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Host cell damage is a key parameter for research in infection biology, drug testing, and substance safety screening. In this study, we introduce a luciferase reporter system as a new and reliable assay to measure cell damage and validate it with the pathogenic yeast, Candida albicans , as a test case. We transduced human epithelial cell lines with a lentiviral vector to stably express an optimized luciferase enzyme, Nanoluc. Upon cell damage, the release of cytoplasmic luciferase into the extracellular space can be easily detected by a luminometer. We used the luciferase reporter system to investigate the damage caused by C. albicans to different newly generated epithelial reporter cell lines. We found that fungus-induced cell damage, as determined by established methods, correlated tightly with the release of the luciferase. The new luciferase reporter system is a simple, sensitive, robust, and inexpensive method for measuring host cell damage and has a sensitivity comparable to the standard assay, release of lactate dehydrogenase. It is suitable for high-throughput studies of pathogenesis mechanisms of any microbe, for antimicrobial drug screening, and many other applications.IMPORTANCEWe present a quick, easy, inexpensive, and reliable assay to measure damage to mammalian cells. To this end, we created reporter cell lines which artificially express luciferase, an enzyme that can be easily detected in the supernatant when these cells are damaged. We used infections with the well-investigated fungal pathogen of humans, Candida albicans , as a test case of our system. Using our reporter, we were able to recapitulate the known effects of strain variability, gene deletions, and antifungal treatments on host cell damage. This easily adaptable reporter system can be used to screen for damage in infection models with different microbial species, assay cell-damaging potential of substances, discover new non-toxic antibiotics, and many other damage-based applications., Competing Interests: Work from this paper has been used to register the patent "Cytotoxicity assay for detecting cellular damage" (L31002DE) with the Deutsches Patent-und Markenamt authors R.V., M.T., V.T., S.B., and B.H. and Leibniz-HKI as beneficiaries.

Nipah virus (NiV) causes near-annual outbreaks of fatal encephalitis and respiratory disease in South Asia with a high mortality rate (∼70%). Since there are no approved therapeutics for NiV disease in humans, the WHO has designated NiV and henipaviral diseases priority pathogens for research and development. We generated a new recombinant green fluorescent reporter NiV of the circulating Bangladesh genotype (rNiV-B-ZsG) and optimized it alongside our previously generated Malaysian genotype reporter counterpart (rNiV-M-ZsG) for antiviral screening in primary-like human respiratory cell types. Validating our platform for rNiV-B-ZsG with a synthetic compound library directed against viral RNA-dependent RNA polymerases, we identified a hit compound and confirmed its sub-micromolar activity against wild-type NiV, green fluorescent reporter, and the newly constructed bioluminescent red fluorescent double reporter (rNiV-B-BREP) NiV. We furthermore demonstrated that rNiV-B-ZsG and rNiV-B-BREP viruses showed pathogenicity comparable to wild-type NiV-B in the Syrian golden hamster model of disease, supporting additional use of these tools for both pathogenesis and advanced pre-clinical studies in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

The development of human induced pluripotent stem cell (iPSC)-based regenerative therapies is challenged by the lack of specific cell markers to isolate differentiated cell types and improve differentiation protocols. This issue is particularly critical for notochordal-like cells and chondrocytes, which are crucial in treating back pain and osteoarthritis, respectively. Both cell types produce abundant proteoglycan aggrecan (ACAN), crucial for the extracellular matrix. We generated two human iPSC lines containing an ACAN-2A-mScarlet reporter. The reporter cell lines were validated using CRISPR-mediated transactivation and functionally validated during notochord and cartilage differentiation. The ability to isolate differentiated cell populations producing ACAN enables their enrichment even in the absence of specific cell markers and allows for comprehensive studies and protocol refinement. ACAN's prevalence in various tissues (e.g., cardiac and cerebral) underscores the reporter's versatility as a valuable tool for tracking matrix protein production in diverse cell types, benefiting developmental biology, matrix pathophysiology, and regenerative medicine.

Various techniques using fluorescent reporter probes have been developed, such as GFP transgenic mouse lines that are used to detect spatial-temporal expression levels of genes. Although GFP expression is largely considered non-toxic, recent reports have indicated that under certain conditions GFP can display cellular toxicity. We hereby report the nuclear toxicity of H2B-GFP using a K14 specific Tet-on reporter mouse system. Using this system, GFP accumulates in the nucleus of all K14 expressing cells, such as the ocular surface epithelia and ocular adnexa. Expression of high levels of nuclear GFP during embryonic stages led to an eye open-at-birth (EOB) phenotype and abnormal ocular adnexa development and during adult and aging stages showed notable toxicity to ocular tissues. Other tissues, such as skin, also presented multiple defects associated with H2B-GFP expression. This toxicity was found to be concentration dependent, with homozygous mice presenting extremely high toxicity, while heterozygous mice presented limited toxicity. Upon induction, the accumulation of H2B-GFP in the nucleus of homozygous mice led to apoptosis within 2 weeks. This study therefore shows that although the use of nuclear GFP reporter mice is a valuable tool, at high levels, nuclear GFP can be toxic, leading to cell death and affecting tissue function., (© 2024. The Author(s).)

Cell-free transcription-translation (TXTL) systems expressing genes from linear dsDNA enable the rapid prototyping of genetic devices while avoiding cloning steps. However, repetitive inclusion of a reporter gene is an incompressible cost and sometimes accounts for most of the synthesized DNA length. Here we present reporter systems based on split-GFP systems that reassemble into functional fluorescent proteins and can be used to monitor gene expression in E. coli TXTL. The 135 bp GFP10-11 fragment produces a fluorescent signal comparable to its full-length GFP counterpart when reassembling with its complementary protein synthesized from the 535 bp fragment expressed in TXTL. We show that split reporters can be used to characterize promoter libraries, with data qualitatively comparable to full-length GFP and matching in vivo expression measurements. We also use split reporters as small fusion tags to measure the TXTL protein and peptide production yield. Finally, we generalize our concept by providing a luminescent split reporter based on split-nanoluciferase. The ∼80% gene sequence length reduction afforded by split reporters lowers synthesis costs and liberates space for testing larger devices while producing a reliable output. In the peptide production context, the small size of split reporters compared with full-length GFP is less likely to bias peptide solubility assays. We anticipate that split reporters will facilitate rapid and cost-efficient genetic device prototyping, protein production, and interaction assays.

Internal ribosomal entry sites (IRESs) recruit the ribosome to promote translation, typically in an m7G cap-independent manner. Although IRESs are well-documented in viral genomes, they have also been reported in mammalian transcriptomes, where they have been proposed to mediate cap-independent translation of mRNAs. However, subsequent studies have challenged the idea of these "cellular" IRESs. Current methods for screening and discovering IRES activity rely on a bicistronic reporter assay, which is prone to producing false positive signals if the putative IRES sequence has a cryptic promoter or cryptic splicing sites. Here, we report an assay for screening IRES activity using a genetically encoded circular RNA comprising a split nanoluciferase (nLuc) reporter. The circular split nLuc reporter is less susceptible to the various sources of false positives that adversely affect the bicistronic IRES reporter assay and provides a streamlined method for screening IRES activity. Using the circular split nLuc reporter, we find that nine reported cellular IRESs have minimal IRES activity. Overall, the circular split nLuc reporter offers a simplified approach for identifying and validating IRESs and exhibits reduced propensity for producing the types of false positives that can occur with the bicistronic reporter assay., (© 2024 Unti et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Reporter alleles are essential for advancing research with human induced pluripotent stem cells (hiPSCs), notably in developmental biology and disease modeling. This study investigates the state-of-the-art gene-editing techniques tailored for generating reporter alleles in hiPSCs, emphasizing their effectiveness in investigating cellular dynamics and disease mechanisms. Various methodologies, including the application of CRISPR/Cas9 technology, are discussed for accurately integrating reporter genes into the specific genomic loci. The synthesis of findings from the studies utilizing these reporter alleles reveals insights into developmental processes, genetic disorder modeling, and therapeutic screening, consolidating the existing knowledge. These hiPSC-derived models demonstrate remarkable versatility in replicating human diseases and evaluating drug efficacy, thereby accelerating translational research. Furthermore, this review addresses challenges and future directions in refining the reporter allele design and application to bolster their reliability and relevance in biomedical research. Overall, this investigation offers a comprehensive perspective on the methodologies, applications, and implications of reporter alleles in hiPSC-based studies, underscoring their essential role in advancing both fundamental scientific understanding and clinical practice.

