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Luciferase is one of the bioluminescence-producing agents, which was widely used as a reporter enzyme for constructing bioassay systems to study gene expression with high accuracy and within a broad dynamic spectrum. Perception of pathogen associated molecular patterns (PAMPs) in plants often lead to significant transcriptional changes. The transcriptional changes of defense-related genes are often used as a marker to assay PAMP-triggered plant immune response. In this study, we showed that the marker gene CYP71D20 was rapidly activated in Nicotiana benthamiana upon treatment with the bacterial PAMP flg22 and the Phytophthora elicitin INF1. In addition, we generated transgenic N. benthamiana using the luciferase as a reporter gene to analyze CYP71D20 gene expression upon PAMP treatment. The transgenic line carrying the luciferase gene driven by CYP71D20 promoter was treated with the bacterial PAMP flg22 or Phytophthora elicitin INF1. Transcriptional activation of CYP71D20 was measured by monitoring the luciferase activity. The results showed that the LUC activity was increased after treatment with different PAMPs, indicating that the CYP71D20 -promotor luciferase assay can be used to study the PAMP-triggered gene expression in N. benthamiana .

Objective: Various plants have been reported to contain compounds that promote the transcriptional activity of Nuclear factor erythroid 2-related factor 2 (Nrf2) to induce a set of xenobiotic detoxifying enzymes, such as NAD(P)H-quinone acceptor oxidoreductase 1 (NQO1), via the antioxidant response element (ARE). While conventional methods for evaluating Nrf2 induction potency include measurement of NQO1 activity, an ARE luciferase reporter assay was recently developed to specifically assess Nrf2 induction potency of compounds of interest. In this study, we compared the abilities of these two assays to evaluate and determine Nrf2 induction potency of plant-derived compounds., Results: Although the compounds exhibited a high degree of consistency between assays, several compounds did not. These results suggest that although the NQO1 assay can be used as an evaluation method to estimate Nrf2 induction potency of a compound, an ARE luciferase reporter approach may offer greater precision. In summary, inconsistency in Nrf2 induction potency evaluated by the reporter and NQO1 assays of plant-derived compounds, including resveratrol, may be due to a variety of factors that may regulate NQO1 gene expression other than Nrf2 and/or directly modulate NQO1 enzymatic activity or protein levels, with each compound having a different degree of effect on these factors., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Inducible promoters are essential for precise control of target gene expression in synthetic biological systems. However, engineering eukaryotic promoters is often more challenging than engineering prokaryotic promoters due to their greater mechanistic complexity. In this study, we describe a simple and reliable approach for constructing strongly inducible synthetic promoters with minimum leakiness in yeasts. The results indicate that the leakiness of yeast-inducible synthetic promoters is primarily the result of cryptic transcriptional activation of heterologous sequences that may be avoided by appropriate insulation and operator mutagenesis. Our promoter design approach has successfully generated robust, inducible promoters that achieve a > 10 3 -fold induction in reporter gene expression. The utility of these promoters is demonstrated by using them to produce various biologics with titers up to 2 g/L, including antigens designed to raise specific antibodies against a SARS-CoV-2 omicron variant through chicken immunization., Competing Interests: Competing interests: M.S. and Y.Shoya are employees of Pharma Foods International Co. Ltd. M.T., K.N., A.K., and J.I. are inventors of pending patent applications submitted by Kobe University that cover the inducible synthetic promoters described in this study (PCT/JP2021/044573, PCT/JP2021/044574, and JP2020202234). Y.I. is the inventor of one of the above pending patent applications (PCT/JP2021/044573). M.T., Y.I., M.M.-K., M.S., Y.Shoya., A.K., and J.I. are inventors of the pending patent application submitted by Kobe University and Pharma Foods International Co. Ltd. that covers the IgY antibody obtained in this study (JP2023188697). The other authors declare no competing interests., (© 2024. The Author(s).)

Incidences of fluconazole (FLC) resistance among Candida glabrata clinical isolates are a growing issue in clinics. The pleiotropic drug response network in C. glabrata confers azole resistance and is defined primarily by the Zn 2 Cys 6 zinc cluster-containing transcription factor Pdr1 and target genes such as CDR1 , which encodes an ATP-binding cassette transporter protein thought to act as an FLC efflux pump. Mutations in the PDR1 gene that render the transcription factor hyperactive are the most common cause of fluconazole resistance among clinical isolates. The phenothiazine class drug fluphenazine and a molecular derivative, CWHM-974, which both exhibit antifungal properties, have been shown to induce the expression of Cdr1 in Candida spp. We have used a firefly luciferase reporter gene driven by the CDR1 promoter to demonstrate two distinct patterns of CDR1 promoter activation kinetics: gradual promoter activation kinetics that occur in response to ergosterol limitations imposed by exposure to azole and polyene class antifungals and a robust and rapid CDR1 induction occurring in response to the stress imposed by fluphenazines. We can attribute these different patterns of CDR1 induction as proceeding through the promoter region of this gene since this is the only segment of the gene included in the luciferase reporter construct. Genetic analysis indicates that the signaling pathways responsible for phenothiazine and azole induction of CDR1 overlap but are not identical. The short time course of phenothiazine induction suggests that these compounds may act more directly on the Pdr1 protein to stimulate its activity., Importance: Candida glabrata has emerged as the second-leading cause of candidiasis due, in part, to its ability to acquire high-level resistance to azole drugs, a major class of antifungal that acts to block the biosynthesis of the fungal sterol ergosterol. The presence of azole drugs causes the induction of a variety of genes involved in controlling susceptibility to this drug class, including drug transporters and ergosterol biosynthetic genes such as ERG11. We found that the presence of azole drugs leads to an induction of genes encoding drug transporters and ERG11, while exposure of C. glabrata cells to antifungals of the phenothiazine class of drugs caused a much faster and larger induction of drug transporters but not ERG11. Coupled with further genetic analyses of the effects of azole and phenothiazine drugs, our data indicate that these compounds are sensed and responded to differentially in the yeast cell., Competing Interests: The authors declare no conflict of interest.

Feline herpesvirus type 1 (FHV-1), a member of the Herpesviridae family, is one of the most important pathogens that causes upper respiratory tract disease in felines. Following infection, FHV-1 can spread retrogradely to the trigeminal ganglia, establishing a life-long latency. Although vaccines are available for routine feline vaccination, FHV-1 is still an agent that poses a serious threat to feline health. There are currently no specific drugs for the treatment of FHV-1. To facilitate the screening of antiviral drugs, we constructed a reporter FHV-1 virus, which expresses a secreted Gaussia luciferase (GLuc) and a bright green fluorescent protein, mNeonGreen. The reporter virus shows slower growth than does the wild-type FHV-1. The expression of the two reporter genes, Gluc and mNeonGreen, was consistent with viral propagation and remained stable during continuous passage in CRFK cells, even after twenty rounds. In addition, the known inhibitor ganciclovir was used to confirm the characteristics of the reporter virus for drug screening. We found that the reporter FHV-1 is suitable for antiviral screening assays. Overall, our work provides a useful tool for screening drugs to combat FHV-1., Competing Interests: Declarations. Competing interests: The authors declare that they have no competing interests., (© 2024. The Author(s).)

