISPR-Cas12a vector, making it a valuable tool for selecting effective gRNAs for gene editing.

Andrographolide suppresses SARS-CoV-2 infection by downregulating ACE2 expression: A mechanistic study.

Angiotensin-converting enzyme 2 (ACE2) is the receptor that enables SARS-CoV-2 to invade host cells. Previous studies have reported that reducing ACE2 expression may have an anti-SARS-CoV-2 effect. In this study, we constructed a pGL4.10-F2-ACE2 vector with double luciferase genes (firefly and Renilla luciferase) under the control of the ACE2 promoter and used it to screen compounds from Chinese traditional medicinal herbs (CTMHs) that can inhibit ACE2 transcription in human cells. We transfected HEK293T cells with pGL4.10-F2-ACE2 and treated them with CTMH compounds and then measured fluorescence to evaluate the indirect inhibition of ACE2 transcription. Out of 37 compounds tested, andrographolide demonstrated a dose-dependent inhibition of ACE2 transcription. We further confirmed by RT-qPCR and Western blot assays that andrographolide also reduced ACE2 expression in BEAS-2B cells in a dose-dependent manner. Moreover, pseudovirus infection assays in BEAS-2B cells demonstrated that andrographolide can inhibit SARS-CoV-2 infection in a dose-dependent manner. These results suggest that andrographolide has potential anti-SARS-CoV-2 activity and could be a candidate drug for COVID-19 prevention and treatment.

Protein kinase SnRK2.6 phosphorylates transcription factor bHLH3 to regulate anthocyanin homeostasis during strawberry fruit ripening.

Strawberry (Fragaria × ananassa) is a model plant for studying non-climacteric fruit ripening regulated by abscisic acid (ABA); however, the signaling of ABA in the regulation of fruit coloration is not fully understood. In this study, we identified the transcription factor BASIC HELIX-LOOP-HELIX 3 (bHLH3) as being key to fruit coloration via yeast two-hybrid library screening using the bait SUCROSE NONFERMENTING 1 (SNF1)-RELATED PROTEIN KINASE 2 (SnRK2.6), which is a core ABA signaling component that negatively regulates ripening. The interaction was also confirmed by firefly luciferase complementation assays and pull-down assays. RT-qPCR and western blot analysis confirmed that bHLH3 is expressed ubiquitously in strawberry tissues, and it is expressed stably during fruit development. Overexpression and RNAi of both bHLH3 and SnRK2.6 demonstrated that bHLH3 and SnRK2.6 promote and inhibit strawberry fruit coloration, respectively. Using EMSAs, we showed that bHLH3 promotes the expression of UDP-GLUCOSE: FLAVONOL-O-GLUCOSYLTRANSFERASE (UFGT), a key gene for anthocyanin biosynthesis, by directly binding to its promoter. We determined that SnRK2.6 can phosphorylate bHLH3 and that this inhibits its binding to the UFGT promoter, consequently suppressing expression. Altogether, we propose that increased ABA content during strawberry fruit ripening leads to decreased expression of SnRK2.6, which in turn releases the phosphorylation of bHLH3 and thereby enhances UFGT expression, ultimately promoting the coloration of the fruit.

Cycloastragenol induces apoptosis and protective autophagy through AMPK/ULK1/mTOR axis in human non-small cell lung cancer cell lines

ObjectiveStudies have demonstrated that cycloastragenol induces antitumor effects in prostate, colorectal and gastric cancers; however, its efficacy for inhibiting the proliferation of lung cancer cells is largely unexplored. This study explores the efficacy of cycloastragenol for inhibiting non-small cell lung cancer (NSCLC) and elucidates the underlying molecular mechanisms. MethodsThe effects of cycloastragenol on lung cancer cell proliferation were assessed using an adenosine triphosphate monitoring system based on firefly luciferase and clonogenic formation assays. Cycloastragenol-induced apoptosis in lung cancer cells was evaluated using dual staining flow cytometry with an annexin V-fluorescein isothiocyanate/propidium iodide kit. To elucidate the role of cycloastragenol in the induction of apoptosis, apoptosis-related proteins were examined using Western blots. Immunofluorescence and Western blotting were used to determine whether cycloastragenol could induce autophagy in lung cancer cells. Genetic techniques, including small interfering RNA technology, were used to investigate the underlying mechanisms. The effects against lung cancer and biosafety of cycloastragenol were evaluated using a mouse subcutaneous tumor model. ResultsCycloastragenol triggered both autophagy and apoptosis. Specifically, cycloastragenol promoted apoptosis by facilitating the accumulation of phorbol-12-myristate-13-acetate-induced protein 1 (NOXA), a critical apoptosis-related protein. Moreover, cycloastragenol induced a protective autophagy response through modulation of the adenosine 5ʹ-monophosphate-activated protein kinase (AMPK)/unc-51-like autophagy-activating kinase (ULK1)/mammalian target of rapamycin (mTOR) pathway. ConclusionOur study sheds new light on the antitumor efficacy and mechanism of action of cycloastragenol in NSCLC. This insight provides a scientific basis for exploring combination therapies that use cycloastragenol and inhibiting the AMPK/ULK1/mTOR pathway as a promising approach to combating lung cancer.Please cite this article as follows: Zhu LH, Liang YP, Yang L, Zhu F, Jia LJ, Li HG. Cycloastragenolinduces apoptosis and protective autophagy through AMPK/ULK1/mTOR axis in human non-small celllung cancer cell lines. J Integr Med. 2024; 22(4): 504–515.