The dCas9-based Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi) gene regulation technique requires two components: a catalytically inactive Cas9 protein (dCas9) and a single-guide RNA that targets the gene of interest. This system is commonly activated by expressing dCas9 through an inducible gene promoter, but these inducers may affect cellular physiology, and accessibility and permeability of the inducer are limited in relevant model systems. Here, we have developed an alternative approach for CRISPRi activation in the clinical isolate Staphylococcus aureus USA300 LAC, where dCas9 was expressed through endogenous virulence gene promoters (vgp); coagulase, autolysin, or fibronectin-binding protein A. Additionally, we integrated a fluorescent reporter gene into the vgp-CRISPRi system to monitor the activity of the dcas9 -controlling promoter. Testing the efficacy of vgp-CRISPRi by inducing growth arrest (when targeting penicillin-binding protein 1), downregulating target gene expression, or blocking coagulase-dependent coagulation of blood plasma, we provide a proof-of-concept demonstration that the virulence gene promoter-driven CRISPRi system is functional in S. aureus .IMPORTANCEThe presented inducer-free, endogenous virulence gene promoter-induced, dCas9-based Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) interference (CRISPRi system addresses several shortcomings related to the use of inducer-dependent systems such as effects on cell physiology or limitations in permeability, and it avoids the high, putatively toxic levels of dCas9 in CRISPRi systems controlled by strong, constitutive promoters., Competing Interests: The authors declare no conflict of interest.

The development of therapies to combat neurodegenerative diseases is widely recognized as a research priority. Despite recent advances in understanding their molecular basis, there is a lack of suitable early biomarkers to test selected compounds and accelerate their translation to clinical trials. We have investigated the utility of in vivo reporters of cytoprotective pathways (e.g. NRF2, p53) as surrogate early biomarkers of the ALS degenerative disease progression. We hypothesized that cellular stress observed in a model of ALS may precede overt cellular damage and could activate our cytoprotective pathway reporters. To test this hypothesis, we generated novel ALS-reporter mice by crossing the hTDP-43tg model into our oxidative stress/inflammation (Hmox1; NRF2 pathway) and DNA damage (p21; p53 pathway) stress reporter models. Histological analysis of reporter expression in a homozygous hTDP-43tg background demonstrated a time-dependent and tissue-specific activation of the reporters in tissues directly associated with ALS, before moderate clinical signs are observed. Further work is warranted to determine the specific mechanisms by which TDP-43 accumulation leads to reporter activation and whether therapeutic intervention modulates reporters' expression. We anticipate the reporter strategy could be of great value in developing treatments for a range of degenerative disorders.

Studying influenza A viruses (IAVs) requires secondary experimental procedures to detect the presence of the virus in infected cells or animals. The ability to generate recombinant (r)IAV using reverse genetics techniques has allowed investigators to generate viruses expressing foreign genes, including fluorescent and luciferase proteins. These rIAVs expressing reporter genes have allowed for easily tracking viral infections in cultured cells and animal models of infection without the need for secondary approaches, representing an excellent option to study different aspects in the biology of IAV where expression of reporter genes can be used as a readout of viral replication and spread. Likewise, these reporter-expressing rIAVs provide an excellent opportunity for the rapid identification and characterization of prophylactic and/or therapeutic approaches. To date, rIAV expressing reporter genes from different viral segments have been described in the literature. Among those, rIAV expressing reporter genes from the viral NS segment have been shown to represent an excellent option to track IAV infection in vitro and in vivo, eliminating the need for secondary approaches to identify the presence of the virus. Here, we summarize the status on rIAV expressing traceable reporter genes from the viral NS segment and their applications for in vitro and in vivo influenza research.

Estrogen receptor alpha (ERα) is a nuclear receptor that is expressed mainly in the breast, uterus, and ovary, among several other organs. ERα plays important roles in reproduction, mammary gland formation, and glucose homeostasis. Disruption of ERα may result in adverse outcomes, such as cancer, impaired fertility, and abnormal fetal growth. Therefore, identifying compounds that modulate ERα is of great interest due to their potential-endocrine disrupting capability and pharmaceutical applications. To rapidly test tens of thousands of compounds, high-throughput screening assays are essential. Here, we describe high-throughput screening methods, including plating and treatment of cells in 384-well and 1536-well plates and analysis of the resulting data. The two cell lines used, MCF7-VM7Luc4E2 and HEK293-ERα-bla, have been described previously. MCF7-VM7Luc4E2 cells are a stable luciferase reporter gene cell line expressing full-length endogenous estrogen receptor in the MCF7 cell line background, and HEK293-ERα-bla cells stably express an ERα ligand-binding domain/GAL4 DNA-binding domain fusion regulating a UAS β-lactamase reporter gene. These cell lines can be used to identify and confirm ERα modulators. Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Establishment of a high-throughput ERα reporter gene assay with luminescence readout to identify activators and inhibitors of estrogen receptor α Basic Protocol 2: Use of an orthogonal assay with fluorescence readout to confirm potential estrogen receptor activators or inhibitors., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Current Protocols published by Wiley Periodicals LLC.)

The activation of yes-associated protein 1 (YAP1) and transcriptional co-activator with PDZ-binding motif (TAZ) has been implicated in both regeneration and tumorigenesis, thus representing a double-edged sword in tissue homeostasis. However, how the activity of YAP1/TAZ is regulated or what leads to its dysregulation in these processes remains unknown. To explore the upstream stimuli modulating the cellular activity of YAP1/TAZ, we developed a highly sensitive YAP1/TAZ/TEAD-responsive DNA element (YRE) and incorporated it into a lentivirus-based reporter cell system to allow for sensitive and specific monitoring of the endogenous activity of YAP1/TAZ in terms of luciferase activity in vitro and Venus fluorescence in vivo. Furthermore, by replacing YRE with TCF- and NF-κB-binding DNA elements, we demonstrated the applicability of this reporter system to other pathways such as Wnt/β-catenin/TCF- and IL-1β/NF-κB-mediated signaling, respectively. The practicality of this system was evaluated by performing cell-based reporter screening of a chemical compound library consisting of 364 known inhibitors, using reporter-introduced cells capable of quantifying YAP1/TAZ- and β-catenin-mediated transcription activities, which led to the identification of multiple inhibitors, including previously known as well as novel modulators of these signaling pathways. We further confirmed that novel YAP1/TAZ modulators, such as potassium ionophores, Janus kinase inhibitors, platelet-derived growth factor receptor inhibitors, and genotoxic stress inducers, alter the protein level or phosphorylation of endogenous YAP1/TAZ and the expression of their target genes. Thus, this reporter system provides a powerful tool to monitor endogenous signaling activities of interest (even in living cells) and search for modulators in various cellular contexts., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Getah virus (GETV) is a re-emerging mosquito-borne RNA virus that induces fever, hind limb edema, swollen submandibular lymph nodes, and urticaria in horses. In pigs, the virus often results in stillbirths among pregnant sows, and neurological symptoms leading to death in piglets. Currently, there are no specific treatments or drugs available for GETV infection. The use of reporter viruses to monitor viral replication and spread in real-time within infected cells and animals provides a powerful tool for targeting antiviral drugs throughout the viral life cycle. Their fluorescence-tracked characteristics greatly facilitate virus neutralization tests (VNTs). In this study, we engineered two recombinant viruses by inserting different reporter protein genes at the 3' end of the structural protein gene, an unreported location that can accommodate exogenous genes. The rGEEiLOV and rGEEGFP viruses demonstrated genetic stability for at least five passages and replicated at a rate similar to that of the parental virus in BHK-21 cells. The rGEEGFP virus facilitated viral neutralization testing. Additionally, we used the reporter virus rGEEGFP to confirm ivermectin, a broad-spectrum antiparasitic agent, as a potential inhibitor of GETV in vitro. Ivermectin appears to inhibit the early replication stages of the virus and can block cell-to-cell viral transmission. In conclusion, rGEEGFP holds significant potential for antiviral screening to identify specific inhibitors against GETV and for use in viral neutralization tests., Competing Interests: Declaration of competing interest The research was conducted in the absence of any commercial or financial relationships., (Copyright © 2024 Elsevier Inc. All rights reserved.)