The cellular nonsense-mediated decay (NMD) pathway recognizes and degrades mRNAs with unusual structural features, such as long 3' UTRs or overlapping reading frames, and therefore serves as a transcript quality control mechanism. A broad spectrum of today's knowledge about the nonsense-mediated mRNA decay pathway has been discovered using NMD reporter systems, mostly consisting of multiple exons, with a wild-type and a premature termination codon-containing variant. In a preliminary NMD study, we used the seven-exon triose phosphate isomerase (TPI) reporter and observed that in this well-known NMD reporter, surprisingly, not all splice sites are used constitutively, but additional cryptic splice sites are used. As this is more frequently observed in the construction of minigenes, especially when unknown splicing regulatory elements (SREs) are removed, for example, by shortening introns, this may affect the reliability of such reporters. To demonstrate how such minigenes can be improved in general with respect to constitutive splice site recognition, we restored an intron length in the TPI reporter or made bioinformatic adjustments to SREs or intrinsic strength of the splice sites themselves. As a result, this NMD reporter could be made more robust and specific for the evaluation of NMD sensitivity within a single transcript. The modifications of the TPI reporter shown here as examples can generally be used for the transfer of cellular multiexon transcripts to minigenes., (© 2025 Peter et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

IFN-β is a potent antiviral cytokine and the first member of the type I IFN family of cytokines to be induced during the antiviral response. IFN-β plays an essential protective role in host defense against virus infections, as well as a pathogenic role in numerous autoimmune and autoinflammatory disorders. However, contemporary tools to study the induction, kinetics, and behavior of IFN-β are lacking. In this study, we describe a knockin Ifnb-IRES-TdTomato-Cre reporter mouse to track IFN-β-expressing cells. We demonstrate pathway-specific induction of the TdTomato reporter and show that the linked Cre recombinase enables permanent marking of cells that express IFN-β. We identify a robust MAVS-dependent IFN-β response in lung epithelial cells following Sendai virus infection in vivo. Finally, we find that activation of RNase L in macrophages by RNA ligands of the RIG-I-like receptors prevents protein translation of IFN-β and the TdTomato reporter. Our mouse model provides a powerful tool to study the biology of type I IFN induction and the antiviral response., (Copyright © 2024 by The American Association of Immunologists, Inc.)

ER-phagy is an evolutionarily conserved mechanism crucial for maintaining cellular homeostasis. However, significant gaps persist in our understanding of how ER-phagy and the ER network vary across cell subtypes, tissues, and organs. Furthermore, the pathophysiological relevance of ER-phagy remains poorly elucidated. Addressing these questions requires developing quantifiable methods to visualize ER-phagy and ER architecture in vivo. We generated two transgenic mouse lines expressing an ER lumen-targeting tandem RFP-GFP (ER-TRG) tag, either constitutively or conditionally. This approach enables precise spatiotemporal measurements of ER-phagy and ER structure at single-cell resolution in vivo. Systemic analysis across diverse organs, tissues, and primary cultures derived from these ER-phagy reporter mice unveiled significant variations in basal ER-phagy, both in vivo and ex vivo. Furthermore, our investigation uncovered substantial remodeling of ER-phagy and the ER network in different tissues under stressed conditions such as starvation, oncogenic transformation, and tissue injury. In summary, both reporter models represent valuable resources with broad applications in fundamental research and translational studies., (© 2024 Sang et al.)

The human CPAMD8 gene encodes proteins in the A2M/C3 (alpha-2-macroglobulin/complement 3) family, predominantly expressed in the distal tips of the retinal neuroepithelium that form the iris and ciliary body. Mutations in CPAMD8 have been linked to anterior segment dysplasia and congenital glaucoma. Using CRISPR/Cas9 editing, we inserted a 3*EAAAK-EGFP fluorescent tag into the CPAMD8 gene, enabling real-time observation of its expression and providing insights into its biological functions. The resulting gene-edited cell line retained normal stem cell morphology and karyotype, expressed essential pluripotency markers, and exhibited differentiation potential., 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 Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Existing analyses with conventional assays have generated significant insights into static states of DNA methylation but were unable to visualize the dynamics of epigenetic regulation., Materials & Results: We utilized a genomic DNA methylation reporter (GMR) system carrying Snrpn minimal promoter and CpG regions of Cdk1 (Cyclin-dependent kinase 1) or Sox2 (SRY-Box Transcription Factor 2). Mouse Sox2 GMR iPSCs rapidly lost fluorescent reporter signal upon the induction of cardiac differentiation. Cdk1 GMR reporter signal was strong in undifferentiated iPSCs, and gradually decreased during cardiomyocyte differentiation. RT-qPCR and pyrosequencing demonstrated that the reduction of Sox2 and Cdk1 was regulated by hypermethylation of their promoters' CpG regions during cardiac differentiation., Conclusion: The GMR reporter system can be useful for monitoring real-time epigenetic DNA modification at single-cell resolution.

An ability to monitor cell cycle progression in real time has numerous applications in pluripotent stem cell-based models and therapeutics development. Fluorescence Ubiquitination-based Cell Cycle Indicator (FUCCI) systems enable live monitoring of the cell cycle in stem cells and their differentiated progenies in a non-invasive manner. We describe the generation and characterisation of a cell line with a doxycycline-inducible reporter system, iFUCCI, in the human embryonic stem cell (hESC) line MEL-1. MEL-1 iFUCCI hESCs exhibited a normal karyotype, expressed markers of the undifferentiated state, and demonstrated expected differentiation potential by giving rise to cell populations from all three germ layers., 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. Published by Elsevier B.V.)

The SFTPC gene is responsible for the production of the pulmonary surfactant protein C (SPC), a highly hydrophobic molecule that plays a crucial role in maintaining lung integrity through its influence on the synthesis of alveolar surfactant proteins. In this study, we harnessed the CRISPR/Cas9 system for precise genome editing to create a modified H1 human embryonic stem cell (hESC) line, incorporating the SFTPC-mCherry reporter construct. Therefore, the engineered SFTPC-mCherry knock-in (KI) hESC line can serve as an effective tool for tracking the expression patterns of the SFTPC gene as alveolar type 2 cells differentiate from hESCs., 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 Author(s). Published by Elsevier B.V. All rights reserved.)

Cell-based assays are widely exploited for drug screening and biosensing, providing useful information about bioactivity of target analytes and complex biological samples. It is well recognized that 3D cell models are required to achieve highly valuable information, also from the perspective of replacing animal models. However, bioassays relying on 3D cell models are generally highly demanding in terms of facilities, equipment, and skilled personnel requirements. To reduce cost, increase sustainability, and provide a flexible 3D cell-based platform for bioassays, we here report a novel approach based on a 3D-printed microtissue device. To assess the suitability of this strategy for reporter gene technology, we selected to monitor two molecular pathways which were of interest in several applications, hypoxia signaling and the p53 pathway. The investigation of such pathways is highly relevant in fields spanning from drug screening to bioactivity monitoring for industrial by-product valorization. Microtissues of human hepatocarcinoma (HepG2) and human embryonic kidney (Hek293T) cell lines were obtained with a low-cost and sustainable chip platform and bioassays were developed to monitor the hypoxia-inducible factors (HIFs) and the p53 tumor suppressor pathway. HepG2 and Hek293T 3D cell models were genetically engineered to express the Luc2P from Photinus pyralis firefly either under the regulation of p53 or HIF response elements. The bioassays allowed quantitative assessment of hypoxia and tumoral activity with 1,10-phenanthroline for HIF and with doxorubicin for p53 pathway activation, respectively, showing good potential for applications of this sustainable and low-cost 3D-printed microfluidic platform for bioactivity analyses, drug screening, and precision medicine., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Interferons (IFNs) are a critical component of innate immune defenses and limit viral disease severity. To advance studies on IFNs and their neutralization by pathogenic autoantibodies, we generated a Renilla luciferase-based reporter cell line capable of detecting the activities of IFN-Is, IFN-II, and IFN-IIIs. The reporter cell line exhibits a 125- to 2000-fold higher sensitivity to IFNs than a commonly used alternative biological reporter system and allows for a rapid and simple live-cell workflow for detecting low titer amounts of neutralizing anti-IFN antibodies., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

