Cardiovascular disease remains the leading cause of death worldwide, with coronary artery disease being the primary contributor. Our recent studies suggest that activation of LepRs (leptin receptors) in the brain can improve cardiac function after myocardial infarction. However, the mechanism by which this cardioprotective effect is transmitted from the brain to the heart remains unclear. We hypothesize that brain LepR activation stimulates brown adipose tissue (BAT) to secrete extracellular vesicles (EVs) enriched with cardioprotective factors. These EVs may safeguard the heart by modulating cardiac mitochondrial function and collagen deposition. Sprague-Dawley rats with BAT intact, BAT ablation, or BAT sympathetic denervation were implanted with an intracerebroventricular cannula for continuous leptin or vehicle delivery over 28 days after cardiac ischemia-reperfusion injury. Cardiac function was assessed weekly via echocardiography and by ventricular catheterization at the end of the protocol. EVs were isolated from BAT for analysis. Rab27a, a protein required for EV release, was knocked down using adeno-associated virus, and EV tracking was conducted using a double fluorescent reporter mouse model. Our findings indicate that BAT ablation or BAT sympathetic denervation diminishes the cardioprotective effects of brain LepR activation. We also observed an increased concentration of EVs within the BAT of rats treated with intracerebroventricular leptin compared with vehicle-treated controls, an effect abolished by BAT denervation. Furthermore, knockdown of Rab27a in BAT reduced the cardioprotective benefits of brain LepR activation. MicroRNA-29c-3p was identified as a cargo of leptin-stimulated BAT-derived EVs and appears to play a key role in mitigating cardiac fibrosis after ischemia-reperfusion injury in leptin-treated animals. Activation of LepR in the brain protects the heart after ischemia-reperfusion injury via sympathetic-mediated BAT-derived EVs enriched with microRNA-29c-3p.

CTNNB1, the gene encoding β-catenin, is a frequent target for oncogenic mutations activating the canonical Wnt signaling pathway, typically through missense mutations within a degron hotspot motif in exon 3. Here, we combine saturation genome editing with a fluorescent reporter assay to quantify signaling phenotypes for all 342 possible missense mutations in the mutation hotspot. Our data define the genetic requirements for β-catenin degron function, refine the consensus motif for substrate recognition by β-TRCP and reveal diverse levels of signal activation among known driver mutations. Tumorigenesis in different human tissues involves selection for CTNNB1 mutations spanning distinct ranges of predicted activity. In hepatocellular carcinoma, mutation effect scores distinguish two tumor subclasses with different levels of β-catenin signaling, and weaker mutations predict greater immune cell infiltration in the tumor microenvironment. Our work provides a resource to understand mutational diversity within a pan-cancer mutation hotspot, with potential implications for targeted therapy.

Zhi-Shen Pill ameliorates impaired glucose tolerance by targeting pancreatic β-cell and adipocyte pathways.

Development of carbon monoxide-tolerant fluorescent reporter system functional under anoxic conditions in Eubacterium callanderi KIST612.

Influenza A virus (IAV) is one of the most important zoonotic pathogens and can cause global influenza pandemics and seasonal influenza outbreaks. Generation of recombinant IAV expressing a fluorescent protein will allow the infection to be easily monitored. In this study, we initially constructed a replication-defective H1N1/ΔPB2-GFP and a replication-competent H1N1/NS-GFP. However, these two reporter IAVs exhibited genetic instability. To stabilize the recombinant viral genome, we recoded the gfp sequence (rGFP) using synonymous codons to mimic the high-NP-binding regions involved in NP-vRNA interaction. This approach resulted in the development of replication-defective H1N1/ΔPB2(300)-rGFP and replication-competent H1N1/NS-rGFP, both of which exhibited enhanced stability in GFP expression. By replacing the HA segment from strain A/mink/China/CY 2017 (H5N1), we also generated a replication-defective H5N1/ΔPB2(300)-rGFP, which showed excellent genetic stability. Using these reporter IAVs, the blocking of virus infection by neutralizing antibodies and antivirals can be rapidly detected by the loss of fluorescent reporter expression. Replication-defective reporter IAVs constructed in this study can only infect and replicate in cells expressing PB2, allowing the possibility of manipulation of highly pathogenic IAV and their related reassortant strains in biosafety level-2 laboratories. Our data highlight the importance of NP-vRNA interaction for the stability of IAV genome, and the reporter IAVs generated using this strategy could be powerful tools for both basic and applied influenza virus research.