We genetically manipulated HaCaT cells, a spontaneously immortalised normal keratinocyte cell line, to stably express two different coloured luciferase reporter genes, driven by interleukin 8 (IL-8) and ubiquitin-C (UBC) promoters, respectively. Subsequently, we generated a three-dimensional (3D) skin-like in vitro composite (SLIC) utilising these cells, with the objective of monitoring bioluminescence emitted from the SLIC. This SLIC was generated on non-woven silica fibre membranes in differentiation medium. Immunohistochemical analyses of skin differentiation markers in the SLIC revealed the expression of keratins 2 and 10, filaggrin, and involucrin, indicating mature skin characteristics. This engineered SLIC was employed for real-time bioluminescence monitoring, allowing the assessment of time- and dose-dependent responses to UV stress, as well as to hydrophilic and hydrophobic chemical loads. Notably, evaluation of responses to hydrophobic substances has been challenging with conventional 2D cell culture methods, suggesting the need for a new approach, which this technology could address. Our observations suggest that engineered SLIC with constitutively expressing reporters driven by selected promoters which are tailored to specific objectives, significantly facilitates assays exploring the physiological functions of skin cells based on genetic response mechanisms. It also highlights new avenues for evaluating the physiological impacts of various compounds designed for topical application to human skin., (© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Various reporter genes have been developed to study gene expression pattern and gene regulation. The RUBY reporter gene was recently developed and widely used, because of its visible and noninvasive advantages. However, quantitative analysis of RUBY gene expression levels was lacking. In this study, we introduce a novel betalain quantification method in combination with the tobacco transient expression system. The betalain produced in tobacco leaves was extracted and purified, and its concentration was quantitatively measured. We successfully applied this approach in studying the transcriptional regulation of ARC5 gene by transcription factors CPD25 and CPD45. Furthermore, with this method, we showed that the gene expression of RCA and Rbcs1A gene were regulated by light, transcription factors HY5 and PIFs through G-box and I-box elements. The development of this betalain quantification approach with the tobacco transient expression system offers a cost-effective and intuitive strategy for studying the regulatory mechanism of gene expression., (© 2024 John Wiley & Sons Ltd.)

The objective of the current study is to develop a new method for tracking transplanted human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) using magnetic resonance imaging (MRI). The CRISPR/dCas9 activation system is employed to overexpress ferritin heavy chain (FHC) in hiPSC-CMs. The mRNA and protein expression of FHC in hiPSC and hiPSC-CMs significantly increased after transfection. Iron chloride does not affect the cell viability in a concentration range from 0 to 2000 µm. hiPSCs overexpressing FHC (hiPSC- FHC OE ) and hiPSC-CMs overexpressing FHC (hiPSC-CM-FHC OE ) significantly enhanced cellular uptake of iron chloride but with no changes in electrophysiological properties compared to hiPSC-CM-Control. Furthermore, hiPSC-CM-FHC OE presented robust contrast and lower T 2 * values, signifying their potential as highly effective candidates for cardiac MRI. Next, hiPSC-CM-FHC OE is injected into mouse hearts and after 3 days of transplantation, MR images are obtained. hiPSC-CM-FHC OE cells exhibited clear signals in the hearts with lower T 2 * and rapid signal decay. Collectively, data from this proof-of-concept study demonstrated that endogenous labeling with FHC in hiPSC-CMs can be a potent strategy for enhancing the accuracy of cardiac MRI. This technology represents a significant step forward in tracking the transplanted hiPSC-CMs in the hearts of live animals., (© 2024 Wiley‐VCH GmbH.)

Mitochondria-endoplasmic reticulum contact sites (MERCS) serve as hotspots for important cellular processes, including calcium homeostasis, phospholipid homeostasis, mitochondria dynamics, and mitochondrial quality control. MERCS reporters based on complementation of green fluorescent proteins (GFP) fragments have been designed to visualize MERCS in real-time, but we find that they do not accurately respond to changes in MERCS content. Here, we utilize split LacZ complementing fragments to develop the first MERCS reporter system (termed SpLacZ-MERCS) that continuously integrates the MERCS information within a cell and generates a fluorescent output. Our system exhibits good organelle targeting, no artifactual tethering, and effective, dynamic tracking of the MERCS level in single cells. The SpLacZ-MERCS reporter was validated by drug treatments and genetic perturbations known to affect mitochondria-ER contacts. The signal-integrating nature of SpLacZ-MERCS may enable systematic identification of genes and drugs that regulate mitochondria-ER interactions. Our successful application of the split LacZ complementation strategy to study MERCS may be extended to study other forms of interorganellar crosstalk.

Oropouche fever caused by Oropouche virus (OROV) is a significant zoonosis in Central and South America. Despite its public health significance, we lack high-throughput diagnostics, therapeutics, and a comprehensive knowledge of OROV biology. Reporter viruses are valuable tools to rapidly study virus dynamics and develop neutralization and antiviral screening assays. OROV is a tri-segmented bunyavirus, which makes generating a reporter virus challenging, as introducing foreign elements into the viral genome typically affects fitness. We previously demonstrated that the non-structural gene NSm on the OROV medium (M) segment is non-essential for replication in vitro . Taking advantage of this, we have now generated a recombinant OROV expressing fluorescent protein ZsGreen in place of NSm. This reporter OROV is both stable and pathogenic in IFNAR -/- mice and provides a powerful tool for OROV pathogenesis studies and assay development.IMPORTANCEEmerging and reemerging infectious agents such as zoonotic bunyaviruses are of global health concern. Oropouche virus (OROV) causes recurring outbreaks of acute febrile illness in the Central and South American human populations. Biting midges are the primary transmission vectors, whereas sloths and non-human primates are their reservoir hosts. As global temperatures increase, we will likely see an expansion in arthropod-borne pathogens such as OROV. Therefore, developing reagents to study pathogen biology to aid in identifying druggable targets is essential. Here, we demonstrate the feasibility and use of a fluorescent OROV reporter in mice to study viral dynamics and pathogenesis. We show that this reporter OROV maintains characteristics such as growth and pathogenicity similar to the wild-type virus. Using this reporter virus, we can now develop methods to assist OROV studies and establish various high-throughput assays., Competing Interests: The authors declare no conflict of interest.