New approach methodologies (NAMs) for predicting embryotoxicity and developmental toxicity are urgently needed for generating human relevant data, while reducing turnover time and costs, and alleviating ethical concerns related to the use of animal models. We have previously developed the PluriLum assay, a NKX2.5-reporter gene 3D model using human-induced pluripotent stem cells (hiPSCs) that are genetically modified to enable the assessment of adverse effects of chemicals on the early-stage embryo. Aiming at improving the predictive value of the PluriLum assay for future screening purposes, we sought to introduce standardization steps to the protocol, improving the overall robustness of the PluriLum assay, as well as a shortening of the assay protocol. First, we showed that the initial size of embryoid bodies (EBs) is crucial for a proper differentiation into cardiomyocytes and overall reproducibility of the assay. When the starting diameter of the EBs exceeds 500 µm, robust differentiation can be anticipated. In terms of reproducibility, exposure to the fungicide epoxiconazole at smaller initial diameters resulted in a larger variation of the derived data, compared to more reliable concentration-response curves obtained using spheroids with larger initial diameters. We further investigated the ideal length of the differentiation protocol, resulting in a shortening of the PluriLum assay by 24 h to 7 days. Following exposure to the teratogens all-trans and 13-cis retinoic acid, both cardiomyocyte contraction and measurement of NKX2.5-derived luminescence were recorded with a similar or increased sensitivity after 6 days of differentiation when compared to the original 7 days. Finally, we have introduced an efficient step for enzymatic dissociation of the EBs at assay termination. This allows for an even splitting of the individual EBs and testing of additional endpoints other than the NKX2.5-luciferase reporter, which was demonstrated in this work by the simultaneous assessment of ATP levels. In conclusion, we have introduced standardizations and streamlined the PluriLum assay protocol to improve its suitability as a NAM for screening of a large number of chemicals for developmental toxicity testing., (© 2024. The Author(s).)

Targeted mouse mutants are a common tool used to investigate gene function. The International Knockout Mouse Consortium undertook a large-scale screen of mouse mutants, making use of the knockout-first allele design that contains the En2 splice acceptor sequence coupled to the lacZ reporter gene. Although the knockout-first allele was designed to interfere with splicing and thus disrupt gene function, the En2 sequence has been reported to be transcribed within the host gene mRNA due to a cryptic splice site within the En2 sequence which allows splicing to the next exon of the host gene. In some circumstances, this has the potential to permit translation of a mutant protein. Here, we describe our computational analysis of all the mouse protein-coding genes with established knockout-first embryonic stem cell lines, and our predictions of their transcription outcome should the En2 sequence be included. As part of the large-scale mutagenesis program, mutant mice underwent a broad phenotyping screen, and their phenotypes are available. No wide-scale effects on mouse phenotypes reported were found as a result of the predicted En2 insertion. However, the En2 insertion was found experimentally in the transcripts of 24 of 35 mutant alleles examined, including the five already described, two with evidence of readthrough. Splicing from the cryptic splice site also has the potential to disrupt expression of the lacZ reporter gene. It is recommended that mutant transcripts be checked for this insertion as well as for leaky transcription in studies involving knockout-first alleles., (© 2024. The Author(s).)

The transcription factor SOX2 plays a crucial role in pluripotency during embryogenesis. In this study, we successfully generated porcine induced pluripotent stem cells (piPSC) by transducing porcine fetal fibroblasts with doxycycline-inducible reprogramming lentiviral vectors. To enhance the utility of these piPSCs, we used a CRISPR/Cas9-based homology-directed repair (HDR) system to introduce a nls-zsGreen reporter in-frame and before the stop codon of the SOX2 coding sequence. The incorporation of the zsGreen reporter offers a novel opportunity for real-time monitoring of SOX2 expression and aiding in the characterization of piPSC lines., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Bhanu P. Telugu reports a relationship with RenOVAte Biosciences Inc., that includes: board membership, consulting or advisory, and equity or stocks. Dr. Telugu is a founding member and Chief Scientific Officer of RenOVAte Biosciences Inc. All other authors do not have conflicts to declare., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Activator protein 1 (AP-1) is involved in cell fate determination and function. To monitor the AP-1 activity, we cloned a AP-1 binding sites fragment into the upstream of minimal TATA-box promoter, then a luciferase-GFP reporter (LuciGFP) was designated to the downstream of the promoter. With CRISPR/Cas9, the AP-1-LuciGFP reporter was introduced into AAVS1 locus, a safe harbor for gene editing. Thus, this AP-1-LuciGFP reporter cell line could be subjected to monitor the AP-1 activity during the cell differentiation, cell fate transition and disease modeling., 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 Author(s). Published by Elsevier B.V. All rights reserved.)

Over the past decade, the use of human stem cell-derived β cells (SC-β cells) to model pancreatic β cell development, function and disease has become increasingly common. Though protocols are rapidly improving, current directed differentiation strategies do not yield a pure population of insulin-positive SC-β cells in vitro. Therefore, it is experimentally advantageous to have reporter lines that allow for live sorting of insulin-positive populations. To aid in these studies, we have knocked mNeonGreen fluorescent protein into the endogenous insulin locus of the commonly used H1 (WA01) human embryonic stem cell line., 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.)