CRISPR-Cas-based live-cell imaging has rapidly become a central technology for studying genome dynamics with high specificity and flexibility. By coupling nuclease-deactivated Cas (dCas) with programmable guide RNAs, genomic loci can be tracked in living cells, providing direct insights into nuclear organization and chromatin behavior. While repetitive regions such as telomeres and centromeres are readily visualized, labeling non-repetitive loci remains more challenging due to weak signals and high background. Recent advances, including multicolor labeling strategies, innovative amplification systems based on dCas9 and single-guide RNA (sgRNA) engineering, and integration with novel fluorescent reporters, have markedly expanded the applicability of CRISPR imaging across the genome. These developments have expanded the multiplexing capacity of CRISPR imaging, improved signal-to-background ratios, and even enabled the visualization of non-repetitive genomic loci. Nonetheless, key challenges remain, including cellular toxicity, replication stress, and genomic instability associated with prolonged CRISPR expression. In this review, we summarize recent advances in CRISPR live-cell imaging and highlight key design trade-offs and biological constraints.

The increasing demand for rapid identification of bacteria capable of degrading environmentally relevant organic compounds highlights the need for scalable and selective analytical tools. Cupriavidus necator catabolizes several hydroxybenzoic acids, including 2-hydroxybenzoate (salicylate, 2-HBA), 4-hydroxybenzoate (4-HBA), and 3-hydroxybenzoate (3-HBA), funneling them into central aromatic catabolism via monooxygenation to 2,5-dihydroxybenzoate (gentisate, 2,5-dHBA) and 3,4-dihydroxybenzoate (protocatechuate, 3,4-dHBA) followed by the oxidative cleavage reaction, enabling complete conversion to tricarboxylic acid (TCA) cycle intermediates. To quantify how readily C. necator is able to activate catabolic genes in response to hydroxybenzoic acid, an extracellular ligand, we applied an approach centered on a transcription-factor (TF)-based biosensor that combines ligand-bound regulator activity with a fluorescent reporter. This approach allowed to evaluate the ligand sensitivity by determining gene activation threshold ACmin and half-maximal effective concentration EC50. Amongst studied hydroxybenzoic acids, 2-HBA and 4-HBA sensors from C. necator showed very low thresholds 4.8 and 2.4 μM and EC50 values of 19.91 and 13.06 μM, indicating high sensitivity to these compounds and implicating a scavenging characteristic of associated catabolism. This study shows that the TF-based-biosensor approach applied for mapping functional sensing ranges of hydroxybenzoates combined with the research and informatics of catabolism can advance our understanding of how gene expression regulation systems have evolved to respond differentially to the availability and concentration of carbon sources. Furthermore, it can inform metabolic engineering strategies in the prevention of premature pathway activation or in predicting competitive substrate hierarchies in complex mixed environments.

Derivation and analysis of human somatic sensory neuron subtypes facilitated through fluorescent hPSC reporters.

An innovative triple-channel probing strategy for ultrasensitive detection of quercetin in food matrices based on carbon dots.

The mitochondrial genome (mtDNA) encodes essential subunits of the electron transport chain and ATP synthase. Mutations in these genes impair oxidative phosphorylation, compromise mitochondrial ATP production and cellular energy supply, and can cause mitochondrial diseases. These consequences highlight the importance of mtDNA quality control (mtDNA-QC), the process by which cells selectively maintain intact mtDNA to preserve respiratory function. Here, we developed a high-throughput flow cytometry assay for Saccharomyces cerevisiae to track mtDNA segregation in cell populations derived from heteroplasmic zygotes, in which wild-type (WT) mtDNA is fluorescently labeled and mutant mtDNA remains unlabeled. Using this approach, we observe purifying selection against mtDNA lacking subunits of complex III (COB), complex IV (COX2) or the ATP synthase (ATP6), under fermentative conditions that do not require respiratory activity. By integrating cytometric data with growth assays, qPCR-based mtDNA copy-number measurements, and simulations, we find that the decline of mtDNAΔatp6 in populations derived from heteroplasmic zygotes is largely explained by the combination of its reduced mtDNA copy number—biasing zygotes toward higher contributions of intact mtDNA—and the proliferative disadvantage of cells carrying this variant. In contrast, the loss of mtDNAΔcob and mtDNAΔcox2 cannot be explained by growth defects and copy-number asymmetries alone, indicating an additional intracellular selection against these mutant genomes when intact mtDNA is present. In heteroplasmic cells containing both intact and mutant mtDNA, fluorescent reporters revealed local reductions in ATP levels and membrane potential () near mutant genomes, indicating spatial heterogeneity in mitochondrial physiology that reflects local mtDNA quality. Disruption of the respiratory chain by deletion of nuclear-encoded subunits (RIP1, COX4) abolished these physiological gradients and impaired mtDNA-QC, suggesting that local bioenergetic differences are required for selective recognition. Together, our findings support a model in which yeast cells assess local respiratory function as a proxy for mtDNA integrity, enabling intracellular selection for functional mitochondrial genomes.