Filamentous fungi with their diverse inventory of carbohydrate-active enzymes promise a holistic usage of lignocellulosic residues. A major challenge for application is the inherent repression of enzyme production by carbon catabolite repression (CCR). In the presence of preferred carbon sources, the transcription factor CreA/CRE-1 binds to specific but conserved motifs in promoters of genes involved in sugar metabolism, but the status of CCR is notoriously difficult to quantify. To allow for a real-time evaluation of CreA/CRE-1-mediated CCR at the transcriptional level, we developed a luciferase-based construct, representing a dynamic, highly responsive reporter system that is inhibited by monosaccharides in a quantitative fashion. Using this tool, CreA/CRE-1-dependent CCR triggered by several monosaccharides could be measured in Neurospora crassa, Aspergillus niger and Aspergillus nidulans over the course of hours, demonstrating distinct and dynamic regulatory processes. Furthermore, we used the reporter to visualize the direct impacts of multiple CreA truncations on CCR induction. Our reporter thus offers a widely applicable quantitative approach to evaluate CreA/CRE-1-mediated CCR across diverse fungal species and will help to elucidate the multifaceted effects of CCR on fungal physiology for both basic research and industrial strain engineering endeavours., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) are legacy organic micropollutants (OMPs) that are sporadically detected in drinking water (DW) sources. The European Drinking Water Directive requires EU member states to monitor 5 PAHs in DW and its sources. The Dutch national regulations require 6 additional PAHs to be monitored and 7 polychlorinated biphenyls (PCBs). These indicator compounds act as representatives for large compound classes. PCBs alone comprise 209 congeners, it is evident that conventional chemical target analysis (GC-tQ-MS) alone is not sufficient to monitor these entire compound classes. This study investigated the application of reporter gene assays as effect-based methods (EBMs) to monitor PAHs and PCBs in DW sources. Herein, it was assessed what added value the bioassays can bring compared to the current approach of chemical target analysis for PCBs and PAHs. Regulated and non-regulated PAHs and PCBs were tested in four bioassays to determine the relative potency factors (RPFs) for these compounds. Non-regulated congeners were found to be active in the PAH-CALUX and anti-AR CALUX. An assessment of surface water (SW) spiked with standard mixtures containing PAHs and PCBs confirmed the predictable behavior of the PAH-CALUX. Moreover, the bioassay was able to detect AhR-mediated activity caused by non-regulated PAHs and PCBs, whereas this would have been missed by conventional chemical target analysis. Last, a field study was conducted in Dutch DW sources at six sampling moments. The PAH-CALUX detected AhR-mediated activity at all sampling moments and an ecological effect-based trigger (EBT) value was exceeded on multiple accounts. Combined application of GC-tQ-MS and the PAH-CALUX ensures compliancy with monitoring legislation and provides additional insights into potential hazards to humans and the environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The forkhead box O (FOXO) family of transcription factors translates environmental cues into precise gene expression patterns maintaining cellular equilibrium while influencing critical determinations of cell destiny and differentiation. FOXO proteins exert their effects through specific consensus binding to promoter sites within target genes. Notably, among the array of techniques available for assessing the transcriptional activity of FOXO factors, the utilization of luciferase-based reporters emerges as particularly distinctive. Luciferase, an enzyme sourced from bioluminescent organisms, instigates the oxidation of luciferin, culminating in the generation of oxyluciferin accompanied by discernible luminescence, a quantifiable event readily gauged using a luminometer. The adoption of luciferase activity as a measure in transcriptional assays is widespread due to its numerous advantages including simplicity, remarkable reproducibility, and high sensitivity. Moreover, the continuous advancements witnessed in luciferase-based vectors and measurement reagents bestow notable flexibility upon this methodology. Luciferase-based reporters offer a powerful tool for uncovering constituents within the signaling pathways governing FOXO factor function. Furthermore, these assays are also suitable for evaluating the efficacy of FOXO-targeting agents, whether they be inhibitors or activators. Here, we present a comprehensive, step-by-step elucidation of a commonly employed assay, adeptly quantifying the potential of small molecular compounds to amplify FOXO-specific transcriptional activity in U2OS cells., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Here, I describe the methods to perform dual luciferase reporter assay in Schizosaccharomyces pombe. The experiment requires the generation of a reporter plasmid construct, with firefly luciferase gene controlled under the genetic element of interest (such as transcriptional promoter or translation initiation signal preceded by a defined promoter) and Renilla luciferase gene expressed under a fixed promoter. S. pombe transformants carrying the plasmids with varied control elements are grown in a selective medium and chilled on ice. Small amounts of cells are pelleted and subjected to the commercially available dual luciferase assay., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

RNA binding proteins (RBPs) and their associated partners are key factors of posttranscriptional control of gene expression. To study and manipulate the functional consequences of binding of these regulators to their targets, several tethering assays have been developed, in which a protein of interest is brought to a reporter mRNA through heterologous RNA-protein interaction motifs. The effect of such constrained binding is then monitored by measuring the accumulation of the reporter protein and mRNA. This chapter describes a protocol for the λN-BoxB tether system in transiently transfected mammalian cells. Combining the luciferase reporter technology to quantify protein amounts by light measurement and RNA amounts by RT-qPCR, this assay provides a simple and robust way to analyze the consequences of any protein binding in a controlled and defined manner., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Luciferases catalyze a reaction that involves the emission of light, a phenomenon referred to as "bioluminescence". The calcium-sensing receptor (CaSR), a G protein-coupled receptor (GPCR), induces characteristic signaling pathways that stimulate extracellular signal-regulated kinase 1/2 (ERK1/2) and Ca 2+ mobilization from the endoplasmic reticulum. ERK1/2 causes an activation of the serum response element (SRE), whereas Ca 2+ causes an activation of the nuclear factor of activated T-cells response element (NFAT-RE). Transfection of cells with a vector containing a firefly luciferase reporter gene under the control of the SRE or NFAT-RE allows the monitoring of ERK1/2 activation and Ca 2+ mobilization, respectively, by measuring luminescence. In a dual luciferase assay, firefly luminescence is normalized by co-transfecting an internal control vector, which contains a constitutively active promoter driving the expression of a second luciferase, namely, Renilla luciferase, whose activity can be quantified within the same sample. Here, a protocol for the analysis of CaSR signaling using dual luciferase assays in HEK293 cells is provided. The assays can, for example, be used to investigate functional consequences of mutations in the CaSR gene., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Luciferase reporter systems are commonly used in scientific research to investigate a variety of biological processes, including antiviral innate immunity. These systems employ the use of luciferase enzymes derived from organisms such as fireflies or renilla reniformis, which emit light upon reaction with a substrate. In the context of antiviral innate immunity, the luciferase reporter systems offer a noninvasive and highly sensitive approach for real-time monitoring of immune responses in vitro and in vivo, enabling researchers to delve into the intricate interactions and signaling pathways involved in host-virus dynamic interactions. Here, we describe the methods of the promoter-luciferase reporter and enhancer-luciferase reporter, which provide insights into the transcriptional and post-transcriptional regulation of antiviral innate immunity. Additionally, we outline the split-luciferase complementary reporter method, which was designed to explore protein-protein interactions associated with antiviral immunity. These methodologies offer invaluable knowledge regarding the molecular mechanisms underlying antiviral immune pathways and have the potential to support the development of effective antiviral therapies., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The classic dual luciferase reporter assay has been widely used to rapidly and accurately determine the transcriptional activity of a given promoter induced by certain signal pathways in the cells. In particular, the sensitive characteristics of luciferase highlight its significance in many experiments, such as weak promoter analysis, transfection studies using small amounts of DNA, and detection in cell lines with low transfection efficiency. This chapter presents detailed information and experimental procedures for measuring interferon (IFN)-induced Interferon-Stimulated Response Element (ISRE) promoter activity using the dual luciferase reporter assay., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