Background and Aims: Most experimental studies of allograft vasculopathy (AV) have relied on transplantation between major histocompatibility complex-mismatched inbred mouse strains, but this leads to the complete eradication of donor smooth muscle cells (SMCs) and lesions formed by recipient cells. This is unlike human AV which is thought to form mainly by donor SMCs. Here, we studied sources of neointimal cells in a minor histocompatibility antigen-mismatched AV model by combining male-to-female orthotopic carotid transplantations and lineage tracing by SMC-specific expression of fluorescent proteins., Methods: To track SMC-derived cells in allograft vasculopathy, we used male donor mice with SMC-restricted Cre recombination of the mT/mG reporter transgene, which switches expression of membrane-bound red fluorescent protein (RFP) to green fluorescent protein (GFP), or the stochastically recombining Confetti reporter transgene, which yields a mosaic expression of four fluorescent proteins. Donor carotid segments were harvested and orthotopically allografted to female recipients that were wildtype or had non-recombined reporter transgenes. Inhibition of T cell responses by CTLA4Ig was used in some experiments. Sections of lesions harvested after 4 weeks were analyzed by fluorescence microscopy., Results: Donor-derived SMCs survived and gave rise to part of the neointimal cells in experiments where carotid segments from recombined mT/mG male mice were transplanted into wild-type or non-recombined mT/mG female mice. Sex-mismatched transplants developed significant lesions, increasing the intimal and medial area 4.6-fold (p = 0.038) and 2.0-fold (p = 0.024) compared to sex- and fluorescence-matched controls, respectively. Interestingly, sex-matched fluorescence-positive transplants developed intimal lesions in 50% of fluorescence-naïve recipient controls. To study the clonal structure of the neointimal donor-derived SMC lineage cells, we then transplanted male carotids with heterozygous or homozygous recombined Confetti transgenes into female recipients. These transplants developed lesions with few surviving donor SMCs, indicating that expression of the Confetti reporter increased rejection and donor-specific SMC death. Some of the few remaining donor SMCs underwent clonal expansion. CTLA4Ig administration at the time of surgery did not improve SMC survival in mT/mG or Confetti transplants., Conclusion: Male-to-female transplant models feature donor-derived SMCs, some of which undergo clonal expansion, but immune rejection to fluorescence reporters appears to bias results in lineage tracing models. Overcoming these challenges with alternative reporter transgenes or tolerant recipients is necessary to study the mechanisms by which donor SMCs contribute to allograft vasculopathy., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Current methodologies for hepatocyte induction from human induced pluripotent stem cells (hiPSCs) have limited efficacy due to lack of a functional hepatocyte reporter. To address this, we developed an endogenous albumin (ALB)-sfGFP reporter system in hiPSCs using homologous directed recombination (HDR)-mediated knock-in. The hiPSCs maintained the characteristic morphology, pluripotency, and normal karyotype while demonstrating successful differentiation into all three germ layers both in vitro and in vivo. Co-expression of EGFP and ALB was observed in the derived hepatocyte-like cells (HLCs). This reporter system holds promise for functional hepatocyte induction., 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 Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: This study aimed to evaluate a novel technique for cell tracking by visualising the activity of the human sodium/iodide symporter (hNIS) after transplantation of hNIS-expressing multilayered cell sheets in a rat model of chronic myocardial infarction., Methods: Triple-layered cell sheets were generated from mouse embryonic fibroblasts (MEFs) derived from mice overexpressing hNIS (hNIS-Tg). Myocardial infarction was induced by permanent ligation of the left anterior descending coronary artery in F344 athymic rats, and a triple-layered MEFs sheets were transplanted to the infarcted area two weeks after surgery. To validate the temporal tracking and kinetic analysis of the transplanted MEFs sheets, sequential cardiac single-photon emission computed tomography (SPECT) examinations with a 99m TcO 4 - injection were performed. The cell sheets generated using MEFs of wild-type mice (WT) served as controls., Results: A significantly higher amount of 99m TcO 4 - was taken into the hNIS-Tg MEFs than into WT MEFs (146.1 ± 30.9-fold). The obvious accumulation of 99m TcO 4 - was observed in agreement with the region where hNIS-Tg MEFs were transplanted, and these radioactivities peaked 40-60 min after 99m TcO 4 - administration. The volume of distribution of the hNIS-Tg MEF sheets declined gradually after transplantation, implying cellular malfunction and a loss in the number of transplanted cells., Conclusion: The reporter gene imaging with hNIS enables the serial tracking and quantitative kinetic analysis of cell sheets transplanted to infarcted hearts., Competing Interests: Declarations. Competing interests: The authors have no relevant financial or nonfinancial interests to disclose. Ethics approval: The animal study was approved by the National Cerebral and Cardiovascular Center Ethics Committee for Animal Experiments and was conducted in accordance with the guidelines of the Physiological Society of Japan., (© 2024. The Author(s).)

Most protocols used to study the dynamics of calcium (Ca 2+ ) in the malaria parasite are based on dyes, which are invasive and do not allow discrimination between the signal from the host cell and the parasite. To avoid this pitfall, we have generated a parasite line expressing the genetically encoded calcium sensor GCaMP3. The PfGCaMP3 parasite line is an innovative tool for studying spontaneous intracellular Ca 2+ oscillations without external markers. Using this parasite line, we demonstrate the occurrence of spontaneous Ca 2+ oscillations in the ring, trophozoite, and schizont stages in Plasmodium falciparum. Using the Fourier transform to fluorescence intensity data extracted from different experiments, we observe cytosolic Ca 2+ fluctuations. These spontaneous cytosolic Ca 2+ oscillations occur in the three intraerythrocytic stages of the parasite, with most oscillations occurring in the ring and trophozoite stages. A control parasite line expressing only a GFP control did not reveal such fluctuations, demonstrating the specificity of the observations. Our results clearly show dynamic, spontaneous Ca 2+ oscillations during the asexual stage in P. falciparum, independent from external stimuli., 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.)

Cellular responses to stimuli underpin discoveries in drug development, synthetic biology, and general life sciences. We introduce a library comprising 6144 synthetic promoters, each shorter than 250 bp, designed as transcriptional readouts of cellular stimulus responses in massively parallel reporter assay format. This library facilitates precise detection and amplification of transcriptional activity from our promoters, enabling the systematic development of tunable reporters with dynamic ranges of 50-100 fold. Our library proved functional in numerous cell lines and responsive to a variety of stimuli, including metabolites, mitogens, toxins, and pharmaceutical agents, generating robust and scalable reporters effective in screening assays, biomarkers, and synthetic circuits attuned to endogenous cellular activities. Particularly valuable in therapeutic development, our library excels in capturing candidate reporters to signals mediated by drug targets, a feature we illustrate across nine diverse G-protein coupled receptors (GPCRs), critical targets in drug development. We detail how this tool isolates and defines discrete signaling pathways associated with specific GPCRs, elucidating their transcriptional signatures. With its ease of implementation, broad utility, publicly available data, and comprehensive documentation, our library will be beneficial in synthetic biology, cellular engineering, ligand exploration, and drug development., Competing Interests: Competing interests: W.S.O., A.N.G., J.S.B., S.K., and H.C. hold equity in and/or are employed by Octant Inc., a company where similar types of assays are used for its drug discovery efforts. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Introduction: The sufCDSUB gene cluster, encoding the sole iron-sulfur (Fe-S) cluster assembly system in S. mutans , was recently shown to be up-regulated in response to oxidative stressors and Fe limitation., Methods: In this study, luciferase reporter fusion assays, electrophoretic gel mobility shift assays (EMSA) and in vitro transcription assays (IVT) were used to dissect the cis- and trans- acting factors that regulate the expression of sufCDSUB ., Results and Discussion: Results showed deletion of perR , for the only Fur-family transcriptional regulator in S. mutans , resulted in >5-fold increases in luciferase activity under the control of the sufCDSUB promoter (P<0.01), as compared to the parent strain, UA159 when the reporter strains were grown in medium with no supplemental iron. Site-directed mutagenesis of a PerR-box in the promoter region led to elevation of the reporter activity by >1.6-fold ( P <0.01). In an EMSA, recombinant PerR (rPerR) was shown to bind to the cognate sufCDSUB promoter leading to mobility retardation. On the other hand, the reporter activity was increased by >84-fold (P<0.001) in response to the addition of cysteine at 4 mM to the culture medium. Deletion of cysR , for a LysR-type of transcriptional regulator, led to reduction of the reporter activity by >11.6-fold ( P <0.001). Addition of recombinant CysR (rCysR) to an EMSA caused mobility shift of the sufCDSUB promoter probe, indicative of rCysR-promoter interaction, and rCysR was shown to enhance sufC transcription under the direction of sufCDSUB promoter in vitro . These results suggest that multiple factors are involved in the regulation of sufCDSUB expression in response to environmental cues, including cysteine and Fe availability, consistent with the important role of sufCDSUB in S. mutans physiology., 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. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Ellepola, Guillot, Comeaux, Han, Kajfasz, Bitoun, Spatafora, Lemos and Wen.)

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.