Autophagy is a conserved catabolic pathway that preserves cellular homeostasis through lysosomal degradation. Beyond its general role in proteostasis, selective autophagy mediates the clearance of selective cellular targets such as persistent stress granules (SGs), in a process termed granulophagy. SGs are dynamic cytoplasmic assemblies that normally disassemble after stress relief; however, their aberrant persistence has arisen as a pathological feature of neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS). However, the molecular regulation of granulophagy remains incompletely understood. Here, we established a tandem fluorescent SG reporter system with mCherry-pHluorin-FUSP525L, enabling live-cell visualization of granulophagic flux. Using this system, we screened a chemical library and identified VR23, a proteasome inhibitor, as a potent inducer of granulophagy. VR23 promoted SG clearance through autophagic mechanisms, as evidenced by enhanced LC3 colocalization, lysosome-dependent degradation, and Bafilomycin A1-sensitive flux. Notably, disruption of SG assembly via G3BP1 inhibition abolished VR23-induced clearance, confirming its SG selectivity. These findings suggest a link between proteasome inhibition and granulophagy, highlighting VR23 as a valuable tool compound to dissect the mechanisms of SG turnover, and provide a platform for discovering modulators of pathological SG clearance in protein aggregation.

Neuromodulatory systems regulate neural circuits across broad regions of the brain, and disruption of the dopaminergic system contributes to psychiatric and neurodegenerative disorders. Engineered viral vectors have been used to target the neuromodulatory systems of the nonhuman primate brain (Y. Chen et al., 2023; El-Shamayleh et al., 2016; Gray et al., 2010; Lerchner et al., 2014; Perez et al., 2022). However, a conspicuous obstacle to the isolation and modulation of specific pathways is the inability of many retrogradely infecting viruses to transduce dopaminergic (DA) cells efficiently (Tervo et al., 2016; Cushnie et al., 2020; Weiss et al., 2020). We compare the DA neuron retrograde transduction efficacy of four viral vectors after injection into the striatum of nonhuman primates (NHP). Selectivity was assessed by comparing the neuronal co-expression of fluorescent reporter protein and tyrosine-hydroxylase (TH) antibody in substantia nigra pars compacta (SNc). The rabies pseudotyped lentiviral vector, FuG-B2, produced superior retrograde transduction of DA cells to FuG-C or FuG-E. AAV2.retro was the least effective.

Abstract Introduction and aims Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disease with over 800 causative mutations identified in the COL7A1 gene. eePASSIGE is a breakthrough DNA editing technology that pairs prime editing (PE) with serine integrase technology, allowing for targeted integration of large DNA constructs. We aim (i) to use eePASSIGE to integrate a full-length cDNA of COL7A1 into the AAVS1 safe harbour locus, and (ii) to develop lipid-based nanoparticles (LNPs) capable of delivering eePASSIGE machinery to human skin cells. Together, these two strategies form the basis for a proposed permanent ‘one-size-fits-all’ DEB cure. Methods We have used lipofection of HEK293T, N/TERT, and human fibroblast cells to provide proof of concept. For PE and eePASSIGE, lipofectamine was used to deliver plasmid DNA encoding editing constructs and pegRNAs. For PE, Sanger sequencing of target site amplicons was used to identify successful edits. For eePASSIGE, puromycin selection was used to isolate successfully edited cells. For LNP studies, fluorescent reporter mRNA was delivered to N/TERTs and fibroblasts; successful transfection was quantified with flow cytometry. Results In N/TERT cells, we achieved up to 17% PE efficiency in transfected cells, although only 1% of cells were successfully transfected. eePASSIGE was used to successfully integrate a puromycin resistance gene into HEK293T cells at the AAVS1 safe harbour locus. Lipid nanoparticles were used to successfully transfect human fibroblasts and keratinocytes in submerged culture with reporter constructs with up to 80% efficiency and with lower toxicity than lipofectamine. Conclusions Our experiments thus far have laid the groundwork for future study of eePASSIGE to integrate large coding constructs into disease-relevant cells. Our next steps are (i) to improve transfection of eePASSIGE machinery in cells of interest, and (ii) to optimize LNP formulations for use in three-dimensional culture and in vivo mouse skin.