RNA virus polymerases carry out multiple functions necessary for successful genome replication and transcription. A key tool for molecular studies of viral RNA-dependent RNA polymerases (RdRps) is a 'minigenome' or 'minireplicon' assay, in which viral RdRps are reconstituted in cells in the absence of full virus infection. Typically, plasmids expressing the viral polymerase protein(s) and other co-factors are co-transfected, along with a plasmid expressing an RNA encoding a fluorescent or luminescent reporter gene flanked by viral untranslated regions containing cis -acting elements required for viral RdRp recognition. This reconstitutes the viral transcription/replication machinery and allows the viral RdRp activity to be measured as a correlate of the reporter protein signal. Here, we report on the development of a 'first-generation' plasmid-based minigenome assay for species A rotavirus using a firefly luciferase reporter gene.

The significant advances in the differentiation of human pluripotent stem (hPS) cells into pancreatic endocrine cells, including functional β-cells, have been based on a detailed understanding of the underlying developmental mechanisms. However, the final differentiation steps, leading from endocrine progenitors to mono-hormonal and mature pancreatic endocrine cells, remain to be fully understood and this is reflected in the remaining shortcomings of the hPS cell-derived islet cells (SC-islet cells), which include a lack of β-cell maturation and variability among different cell lines. Additional signals and modifications of the final differentiation steps will have to be assessed in a combinatorial manner to address the remaining issues and appropriate reporter lines would be useful in this undertaking. Here we report the generation and functional validation of hPS cell reporter lines that can monitor the generation of INS + and GCG + cells and their resolution into mono-hormonal cells (INS eGFP , INS eGFP /GCG mCHERRY ) as well as β-cell maturation (INS eGFP /MAFA mCHERRY ) and function (INS GCaMP6 ). The reporter hPS cell lines maintained strong and widespread expression of pluripotency markers and differentiated efficiently into definitive endoderm and pancreatic progenitor (PP) cells. PP cells from all lines differentiated efficiently into islet cell clusters that robustly expressed the corresponding reporters and contained glucose-responsive, insulin-producing cells. To demonstrate the applicability of these hPS cell reporter lines in a high-content live imaging approach for the identification of optimal differentiation conditions, we adapted our differentiation procedure to generate SC-islet clusters in microwells. This allowed the live confocal imaging of multiple SC-islets for a single condition and, using this approach, we found that the use of the N21 supplement in the last stage of the differentiation increased the number of monohormonal β-cells without affecting the number of α-cells in the SC-islets. The hPS cell reporter lines and the high-content live imaging approach described here will enable the efficient assessment of multiple conditions for the optimal differentiation and maturation of SC-islets., (© 2024. The Author(s).)

To investigate the impact of different 5' untranslated regions (UTRs) on mRNA vaccine translation efficiency, five dual-reporter gene expression plasmids with different 5'UTRs were constructed. The corresponding mRNA transcripts were transcribed and capped in vitro. By comparing the expression levels of reporter genes with different 5'UTRs, we identified the 5'UTR associated with the highest expression level. Subsequently, HIVgp145 mRNA vaccines containing various 5'UTRs were constructed and verified. The results demonstrated that mRNA 3 (β-globin 5'UTR) displayed the greatest number of green fluorescence-positive cells and the highest luciferase fluorescence intensity in the reporter gene expression system. Further, among the HIVgp145 mRNA vaccines with different 5'UTRs, mRNA 7 (β-globin 5'UTR) exhibited the highest level of expression. These findings indicate that it is feasible to use the 5'UTR of β-globin in an mRNA vaccine, laying the foundation for animal immunogenicity testing., (© 2024. The Author(s).)

Mechanistic investigations are of paramount importance in elucidating the modes of action of antibiotics and facilitating the discovery of novel drugs. We reported a luciferase-based reporter system using bacterial cells to unveil mechanisms of antimicrobials targeting transcription and translation. The reporter gene Nluc encoding NanoLuciferase (NanoLuc) was integrated into the genome of the Gram-positive model organism, Bacillus subtilis , to generate a reporter strain BS2019. Cellular transcription and translation levels were assessed by quantifying the amount of Nluc mRNA as well as the luminescence catalyzed by the enzyme NanoLuc. We validated this system using three known inhibitors of transcription (rifampicin), translation (chloramphenicol), and cell wall synthesis (ampicillin). The B. subtilis reporter strain BS2019 successfully revealed a decline in Nluc expression by rifampicin and NanoLuc enzyme activity by chloramphenicol, while ampicillin produced no observable effect. The assay was employed to characterize a previously discovered bacterial transcription inhibitor, CUHK242, with known antimicrobial activity against drug-resistant Staphylococcus aureus . Production of Nluc mRNA in our reporter BS2019 was suppressed in the presence of CUHK242, demonstrating the usefulness of the construct, which provides a simple way to study the mechanism of potential antibiotic candidates at early stages of drug discovery. The reporter system can also be modified by adopting different promoters and reporter genes to extend its scope of contribution to other fields of work., Importance: Discovering new classes of antibiotics is desperately needed to combat the emergence of multidrug-resistant pathogens. To facilitate the drug discovery process, a simple cell-based assay for mechanistic studies is essential to characterize antimicrobial candidates. In this work, we developed a luciferase-based reporter system to quantify the transcriptional and translational effects of potential compounds and validated our system using two currently marketed drugs. Reporter strains generated in this study provide readily available means for identifying bacterial transcription inhibitors as prospective novel antibacterials. We also provided a series of plasmids for characterizing promoters under various conditions such as stress., Competing Interests: The authors declare no conflict of interest.