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.

Diagnosis of Nonalcoholic Fatty Liver Disease via a H2S-Responsive Bioluminescent Probe Combined with Firefly Luciferase mRNA Delivery.

The early detection of nonalcoholic fatty liver disease (NAFLD) through bioluminescent probes is of great significance. However, there remains a challenge to apply them in nontransgenic natural animals due to the lack of exogenous luciferase. To address this issue, we herein report a new strategy for in situ monitoring of endogenous hydrogen sulfide (H2S) in the liver of NAFLD mice by leveraging a H2S-responsive bioluminescent probe (H-Luc) combined with firefly luciferase (fLuc) mRNA delivery. The probe H-Luc was created by installing a H2S recognition moiety, 2,4-dinitrophenol, onto the luciferase substrate (d-luciferin), which is allowed to release cage-free d-luciferin in the presence of H2S via a nucleophilic aromatic substitution reaction. In the meantime, the intracellular luciferase was introduced by lipid nanoparticle (LNP)-mediated fLuc mRNA delivery, rendering it suitable for bioluminescence (BL) imaging in vitro and in vivo. Based on this luciferase-luciferin system, the endogenous H2S could be sensitively and selectively detected in living cells, showing a low limit of detection (LOD) value of 0.72 μM. More importantly, after systematic administration of fLuc mRNA-loaded LNPs in vivo, H-Luc was able to successfully monitor the endogenous H2S levels in the NAFLD mouse model for the first time, displaying a 28-fold higher bioluminescence intensity than that in the liver of normal mice. We believe that this strategy may shed new light on the diagnosis of inflammatory liver disease, further elucidating the roles of H2S.

Luciferase-Decorated Gold Nanorods as Dual-Modal Contrast Agents for Tumor-Targeted High-Performance Bioluminescence/Photoacoustic Imaging.

Gold nanorods (AuNRs) have been considered highly compelling materials for early cancer diagnosis and have aroused a burgeoning fascination among the biomedical sectors. By leveraging the versatile tunable optical properties of AuNRs, herein, we have developed a novel tumor-targeted dual-modal nanoprobe (FFA) that exhibits excellent bioluminescence and photoacoustic imaging performance for early tumor diagnosis. FFA has been synthesized by anchoring the recombinant bioluminescent firefly luciferase protein (Fluc) on the folate-conjugated AuNRs via the PEG linker. TEM images and UV-vis studies confirm the nanorod morphology and successful conjugation of the biomolecules to AuNRs. The nanoprobe FFA relies on the ability of the folate module to target the folate receptor-positive tumor cells actively, and simultaneously, the Fluc module facilitates excellent bioluminescent properties in physiological conditions. The success of chemical engineering in the present study enables stronger bioluminescent signals in the folate receptor-positive cells (Skov3, Hela, and MCF-7) than in folate receptor-negative cells (A549, 293T, MCF-10A, and HepG2). Additionally, the AuNRs induced strong photoacoustic conversion performance, enhancing the resolution of tumor imaging. No apparent toxicity was detected at the cellular and mouse tissue levels, manifesting the biocompatibility nature of the nanoprobe. Prompted by the positive merits of FFA, the in vivo animal studies were performed, and a notable enhancement was observed in the bioluminescent/photoacoustic intensity of the nanoprobe in the tumor region compared to that in the folate-blocking region. Therefore, this synergistic dual-modal bioluminescent and photoacoustic imaging platform holds great potential as a tumor-targeted contrast agent for early tumor diagnosis with high-performance imaging information.

Imaging CAR-NK cells targeted to HER2 ovarian cancer with human sodium-iodide symporter-based positron emission tomography

Chimeric antigen receptor (CAR) cell therapies utilize CARs to redirect immune cells towards cancer cells expressing specific antigens like human epidermal growth factor receptor 2 (HER2). Despite their potential, CAR T cell therapies exhibit variable response rates and adverse effects in some patients. Non-invasive molecular imaging can aid in predicting patient outcomes by tracking infused cells post-administration. CAR-T cells are typically autologous, increasing manufacturing complexity and costs. An alternative approach involves developing CAR natural killer (CAR-NK) cells as an off-the-shelf allogeneic product. In this study, we engineered HER2-targeted CAR-NK cells co-expressing the positron emission tomography (PET) reporter gene human sodium-iodide symporter (NIS) and assessed their therapeutic efficacy and PET imaging capability in a HER2 ovarian cancer mouse model.NK-92 cells were genetically modified to express a HER2-targeted CAR, the bioluminescence imaging reporter Antares, and NIS. HER2-expressing ovarian cancer cells were engineered to express the bioluminescence reporter Firefly luciferase (Fluc). Co-culture experiments demonstrated significantly enhanced cytotoxicity of CAR-NK cells compared to naive NK cells. In vivo studies involving mice with Fluc-expressing tumors revealed that those treated with CAR-NK cells exhibited reduced tumor burden and prolonged survival compared to controls. Longitudinal bioluminescence imaging demonstrated stable signals from CAR-NK cells over time. PET imaging using the NIS-targeted tracer 18F-tetrafluoroborate ([18F]TFB) showed significantly higher PET signals in mice treated with NIS-expressing CAR-NK cells.Overall, our study showcases the therapeutic potential of HER2-targeted CAR-NK cells in an aggressive ovarian cancer model and underscores the feasibility of using human-derived PET reporter gene imaging to monitor these cells non-invasively in patients.