Corticospinal motor neurons (CSMNs), also named upper motor neurons, are the giant pyramidal neurons called Betz cells. In mammals, the majority of CSMNs reside within layer V of the primary motor cortex, where they extend long axon bundles named the pyramidal tract into the brainstem and the spinal cord to control voluntary movement. CSMN lesions are implicated in a variety of neurodegenerative disorders, such Amyotrophic Lateral Sclerosis, Primary Lateral Sclerosis and Hereditary Spastic paraplegia. Although FEZF2-CTIP2 genetic axis have been indicated as the cardinal molecular pathway underlying the development of CSMNs, these proteins are transcription factors that are mostly used to label the nuclei of CSMNs in the fixed cells and tissues. Therefore, a fluorescent reporter to mark CSMNs will be invaluable in identifying living CSMNs, including their extended processes, for time-lapse imaging and high-throughput molecular analyses with much more improved specificity. Based on the in-silico analysis, we identified a putative region within the promoter sequence of FEZF2 and assembled it with an indispensable enhancer motif at its downstream of the gene to form a complex promoter that drives the expression of reporter GFP. The plasmid and virus of FEZF2:eGFP reporter constructs were further validated for its use in specifically labeling CSMNs in primary neuronal cultures from the embryonic rat motor cortex, postnatal mouse cortex. This innovative molecular labeling tool has the potential to offer indispensable support in diverse experimental setups, enabling a comprehensive understanding of the susceptibility and specificity of CSMNs in a wide array of neurological disorders., Competing Interests: Declarations Ethics approval The studies in Nantong university reported here were submitted to Ethics Committee on Animal Experimentation of Nantong University, and all procedures were approved (S20200330-005) by the Animal Care and Use Committee of Nantong University. All of the animal experiments in Drexel University were performed in compliance with the NIH’s Guide for the Care and Use of Laboratory Animals (National Academies Press, 2011) and were reviewed and approved by the Institutional Animal Care and Use Committee at Drexel University. The animal work for this study was carried out under the project license 1045165, protocol no. 20861. Efforts were made to minimize the number of animals used and their suffering. Consent to participate Not applicable. Consent for publication Not applicable. Conflict of interest The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Protein synthesis is influenced by the chemical and structural properties of the amino acids incorporated into the polypeptide chain. Motifs containing consecutive prolines can slow the translation speed and cause ribosome stalling. Translation elongation factor P (EF-P) facilitates peptide bond formation in these motifs, thereby alleviating stalled ribosomes and restoring the regular translational speed. Ribosome pausing at various polyproline motifs has been intensively studied using a range of sophisticated techniques, including ribosome profiling, proteomics, and in vivo screening, with reporters incorporated into the chromosome. However, the full spectrum of motifs that cause translational pausing in Escherichia coli has not yet been identified. Here, we describe a plasmid-based dual reporter for rapid assessment of pausing motifs. This reporter contains two coupled genes encoding mScarlet-I and chloramphenicol acetyltransferase to screen motif libraries based on both bacterial fluorescence and survival. In combination with a diprolyl motif library, we used this reporter to reveal motifs of different pausing strengths in an E. coli strain lacking efp . Subsequently, we used the reporter for a high-throughput screen of four motif libraries, with and without prolines at different positions, sorted by fluorescence-associated cell sorting (FACS) and identify new motifs that influence the translational efficiency of the fluorophore. Our study provides an in vivo platform for rapid screening of amino acid motifs that affect translational efficiencies.

Enzymes that produce volatile metabolites can be coded into genetic circuits to report nondisruptively on microbial behaviors in hard-to-image soils. However, these enzyme reporters remain challenging to apply in gene transfer studies due to leaky off states that can lead to false positives. To overcome this problem, we designed a reporter that uses ribozyme-mediated gene-fragment complementation of a methyl halide transferase (MHT) to regulate the synthesis of methyl halide gases. We split the mht gene into two nonfunctional fragments and attached these to a pair of splicing ribozyme fragments. While the individual mht -ribozyme fragments did not produce methyl halides when transcribed alone in Escherichia coli , coexpression resulted in a spliced transcript that translated the MHT reporter. When cells containing one mht -ribozyme fragment transcribed from a mobile plasmid were mixed with cells that transcribed the second mht -ribozyme fragment, methyl halides were only detected following rare conjugation events. When conjugation was performed in soil, it led to a 16-fold increase in methyl halides in the soil headspace. These findings show how ribozyme-mediated gene-fragment complementation can achieve tight control of protein reporter production, a level of control that will be critical for monitoring the effects of soil conditions on gene transfer and the fidelity of biocontainment measures developed for environmental applications.

The expression of therapeutic genes is critical for the efficacy of gene therapy products. However, existing methods such as immunological analysis at the protein level or reverse-transcription PCR at the RNA level are unable to accurately quantify the expression activity of the target gene. Herein, an in vitro RNA editing-based reporter assay was developed to detect specific mRNA. The designed sensor RNA could specifically identify the target mRNA, and the reporter gene was activated in a dose-dependent manner because of RNA editing mediated by endogenous adenosine deaminases acting on RNA. Of note, all sensors that targeted different regions, including the gene of interest, tag sequence, and 3' untranslated region, showed a dose-dependent response pattern. The sensor reporter assay, which was used for quantifying the transcriptional activity of recombinant adeno-associated virus-based gene therapy products, revealed excellent performance in terms of assay specificity, precision (inter-assay relative standard deviation < 15%), accuracy (90-115% recovery), and linearity (R 2 > 0.99). The reporter assay could also be employed for other gene therapy vectors, including mRNA and recombinant lentivirus. Thus, a robust and reliable platform was developed for assessing the transcriptional activity of therapeutic genes, thereby offering a powerful tool for the quality control of gene therapy products.

Primary hepatocytes are widely utilized for investigating drug efficacy and toxicity, yet variations between batches and limited proliferation capacity present significant challenges. HepaRG cells are versatile cells, capable of maintaining an undifferentiated state and differentiating through dimethyl sulfoxide treatment, allowing for molecular analysis of hepatocyte plasticity. To elucidate the underlying molecular mechanisms of HepaRG cell plasticity, we used CYP3A4G/7R HepaRG cells engineered to express DsRed under the control of the fetus-specific CYP3A7 gene and EGFP under the adult-specific CYP3A4 gene promoter. In time-lapse imaging of CYP3A4G/7R HepaRG cells, we observed CYP3A7-DsRed expression transitioning from negative to positive during proliferation period and CYP3A4-GFP expression activating during differentiation. The de-differentiation potency of differentiated CYP3A4G/7R HepaRG cells was assessed using inhibitors and cytokines. It was found that Y-27632 (Y), A-83-01 (A), and CHIR99021 (C) (collectively referred to as YAC), which are known to promote liver regeneration in mice, did not induce CYP3A7-DsRed expression. Instead, these inhibitors increased CYP3A4-GFP expressing population. Furthermore, CHIR99021 alone increased CYP3A4-GFP-positive cells, while Wnt3a treatment increased CYP3A7-DsRed-positive cells, suggesting that Wnt signaling plays distinct roles in HepaRG cells. It was apparent that de-differentiated cells had increased CYP3A4 activity after a second round of differentiation, compared to differentiated cells after the first round. Transcriptomic analysis of HepaRG cells revealed distinct profiles between proliferative, differentiated, and de-differentiated states, highlighting their robust plasticity. Notably, hepatoblastic cells de-differentiated by YAC or C displayed transcriptome patterns similar to undifferentiated cells, whereas CYP3A7-DsRed and CYP3A4-GFP exhibited expression patterns different from those of undifferentiated cells. These findings underscore the dynamic nature of HepaRG cells while cautioning against solely relying on CYP3 family gene expression as a marker of differentiation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Asano 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.)