Replicons, derived from RNA viruses, are genetic constructs retaining essential viral enzyme genes while lacking key structural protein genes. Upon introduction into cells, the genes carried by the replicon RNA are expressed, and the RNA self-replicates, yet viral particle production does not take place. Typically, RNA replicons are transcribed in vitro and are then electroporated in cells. However, it would be advantageous for the replicon to be generated in cells following DNA transfection instead of RNA. In this study, a bacterial artificial chromosome (BAC) DNA encoding a SARS-CoV-2 replicon under control of a T7 promoter was transfected into HEK293T cells engineered to functionally express the T7 RNA polymerase (T7 RNAP). Upon transfection of the BAC DNA, we observed low, but reproducible expression of reporter proteins GFP and luciferase carried by this replicon. Expression of the reporter proteins required linearization of the BAC DNA prior to transfection. Moreover, expression occurred independently of T7 RNAP. Gene expression was also insensitive to remdesivir treatment, suggesting that it did not involve self-replication of replicon RNA. Similar results were obtained in highly SARS-CoV-2 infection-permissive Calu-3 cells. Strikingly, prior expression of the SARS-CoV-2 N protein boosted expression from transfected SARS-CoV-2 RNA replicon but not from the replicon BAC DNA. In conclusion, transfection of a large DNA encoding a coronaviral replicon led to reproducible replicon gene expression through an unidentified mechanism. These findings highlight a novel pathway toward replicon gene expression from transfected replicon cDNA, offering valuable insights for the development of methods for DNA-based RNA replicon applications., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Friedhoff et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A luciferase reporter assay is a cell-based, enzymatic experiment that utilizes an ectopically expressed luciferase in cultured cells or in live animals, with the presence of its substrate luciferin, to generate luminescence in response to gene regulation at the level of transcription. This assay can be easily formatted for high-throughput sample analysis. The reagents are commercially available and routinely used in various applications. We have automated a luciferase reporter assay on the Opentrons OT-2 platform to measure activation of NF-kB in HeLa cells. The Python script-based protocols were developed to perform cell seeding, reporter construct transfection, and enzyme-catalytic reaction to produce detectable signals for quantification with desirable quality of sample handling and minimal hands-on time., Competing Interests: Declaration of competing interests The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Boren Lin and Kinnari Watson are currently employees of Opentrons Labworks Inc, (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Human epidermal growth factor receptor 2 (HER2) is a key player in the pathogenesis and progression of breast cancer and is currently a primary target for breast cancer immunotherapy. Bioactivity determination is necessary to guarantee the safety and efficacy of therapeutic antibodies targeting HER2. Nevertheless, currently available bioassays for measuring the bioactivity of anti-HER2 mAbs are either not representative or have high variability. Here, we established a reliable reporter gene assay (RGA) based on T47D-SRE-Luc cell line that expresses endogenous HER2 and luciferase controlled by serum response element (SRE) to measure the bioactivity of anti-HER2 antibodies. Neuregulin-1 (NRG-1) can lead to the heterodimerization of HER2 on the cell membrane and induce the expression of downstream SRE-controlled luciferase, while pertuzumab can dose-dependently reverse the reaction, resulting in a good dose-response curve reflecting the activity of the antibody. After optimizing the relevant assay parameters, the established RGA was fully validated based on ICH-Q2 (R1), which demonstrated that the method had excellent specificity, accuracy, precision, linearity, and stability. In summary, this robust and innovative bioactivity determination assay can be applied in the development and screening, release control, biosimilar assessment and stability studies of anti-HER2 mAbs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Genetically encoded reporters for magnetic resonance imaging (MRI) offer a valuable technology for making molecular-scale measurements of biological processes within living organisms with high anatomical resolution and whole-organ coverage without relying on ionizing radiation. However, most MRI reporters rely on synthetic contrast agents, typically paramagnetic metals and metal complexes, which often need to be supplemented exogenously to create optimal contrast. To eliminate the need for synthetic contrast agents, we previously introduced aquaporin-1, a mammalian water channel, as a new reporter gene for the fully autonomous detection of genetically labeled cells using diffusion-weighted MRI. In this study, we aimed to expand the toolbox of diffusion-based genetic reporters by modulating aquaporin membrane trafficking and harnessing the evolutionary diversity of water channels across species. We identified a number of new water channels that functioned as diffusion-weighted reporter genes. In addition, we show that loss-of-function variants of yeast and human aquaporins can be leveraged to design first-in-class diffusion-based sensors for detecting the activity of a model protease within living cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

TFEB and TFE3 belong to the MiT/TFE family of transcription factors that bind identical DNA responsive elements in the regulatory regions of target genes. They are involved in regulating lysosomal biogenesis, function, exocytosis, autophagy, and lipid catabolism. Precise control of TFEB and TFE3 activity is crucial for processes such as senescence, stress response, energy metabolism, and cellular catabolism. Dysregulation of these factors is implicated in various diseases, thus researchers have explored pharmacological approaches to modulate MiT/TFE activity, considering these transcription factors as potential therapeutic targets. However, the physiological complexity of their functions and the lack of suitable in vivo tools have limited the development of selective MiT/TFE modulating agents. Here, we have created a reporter-based biosensor, named CLEARoptimized, facilitating the pharmacological profiling of TFEB- and TFE3-mediated transcription. This innovative tool enables the measurement of TFEB and TFE3 activity in living cells and mice through imaging and biochemical techniques. CLEARoptimized consists of a promoter with six coordinated lysosomal expression and regulation motifs identified through an in-depth bioinformatic analysis of the promoters of 128 TFEB-target genes. The biosensor drives the expression of luciferase and tdTomato reporter genes, allowing the quantification of TFEB and TFE3 activity in cells and in animals through optical imaging and biochemical assays. The biosensor's validity was confirmed by modulating MiT/TFE activity in both cell culture and reporter mice using physiological and pharmacological stimuli. Overall, this study introduces an innovative tool for studying autophagy and lysosomal pathway modulation at various biological levels, from individual cells to the entire organism. Abbreviations: CLEAR: coordinated lysosomal expression and regulation; MAR: matrix attachment regions; MiT: microphthalmia-associated transcription factor; ROI: region of interest; TBS: tris-buffered saline; TF: transcription factor; TFE3: transcription factor binding to IGHM enhancer 3; TFEB: transcription factor EB; TH: tyrosine hydroxylase; TK: thymidine kinase; TSS: transcription start site.

Multimodality reporter gene imaging combines the sensitivity, resolution and translational potential of two or more signals. The approach has not been widely adopted by the animal imaging community, mainly because its utility in this area is unproven. We developed a new complementation-based reporter gene system where the large component of split NanoLuc luciferase (LgBiT) presented on the surface of cells (TM-LgBiT) interacts with a radiotracer consisting of the high-affinity complementary HiBiT peptide labeled with a radionuclide. Radiotracer uptake could be imaged in mice using SPECT/CT and bioluminescence within two hours of implanting reporter-gene-expressing cells. Imaging data were validated by ex vivo biodistribution studies. Following the demonstration of complementation between the TM-LgBiT protein and HiBiT radiotracer, we validated the use of the technology in the highly specific in vivo multimodal imaging of cells. These findings highlight the potential of this new approach to facilitate the advancement of cell and gene therapies from bench to clinic.

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., (© 2024. Crown.)

Animal models of authentic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection require operation in biosafety level 3 (BSL-3) containment. In the present study, we established a mouse model employing a single-cycle infectious virus replicon particle (VRP) system of SARS-CoV-2 that can be safely handled in BSL-2 laboratories. The VRP [ΔS-VRP(G)-Luc] contains a SARS-CoV-2 genome in which the spike gene was replaced by a firefly luciferase (Fluc) reporter gene (Rep-Luci), and incorporates the vesicular stomatitis virus glycoprotein on the surface. Intranasal inoculation of ΔS-VRP(G)-Luc can successfully transduce the Rep-Luci genome into mouse lungs, initiating self-replication of Rep-Luci and, accordingly, inducing acute lung injury mimicking the authentic SARS-CoV-2 pathology. In addition, the reporter Fluc expression can be monitored using a bioluminescence imaging approach, allowing a rapid and convenient determination of viral replication in ΔS-VRP(G)-Luc-infected mouse lungs. Upon treatment with an approved anti-SARS-CoV-2 drug, VV116, the viral replication in infected mouse lungs was significantly reduced, suggesting that the animal model is feasible for antiviral evaluation. In summary, we have developed a BSL-2-compliant mouse model of SARS-CoV-2 infection, providing an advanced approach to study aspects of the viral pathogenesis, viral-host interactions, as well as the efficacy of antiviral therapeutics in the future.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly contagious and pathogenic in humans; thus, research on authentic SARS-CoV-2 has been restricted to biosafety level 3 (BSL-3) laboratories. However, due to the scarcity of BSL-3 facilities and trained personnel, the participation of a broad scientific community in SARS-CoV-2 research had been greatly limited, hindering the advancement of our understanding on the basic virology as well as the urgently necessitated drug development. Previously, our colleagues Jin et al. had generated a SARS-CoV-2 replicon by replacing the essential spike gene in the viral genome with a Fluc reporter (Rep-Luci), which can be safely operated under BSL-2 conditions. By incorporating the Rep-Luci into viral replicon particles carrying vesicular stomatitis virus glycoprotein on their surface, and via intranasal inoculation, we successfully transduced the Rep-Luci into mouse lungs, developing a mouse model mimicking SARS-CoV-2 infection. Our model can serve as a useful platform for SARS-CoV-2 pathological studies and antiviral evaluation under BSL2 containment., Competing Interests: The authors declare no conflict of interest.

A major challenge in the fields of biological imaging and synthetic biology is noninvasively visualizing the functions of natural and engineered cells inside opaque samples such as living animals. One promising technology that addresses this limitation is ultrasound (US), with its penetration depth of several cm and spatial resolution on the order of 100 μm. Within the past decade, reporter genes for US have been introduced and engineered to link cellular functions to US signals via heterologous expression in commensal bacteria and mammalian cells. These acoustic reporter genes (ARGs) represent a novel class of genetically encoded US contrast agent, and are based on air-filled protein nanostructures called gas vesicles (GVs). Just as the discovery of fluorescent proteins was followed by the improvement and diversification of their optical properties through directed evolution, here we describe the evolution of GVs as acoustic reporters. To accomplish this task, we establish high-throughput, semiautomated acoustic screening of ARGs in bacterial cultures and use it to screen mutant libraries for variants with increased nonlinear US scattering. Starting with scanning site saturation libraries for two homologues of the primary GV structural protein, GvpA/B, two rounds of evolution resulted in GV variants with 5- and 14-fold stronger acoustic signals than the parent proteins. We anticipate that this and similar approaches will help high-throughput protein engineering play as large a role in the development of acoustic biomolecules as it has for their fluorescent counterparts.

DDX3X regulates the translation of a subset of human transcripts containing complex 5' untranslated regions (5' UTRs). In this study, we developed the helicase activity reporter for translation (HART), which uses DDX3X-sensitive 5' UTRs to measure DDX3X-mediated translational activity in cells. To directly measure RNA structure in DDX3X-dependent mRNAs, we used SHAPE-MaP to determine the secondary structures present in DDX3X-sensitive 5' UTRs and then used HART to investigate how sequence alterations influence DDX3X sensitivity. Additionally, we identified residues 38-44 as potential mediators of DDX3X's interaction with the translational machinery. HART revealed that both DDX3X's association with the translational machinery and its helicase activity are required for its function in promoting the translation of DDX3X-sensitive 5' UTRs. These findings suggest DDX3X plays a crucial role in regulating translation through its interaction with the translational machinery during ribosome scanning and establish the HART reporter as a robust, lentivirally encoded, colorimetric measurement of DDX3X-dependent translation in cells., (© 2024 Wilkins et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Introduction: Screening for effective antiviral compounds from traditional Mongolian medicine not only aids in the research of antiviral mechanisms of traditional medicines, but is also of significant importance for the development of new antiviral drugs targeting influenza A virus. Our study aimed to establish high-throughput, rapid screening methods for antiviral compounds against influenza A virus from abundant resources of Mongolian medicine., Methods: The use of GFP-based reporter viruses plays a pivotal role in antiviral drugs screening by enabling rapid and precise identification of compounds that inhibit viral replication. Herein, a GFP-based reporter influenza A virus was used to identify potent anti-influenza compounds within traditional Mongolian medicine., Results: Our study led to the discovery of three active compounds: Cardamonin, Curcumin, and Kaempferide, all of which exhibited significant antiviral properties in vitro . Subsequent analysis confirmed that their effectiveness was largely due to the stimulation of the antiviral signaling pathways of host cells, rather than direct interference with the viral components, such as the viral polymerase., Discussion: This study showcased the use of GFP-based reporter viruses in high-throughput screening to unearth antiviral agents from traditional Mongolian medicine, which contains rich antiviral compounds and deserves further exploration. Despite certain limitations, fluorescent reporter viruses present substantial potential for antiviral drug screening research due to their high throughput and efficiency., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Nie, Li, Zhang, Zeng, Cai, Peng, Jiang, Shi and Li.)

Increasing evidence suggests that the muscle stem cell (MuSC) pool is heterogeneous. In particular, a rare subset of PAX7-positive MuSCs that has never expressed the myogenic regulatory factor MYF5 displays unique self-renewal and engraftment characteristics. However, the scarcity and limited availability of protein markers make the characterization of these cells challenging. Here, we describe the generation of StemRep reporter mice enabling the monitoring of PAX7 and MYF5 proteins based on equimolar levels of dual nuclear fluorescence. High levels of PAX7 protein and low levels of MYF5 delineate a deeply quiescent MuSC subpopulation with an increased capacity for asymmetric division and distinct dynamics of activation, proliferation, and commitment. Aging primarily reduces the MYF5 Low MuSCs and skews the stem cell pool toward MYF5 High cells with lower quiescence and self-renewal potential. Altogether, we establish the StemRep model as a versatile tool to study MuSC heterogeneity and broaden our understanding of mechanisms regulating MuSC quiescence and self-renewal in homeostatic, regenerating, and aged muscles., Competing Interests: Declaration of interests Except S.C.S., all authors are or were employees of Société des Produits Nestlé SA., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Positron emission tomography (PET) reporter systems are a valuable means of estimating the level of expression of a transgene in vivo. For example, the safety and efficacy of gene therapy approaches for the treatment of neurological and neuropsychiatric disorders could be enhanced via the monitoring of exogenous gene expression levels in the brain. The present study evaluated the ability of a newly developed PET reporter system [ 18 F]fluoroestradiol ([ 18 F]FES) and the estrogen receptor-based PET reporter ChRERα, to monitor expression levels of a small hairpin RNA (shRNA) designed to suppress choline acetyltransferase (ChAT) expression in rhesus monkey brain. The ChRERα gene and shRNA were expressed from the same transcript via lentivirus injected into monkey striatum. In two monkeys that received injections of viral vector, [ 18 F]FES binding increased by 70% and 86% at the target sites compared with pre-injection, demonstrating that ChRERα expression could be visualized in vivo with PET imaging. Post-mortem immunohistochemistry confirmed that ChAT expression was significantly suppressed in regions in which [ 18 F]FES uptake was increased. The consistency between PET imaging and immunohistochemical results suggests that [ 18 F]FES and ChRERα can serve as a PET reporter system in rhesus monkey brain for in vivo evaluation of the expression of potential therapeutic agents, such as shRNAs., Competing Interests: Declaration of interests M.M. has received research funding from AstraZeneca, Kriya (Redpin) Therapeutics, Dompé farmaceutici, and Attune Neurosciences, and is named as an inventor on a patent describing novel DREADD ligands (WO2019/157083) and a U.S. provisional patent application describing novel fluorinated mu opioid receptor agonists (63/452,879)., (Published by Elsevier Inc.)