Corticotropin-releasing hormone (CRH) neurons within the paraventricular hypothalamic nucleus (PVH) play a crucial role in initiating the neuroendocrine response to stress and are also pivotal in coordination of autonomic, metabolic, and behavioral stress reactions. Although the role of parvocellular CRH PVH neurons in activation of the hypothalamic-pituitary-adrenal (HPA) axis is well established, the distribution and function of CRH-expressing neurons across the whole central nervous system are less understood. Stress responses activate complex neural networks, which differ depending on the type of stressor and on the sex of the individual. Because of the technical difficulties of localizing CRH neurons throughout the rodent brain, several CRH reporter mouse lines have recently been developed. In this study, we used Crh-IRES-Cre;Ai9 reporter mice to examine whether CRH neurons are recruited in a stressor- or sex-specific manner, both within and outside the hypothalamus. In contrast to the clear sexual dimorphism of CRH-mRNA-expressing neurons, quantification of CRH-reporting, tdTomato-positive neurons in different stress-related brain areas revealed only subtle differences between male and female subjects. These results strongly imply that sex differences in CRH mRNA expression occur later in development under the influence of sex steroids and reflects the limitations of using genetic reporter constructs to reveal the current physiological/transcriptional status of a specific neuron population. Next, we compared the recruitment of stress-related, tdTomato-expressing (putative CRH) neurons in male and female Crh-IRES-Cre;Ai9 reporter mice that had been exposed to predator odor. In male mice, fox odor triggered more c-Fos in the CRH neurons of the paraventricular hypothalamic nucleus, central amygdala, and anterolateral bed nucleus of the stria terminalis compared to females. These results indicate that male mice are more sensitive to predator exposure due to a combination of hormonal, environmental, and behavioral factors.

The massed-spaced effect is a hallmark feature of memory formation. We now demonstrate this effect in two separate non-neural, immortalized cell lines stably expressing a short-lived luciferase reporter controlled by a CREB-dependent promoter. We emulate training using repeated pulses of forskolin and/or phorbol ester, and, as a proxy for memory, measure luciferase expression at various points after training. Four spaced pulses of either agonist elicit stronger and more sustained luciferase expression than a single "massed" pulse. Spaced pulses also result in stronger and more sustained activation of molecular factors critical for memory formation, ERK and CREB, and inhibition of ERK or CREB blocks the massed-spaced effect. Our findings show that canonical features of memory do not necessarily depend on neural circuitry, but can be embedded in the dynamics of signaling cascades conserved across different cell types., (© 2024. The Author(s).)

CRISPR-Cas9 editing triggers activation of the TP53-p21 pathway, but the impacts of different editing components and delivery methods have not been fully explored. In this study, we introduce a p21-mNeonGreen reporter iPSC line to monitor TP53-p21 pathway activation. This reporter enables dynamic tracking of p21 expression via flow cytometry, revealing a strong correlation between p21 expression and indel frequencies, and highlighting its utility in guide RNA screening. Our findings show that p21 activation is significantly more pronounced with double-stranded oligodeoxynucleotides (ODNs) or adeno-associated viral vectors (AAVs) compared to their single-stranded counterparts. Lentiviral vectors (LVs) and integrase-defective lentiviral vectors induce notably lower p21 expression than AAVs, suggesting their suitability for gene therapy in sensitive cells such as hematopoietic stem cells or immune cells. Additionally, specific viral promoters like SFFV significantly amplify p21 activation, emphasizing the critical role of promoter selection in vector development. Thus, the p21-mNeonGreen reporter iPSC line is a valuable tool for assessing the potential adverse effects of gene editing methodologies and vectors. Highlights Established a p21-mNeonGreen reporter iPSC line to track activation of the TP53-p21 pathway. Found a direct correlation between p21-mNeonGreen expression and indel frequencies, aiding in gRNA screening. Showed that LVs are preferable over AAVs for certain cells due to lower p21 activation, with viral promoter choice impacting p21 response., (© The Author(s) 2024. Published by Oxford University Press.)

Cassava ( Manihot esculenta Crantz) is a staple crop in tropical and subtropical regions, valued for its high starch content in roots. Effective genetic transformation and genome editing of cassava require efficient screening methods for transgenic and edited plants. In this study, a visual selection marker system using an R2R3-MYB transcription factor anthocyanin 1 gene ( HbAN1, LOC110667474) from a rubber tree ( Hevea brasiliensis Müll. Arg .) has been developed to facilitate the identification of transgenic cassava plants. Transgenic cassava lines expressing HbAN1 accumulated anthocyanins in their leaves, allowing for easy visual identification without the need for destructive assays or specialized equipment. Importantly, the accumulation of anthocyanins did not affect the regeneration or transformation efficiency of cassava. Additionally, the AR-CRISPR/Cas9-gRNA system with the HbAN1 gene as a marker produced MeCDD4 gene-edited cassava mutants with purple leaves, demonstrating successful editing. This anthocyanin-based visual reporter (AR) system will provide an effective tool for genetic transformation and genome editing in cassava.

To design a safe cellular system for testing inhibitors targeting the RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2, a genetic construct was engineered containing viral cDNA with two blocks of reporter genes while the genes encoding structural S, E, and M proteins were absent. The first reporter block, consisting of Renilla luciferase and green fluorescent protein (Rluc-GFP), was located upstream of the SARS-CoV-2 5'-UTR. Meanwhile, the second block represented by firefly luciferase and red fluorescent protein (Fluc-RFP) was positioned downstream of the transcription regulatory sequence (TRS-N). While the first block of reporter genes can be transcribed by both viral RdRp and cellular polymerases, the second block can only be transcribed by the viral polymerase according to the Coronaviridae discontinuous transcription mechanism. This allowed us to accurately assess effectiveness of the viral RdRp inhibition. To facilitate the search for nucleoside RdRp inhibitors the cell line was obtained expressing herpes simplex virus thymidine kinase, which provides the first stage of nucleoside phosphorylation. When screening the ability of a number of compounds to inhibit catalytic activity of the SARS-CoV-2 RdRp, we discovered antiviral activity of 2'-amino-2'-deoxyadenosine and adenosine-N1-oxide, which exceeded activity of molnupiravir, a therapeutic agent used in the treatment of COVID-19.

Glycogen, a branched polysaccharide organized into glycogen granules (GGs), is delivered from the cytoplasm to the lysosomes of hepatocytes by STBD1-driven selective autophagy (glycophagy). Recently, we developed Komagataella phaffii yeast as a simple model of GG autophagy and found that it proceeds non-selectively under nitrogen starvation conditions. However, another group, using Saccharomyces cerevisiae as a model, found that glycogen is a non-preferred cargo of nitrogen starvation-induced bulk autophagy. To clarify cargo characteristics of K. phaffii GGs, we used the same glycogen synthase-based reporter (Gsy1-GFP) of GG autophagy in K. phaffii as was used in S. cerevisiae . The K. phaffii Gsy1-GFP marked the GGs and reported on their autophagic degradation during nitrogen starvation, as expected. However, unlike in S. cerevisiae , glycogen synthase-marked GGs were delivered to the vacuole and degraded there with the same efficiency as a cytosolic glycogen synthase in glycogen-deficient cells, suggesting that glycogen is a neutral cargo of bulk autophagy in K. phaffii . We verified our findings with a new set of reporters based on the glycogen-binding CBM20 domain of human STBD1. The GFP-CBM20 and mCherry-CBM20 fusion proteins tagged GGs, reported about the autophagy of GGs, and confirmed that GGs in K. phaffii are neither preferred nor non-preferred substrates of bulk autophagy. They are its neutral substrates.