Iron scavenging is required for full virulence of mycobacterial pathogens. During infection, the host immune response restricts mycobacterial access to iron, which is essential for bacterial respiration and DNA synthesis. The Mycobacterium tuberculosis iron-dependent regulator (IdeR) responds to changes in iron accessibility by repressing iron-uptake genes when iron is available. In contrast, iron-uptake gene transcription is induced when iron is depleted. The ideR gene is essential in M. tuberculosis and is required for bacterial growth. To further study how iron regulates transcription, wee developed an iron responsive reporter system that relies on an IdeR-regulated promoter to drive Cre and loxP mediated recombination in Mycobacterium smegmatis . Recombination leads to the expression of an antibiotic resistance gene so that mutations that activate the IdeR-regulated promoter can be selected. A transposon library in the background of this reporter system was exposed to media containing iron and hemin, and this resulted in the selection of mutants in the antioxidant mycothiol synthesis pathway. We validated that inactivation of the mycothiol synthesis gene mshA results in increased recombination and increased IdeR-regulated promoter activity in the reporter system. Further, we show that vitamin C, which has been shown to oxidize iron through the Fenton reaction, can decrease promoter activity in the mshA mutant. We conclude that the intracellular redox state balanced by mycothiol can alter IdeR activity in the presence of iron.IMPORTANCE Mycobacterium smegmatis is a tractable organism to study mycobacterial gene regulation. We used M. smegmatis to construct a novel recombination-based reporter system that allows for the selection of mutations that deregulate a promoter of interest. Transposon mutagenesis and insertion sequencing (TnSeq) in the recombination reporter strain identified genes that impact iron regulated promoter activity in mycobacteria. We found that the mycothiol synthesis gene mshA is required for IdeR mediated transcriptional regulation by maintaining intracellular redox balance. By affecting the oxidative state of the intracellular environment, mycothiol can modulate iron-dependent transcriptional activity. Taken more broadly, this novel reporter system can be used in combination with transposon mutagenesis to identify genes that are required by Mycobacterium tuberculosis to overcome temporary or local changes in iron availability during infection., Competing Interests: The authors declare no conflict of interest.

The ability to regulate the expression of genes is a central tool for the characterization of fungal genes. This is of particular interest to study genes required for specific processes or the effect of genes expressed only under specific conditions. Saccharomycopsis species show a unique property of necrotrophic mycoparasitism that is activated upon starvation. Here we describe the use of the MET17 promoter of S. schoenii as a tool to regulate gene expression based on the availability of methionine. Conditional expression was tested using lacZ and GFP reporter genes. Gene expression could be strongly down-regulated by the addition of methionine or cysteine to the growth medium and upregulated by starvation for methionine. We used X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) to detect lacZ-expression in plate assays and ONPG (ortho-nitrophenyl-β-galactopyranoside) as a substrate for β-galactosidase in liquid-phase assays. For in vivo expression analyses we used fluorescence microscopy for the detection and localization of a MET17-driven histone H4-GFP reporter gene. With these assays we demonstrated the usefulness of the MET17 promoter to regulate expression of genes based on methionine availability. In silico analyses revealed similar promoter motifs as found in MET3 genes of Saccharomyces cerevisiae and Ashbya gossypii. This suggests a regulation of the MET17 promoter by CBF1 and MET31/MET32 in conjunction with the transcriptional activator MET4, which were also identified in the S. schoenii genome., (© 2024. The Author(s).)

The topology of the transcription factor network (TFN) of Escherichia coli is far from uniform, with 22 global regulator (GR) proteins controlling one-third of all genes. So far, their production rates cannot be tracked by comparable fluorescent proteins. We developed a library of fluorescent reporters for 16 GRs for this purpose. Each consists of a single-copy plasmid coding for green fluorescent protein (GFP) fused to the full-length copy of the native promoter. We tracked their activity in exponential and stationary growth, as well as under weak and strong stresses. We show that the reporters have high sensitivity and specificity to all stresses tested and detect single-cell variability in transcription rates. Given the influence of GRs on the TFN, we expect that the new library will contribute to dissecting global transcriptional stress-response programs of E. coli . Moreover, the library can be invaluable in bioindustrial applications that tune those programs to, instead of cell growth, favor productivity while reducing energy consumption.IMPORTANCECells contain thousands of genes. Many genes are involved in the control of cellular activities. Some activities require a few hundred genes to run largely synchronous transcriptional programs. To achieve this, cells have evolved global regulator (GR) proteins that can influence hundreds of genes simultaneously. We have engineered a library of Escherichia coli strains to track the levels over time of these, phenotypically critical, GRs. Each strain has a single-copy plasmid coding for a fast-maturing green fluorescent protein whose transcription is controlled by a copy of the natural GR promoter. By allowing the tracking of GR levels, with sensitivity and specificity, this library should become of wide use in scientific research on bacterial gene expression (from molecular to synthetic biology) and, later, be used in applications in therapeutics and bioindustries., Competing Interests: The authors declare no conflict of interest.

Developmental signaling pathways associated with growth factors such as TGFb are commonly dysregulated in melanoma. Here we identified a human TGFb enhancer specifically activated in melanoma cells treated with TGFB1 ligand. We generated stable transgenic zebrafish with this TGFb Induced Enhancer driving green fluorescent protein ( TIE:EGFP ). TIE:EGFP was not expressed in normal melanocytes or early melanomas but was expressed in spatially distinct regions of advanced melanomas. Single-cell RNA-sequencing revealed that TIE:EGFP + melanoma cells down-regulated interferon response while up-regulating a novel set of chronic TGFb target genes. ChIP-sequencing demonstrated that AP-1 factor binding is required for activation of chronic TGFb response. Overexpression of SATB2 , a chromatin remodeler associated with tumor spreading, showed activation of TGFb signaling in early melanomas. Confocal imaging and flow cytometric analysis showed that macrophages localize to TIE:EGFP + regions and preferentially phagocytose TIE:EGFP + melanoma cells compared to TIE:EGFP - melanoma cells. This work identifies a TGFb induced immune response and demonstrates the need for the development of chronic TGFb biomarkers to predict patient response to TGFb inhibitors., Competing Interests: HN, AT, JB, MX, CB, SY, KK, AM No competing interests declared, LZ L.I.Z. is a founder and stockholder of Fate Therapeutics, CAMP4 Therapeutics, Amagma Therapeutics, Scholar Rock, and Branch Biosciences. He is a consultant for Celularity and Cellarity, (© 2024, Noonan, Thornock et al.)

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.

Senecavirus A (SVA) is a newly identified picornavirus associated with swine vesicular disease and neonatal mortality. The development of an SVA incorporating an exogenous reporter gene provides a powerful tool for viral research. In this study, we successfully constructed a recombinant SVA expressing Gaussia Luciferase (Gluc), termed rSVA-Gluc. The growth kinetics of rSVA-Gluc in BHK-21 cells were found to be comparable to those of the parental virus, and Gluc activity paralleled the virus growth curve. Genetic analysis revealed stable inheritance of the inserted reporter protein genes for at least six generations. We evaluated the utility of rSVA-Gluc in antiviral drug screening, and the results highlighted its potential as an effective tool for such purposes against SVA. DATA AVAILABILITY STATEMENT: The data that support the findings of this study are available on request from the corresponding author., Competing Interests: Declaration of Competing Interest The research was conducted in the absence of any commercial or financial relationships. The authors declare that they have no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still epidemic around the world. The manipulation of SARS-CoV-2 is restricted to biosafety level 3 laboratories (BSL-3). In this study, we developed a SARS-CoV-2 ΔN-GFP-HiBiT replicon delivery particles (RDPs) encoding a dual reporter gene, GFP-HiBiT, capable of producing both GFP signal and luciferase activities. Through optimal selection of the reporter gene, GFP-HiBiT demonstrated superior stability and convenience for antiviral evaluation. Additionally, we established a RDP infection mouse model by delivering the N gene into K18-hACE2 KI mouse through lentivirus. This mouse model supports RDP replication and can be utilized for in vivo antiviral evaluations. In summary, the RDP system serves as a valuable tool for efficient antiviral screening and studying the gene function of SARS-CoV-2. Importantly, this system can be manipulated in BSL-2 laboratories, decreasing the threshold of experimental requirements., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest., (Copyright © 2024 The Authors. Publishing services by Elsevier B.V. All rights reserved.)