Growing concerns about the environmental impact of ionic liquids (ILs) have spurred research into their (eco)toxic effects, but studies on their mode of toxic action (MOA) still remain limited. However, understanding the MOA and identifying structural features responsible for enhanced toxicity is crucial for characterising the hazard and designing safer alternatives. Therefore, 45 ILs, with systematically varied chemical structures, were tested for cytotoxicity and two specific endpoints in reporter gene assays targeting the Nrf2-ARE mediated oxidative stress response (AREc32) and aryl hydrocarbon receptor activation (AhR-CALUX). While none of the ILs activated the reporter genes, cytotoxicity was high and markedly different between cell lines. Seven and 25 ILs proved more cytotoxic than predicted by baseline toxicity model in the AREc32 and the AhR-CALUX assays, respectively. The length of the side chain and headgroup structures of ILs altered the MOA of ILs. Cellular metabolism of the ILs, investigated by LC-MS/MS, showed side-chain oxidation of the long-chain quaternary ammonium compounds in AhR-CALUX cells and, to a lower extent, in AREc32 cells, however, this transformation could not explain the high cytotoxicity. Effect data for 72 ILs for ten endpoints retrieved from the Tox21 database identified the inhibition of aromatase activity and of mitochondrial membrane potential as potential MOAs. However, in vitro fluorimetric assays for these endpoints demonstrated that effects were activated in a non-specific manner, probably through cytotoxicity. Although many of the ILs tested induced cytotoxicity at concentrations lower than baseline toxicity, the specific MOAs responsible could not be identified. Alternatively, we suggest that the descriptors currently used may fail to define the affinity of ILs for cells. Testing of the affinity of ILs for a diverse range of biomolecules is needed to accurately describe their interactions with 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 Ltd.. All rights reserved.)

The human malaria parasite Plasmodium falciparum causes the most severe form of malaria in endemic regions and is transmitted via mosquito bites. To better understand the biology of this deadly pathogen, a variety of P. falciparum reporter lines have been generated using transgenic approaches to express reporter proteins, such as fluorescent proteins and luciferases. This review discusses the advances in recently generated P. falciparum transgenic reporter lines, which will aid in the investigation of parasite physiology and the discovery of novel antimalarial drugs. Future prospects for the generation of new and superior human malaria parasite reporter lines are also discussed, and unresolved questions in malaria biology are highlighted to help boost support for the development and implementation of malaria treatments., Competing Interests: Declaration of interests Shinya Miyazaki declares grant/research funding from Shionogi & Co., Ltd., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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.)

Clostridium butyricum has emerged as a promising candidate for both industrial and medical biotechnologies, underscoring the key pursuit of stable gene overexpression in engineering C. butyricum. Unlike antibiotic-selective vectors, native-cryptic plasmids can be utilized for antibiotic-free expression systems in bacteria but have not been effectively exploited in C. butyricum to date. This study focuses on leveraging these plasmids, pCB101 and pCB102, in C. butyricum DSM10702 for stable gene overexpression without antibiotic selection via efficient gene integration using the SacB-based allelic exchange method. Integration of reporter IFP2.0 and glucuronidase generated sustained near-infrared fluorescence and robust enzyme activity across successive subcultures. Furthermore, successful secretion of a cellulase, Cel9M, and the human interleukin 10 from pCB102 highlights native-cryptic plasmids' potential in conferring stable gene products for industrial and medical applications in C. butyricum. This work appears to be the first study to harness the Clostridium native-cryptic plasmid for stable gene overexpression without antibiotics, thereby advancing the biotechnological prospects of C. butyricum., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests. Philippe Lambin reports a relationship with Radiomics SA that includes: equity or stocks and funding grants. Philippe Lambin reports a relationship with Convert Pharmaceuticals SA that includes: equity or stocks and funding grants. Philippe Lambin reports a relationship with LivingMed Biotech srl that includes: equity or stocks and funding grants. Philippe Lambin reports a relationship with BHV srl that includes: consulting or advisory and travel reimbursement. Philippe Lambin reports a relationship with Roche that includes: consulting or advisory and travel reimbursement. Philippe Lambin reports a relationship with Comunicare SA that includes: equity or stocks. Philippe Lambin reports a relationship with Bactam srl that includes: equity or stocks. Ludwig J. Dubois reports a relationship with Convert Pharmaceuticals SA that includes: equity or stocks. Ludwig J. Dubois reports a relationship with LivingMed Biotech srl that includes: equity or stocks. Jan Theys reports a relationship with Convert Pharmaceuticals SA that includes: equity or stocks. Yanchao Zhang has patent pending to Maastricht University. Jan Theys has patent pending to Maastricht University. Philippe Lambin has patent pending to Maastricht University. If there are other authors, they 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 GmbH.. All rights reserved.)

Objective: Human embryonic stem cell (hESC; SC)-derived pancreatic β cells can be used to study diabetes pathologies and develop cell replacement therapies. Although current differentiation protocols yield SCβ cells with varying degrees of maturation, these cells still differ from deceased donor human β cells in several respects. We sought to develop a reporter cell line that could be used to dynamically track SCβ cell functional maturation., Methods: To monitor SCβ cell maturation in vitro, we created an IAPP-2A-mScar and INSULIN-2A-EGFP dual fluorescent reporter (INS 2A-EGFP/+ ;IAPP 2A-mScarlet/+ ) hESC line using CRISPR/Cas9. Pluripotent SC were then differentiated using a 7-stage protocol to islet-like cells. Immunohistochemistry, flow cytometry, qPCR, GSIS and electrophysiology were used to characterise resulting cell populations., Results: We observed robust expression of EGFP and mScarlet fluorescent proteins in insulin- and IAPP-expressing cells without any compromise to their differentiation. We show that the proportion of insulin-producing cells expressing IAPP increases over a 4-week maturation period, and that a subset of insulin-expressing cells remain IAPP-free. Compared to this IAPP-free population, we show these insulin- and IAPP-expressing cells are less polyhormonal, more glucose-sensitive, and exhibit decreased action potential firing in low (2.8 mM) glucose., Conclusions: The INS 2A-EGFP/+ ;IAPP 2A-mScarlet/+ hESC line provides a useful tool for tracking populations of maturing hESC-derived β cells in vitro. This tool has already been shared with 3 groups and is freely available to all., 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 GmbH.. All rights reserved.)

Measurement of gene expression in the brain requires invasive analysis of brain tissue or non-invasive methods that are limited by low sensitivity. Here we introduce a method for non-invasive, multiplexed, site-specific monitoring of endogenous gene or transgene expression in the brain through engineered reporters called released markers of activity (RMAs). RMAs consist of an easily detectable reporter and a receptor-binding domain that enables transcytosis across the brain endothelium. RMAs are expressed in the brain but exit into the blood, where they can be easily measured. We show that expressing RMAs at a single mouse brain site representing approximately 1% of the brain volume provides up to a 100,000-fold signal increase over the baseline. Expression of RMAs in tens to hundreds of neurons is sufficient for their reliable detection. We demonstrate that chemogenetic activation of cells expressing Fos-responsive RMA increases serum RMA levels >6-fold compared to non-activated controls. RMAs provide a non-invasive method for repeatable, multiplexed monitoring of gene expression in the intact animal brain., Competing Interests: Competing interests J.O.S. and S.L. are co-inventors on a US patent application that incorporates discoveries described in this paper. The other authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

One of the major processes occurring during the healing of a fractured long bone is chondrogenesis, leading to the formation of the soft callus, which subsequently undergoes endochondral ossification and ultimately bridges the fracture site. Thus, understanding the molecular mechanisms of chondrogenesis can enhance our knowledge of the fracture repair process. One such molecular process is calciun (Ca ++ ) signaling, which is known to play a critical role in the development and regeneration of multiple tissues, including bone, in response to external stimuli. Despite the existence of various mouse models for studying Ca ++ signaling, none of them were designed to specifically examine the skeletal system or the various musculoskeletal cell types. As such, we generated a genetically engineered mouse model that is specific to cartilage (crossed with Col2a1 Cre mice) to study chondrocytes. Herein, we report on the characterization of this transgenic mouse line using conditional expression of GCaMP6f, a Ca ++ -indicator protein. Specifically, this mouse line exhibits increased GCaMP6f fluorescence following Ca ++ binding in chondrocytes. Using this model, we show real-time Ca ++ signaling in embryos, newborn and adult mice, as well as in fracture calluses. Further, robust expression of GCaMP6f in chondrocytes can be easily detected in embryos, neonates, adults, and fracture callus tissue sections. Finally, we also report on Ca ++ signaling pathway gene expression, as well as real-time Ca ++ transient measurements in fracture callus chondrocytes. Taken together, these mice provide a new experimental tool to study chondrocyte-specific Ca ++ signaling during skeletal development and regeneration, as well as various in vitro perturbations., Competing Interests: Declaration of competing interest None., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cell cycle regulation is critical to blood vessel formation and function, but how the endothelial cell cycle integrates with vascular regulation is not well-understood, and available dynamic cell cycle reporters do not precisely distinguish all cell cycle stage transitions in vivo. Here we characterized a recently developed improved cell cycle reporter (PIP-FUCCI) that precisely delineates S phase and the S/G2 transition. Live image analysis of primary endothelial cells revealed predicted temporal changes and well-defined stage transitions. A new inducible mouse cell cycle reporter allele was selectively expressed in postnatal retinal endothelial cells upon Cre-mediated activation and predicted endothelial cell cycle status. We developed a semi-automated zonation program to define endothelial cell cycle status in spatially defined and developmentally distinct retinal areas and found predicted cell cycle stage differences in arteries, veins, and remodeled and angiogenic capillaries. Surprisingly, the predicted dearth of S-phase proliferative tip cells relative to stalk cells at the vascular front was accompanied by an unexpected enrichment for endothelial tip and stalk cells in G2, suggesting G2 stalling as a contribution to tip-cell arrest and dynamics at the front. Thus, this improved reporter precisely defines endothelial cell cycle status in vivo and reveals novel G2 regulation that may contribute to unique aspects of blood vessel network expansion., Competing Interests: Declarations Competing interest The authors declare no competing interests., (© 2024. The Author(s).)

During development, most cells experience a progressive restriction of fate that ultimately results in a fully differentiated mature state. Understanding more about the gene expression patterns that underlie developmental programs can inform engineering efforts for new or optimized forms. Here, we present a four-state integrase-based recorder of gene expression history and demonstrate its use in tracking gene expression events in Arabidopsis thaliana in two developmental contexts: lateral root initiation and stomatal differentiation. The recorder uses two serine integrases to mediate sequential DNA recombination events, resulting in step-wise, history-dependent switching between expression of fluorescent reporters. By using promoters that express at different times along each of the two differentiation pathways to drive integrase expression, we tie fluorescent status to an ordered progression of gene expression along the developmental trajectory. In one snapshot of a mature tissue, our recorder is able to reveal past gene expression with single cell resolution. In this way, we are able to capture heterogeneity in stomatal development, confirming the existence of two alternate paths of differentiation., (© 2024. The Author(s).)

Neuromuscular signal transmission is affected in various diseases including myasthenia gravis, congenital myasthenic syndromes, and sarcopenia. We used an ATF2-luciferase system to monitor the phosphorylation of MuSK in HEK293 cells introduced with MUSK and LRP4 cDNAs to find novel chemical compounds that enhanced agrin-mediated acetylcholine receptor (AChR) clustering. Four compounds with similar chemical structures carrying benzene rings and heterocyclic rings increased the luciferase activities 8- to 30-folds, and two of them showed continuously graded dose dependence. The effects were higher than that of disulfiram, a clinically available aldehyde dehydrogenase inhibitor, which we identified to be the most competent preapproved drug to enhance ATF2-luciferase activity in the same assay system. In C2C12 myotubes, all the compounds increased the area, intensity, length, and number of AChR clusters. Three of the four compounds increased the phosphorylation of MuSK, but not of Dok7, JNK. ERK, or p38. Monitoring cell toxicity using the neurite elongation of NSC34 neuronal cells as a surrogate marker showed that all the compounds had no effects on the neurite elongation up to 1 μM. Extensive docking simulation and binding structure prediction of the four compounds with all available human proteins using AutoDock Vina and DiffDock showed that the four compounds were unlikely to directly bind to MuSK or Dok7, and the exact target remained unknown. The identified compounds are expected to serve as a seed to develop a novel therapeutic agent to treat defective NMJ signal transmission., Competing Interests: Declaration of competing interest The authors declare that none of the authors have conflict of interest in this manuscript., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Genes are often regulated by multiple enhancers. It is poorly understood how the individual enhancer activities are combined to control promoter activity. Anecdotal evidence has shown that enhancers can combine sub-additively, additively, synergistically, or redundantly. However, it is not clear which of these modes are more frequent in mammalian genomes. Here, we systematically tested how pairs of enhancers activate promoters using a three-way combinatorial reporter assay in mouse embryonic stem cells. By assaying about 69,000 enhancer-enhancer-promoter combinations we found that enhancer pairs generally combine near-additively. This behaviour was conserved across seven developmental promoters tested. Surprisingly, these promoters scale the enhancer signals in a non-linear manner that depends on promoter strength. A housekeeping promoter showed an overall different response to enhancer pairs, and a smaller dynamic range. Thus, our data indicate that enhancers mostly act additively, but promoters transform their collective effect non-linearly., Competing Interests: MM, Bv No competing interests declared, FC co-founder of enGene Statistics GmbH, (© 2023, Martinez-Ara et al.)

The crustacean Daphnia magna is an emerging model for ecological and toxicological genomics. However, the lack of methods for spatial and temporal control of gene expression has impaired the elucidation of molecular mechanisms underlying responses to environments in vivo. Here we report local activation of the hsp70 promoter-driven gene cassette in D. magna by the infrared laser-evoked gene operator (IR-LEGO), a method for heating the target cells with infrared irradiation. We identified the heat-inducible promoter upstream of the D. magna hsp70-A gene. Using this promoter, we generated a transgenic Daphnia harboring the heat-shock responsive GFP reporter gene and confirmed that the GFP gene responds to heat treatment not only in juveniles and adults but also in embryos. We collected embryos from the reporter line and irradiated four different regions of interest in the embryos: a proximal region of the third thoracic segment, a part of the midline, a second maxilla, and a distal region of the endopodite of the second antenna, all of which increased GFP fluorescence with an infrared laser. Our results suggest that the IR-LEGO method is useful for spatial and temporal control of gene expression and would advance the functional genomics in D. magna., (© 2024. The Author(s).)

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.