During herpesvirus replication, capsids are assembled inside the nucleus and translocated into the cytosol by a non-canonical nucleocytoplasmic export process termed nuclear egress. Traditional methods of measuring nuclear egress rely on imaging-based technologies such as confocal and electron microscopy. These techniques are labor-intensive, limited by the number of cells that can be examined, and may not accurately represent the entire population, generating a potential bias during data interpretation. To overcome these problems, we have developed a flow cytometry–based method to measure HSV-1 nuclear egress that we termed FLARE (FLow cytometry–based Assay of nucleaR Egress). This assay uses a double fluorescent reporter system, utilizing HSV-1-tdTomato to identify infected cells and an Alexa Fluor-488-conjugated, capsid-specific antibody to detect cytosolic capsids, thereby distinguishing infected cells with nuclear egress from those without it. This assay provides more quantitative results than traditional methods, enables large-scale high throughput, and can be adapted for use with other herpesviruses.Key features• Quantification of HSV-1 nuclear egress by flow cytometry using a double fluorescent reporter system.• The assay is suitable for large-scale high-throughput screens, e.g., CRISPR/Cas9.• The assay can be adapted for use with other herpesviruses, provided a mature capsid-specific antibody is available.

Cationic Amphiphilic Drugs (CADs) severely disrupt lysosomal function, which leads to a cellular pathology characterized by excess phospholipids called phospholipidosis. Through a forward genetic screen and mining of published datasets, we discovered that CADs induce the expression of the CYP-35B family of cytochrome P450s and the PGP-13 p-glycoprotein pump via the nuclear receptors NHR-70 and NHR-107 in the nematode C. elegans. A pgp-13 fluorescent reporter revealed hundreds of human drugs that upregulate the CAD defense system in vivo. Chemoinformatic analyses indicate that the pgp-13 reporter may be useful in identifying CADs that have pathogenic potential in humans. Mutant analyses coupled to metabolomics and structural modeling show that the CYP-35Bs are necessary and sufficient for CAD metabolism, and that CYP-35B2 D311 is key in mediating electrostatic interactions with the positively charged CADs. We also show that CAD metabolites are effluxed via PGP-13 acting partially redundantly with PGP-14 and that an intact defense system is necessary to resist CAD-induced pathology. Finally, we demonstrate that bacteria that likely cohabitate with C. elegans in nature trigger the CAD defense system, providing a plausible explanation for why a pathway that protects against anthropogenic small molecules exists in nematodes.

Preparation of a Golgi-targetable and photo-triggered NO donor and its application in living cells and zebrafish imaging.

.SignificanceThe combination of two-photon calcium imaging and targeted two-photon optogenetic stimulation, termed all-optical interrogation, provides spatial and temporal precision when recording and manipulating neural circuit activity in vivo. All-optical experiments often use red-shifted opsins in combination with green fluorescent reporters of neuronal activity. However, their excitation spectra still partially overlap, meaning that the imaging laser can excite the opsin. Although some care has been taken in the past to understand the effects of this spectral overlap; further work is required to understand its impact on the findings of all-optical studies.AimWe aimed to investigate whether two-photon imaging of the green fluorescent calcium reporter GCaMP6s at 920 nm increase the rate of response desensitization in neurons targeted for two-photon stimulation at 1035 nm expressing the red-shifted opsin C1V1.ApproachWe systematically varied either the inter-stimulus interval or the duration of two-photon calcium imaging during targeted two-photon optogenetic stimulation of mouse layer 2/3 barrel cortex or visual cortex neurons.ResultsWe found that two-photon imaging at 920 nm decreases trial-by-trial photostimulation responses in targeted C1V1-expressing neurons—an effect that is exacerbated at shorter inter-stimulus intervals. This is consistent with the imaging laser increasing the rate of opsin desensitization. Reduced photostimulation responses are not limited to targeted cells and are found across the field of view. Such network effects are less pronounced at shorter imaging doses.ConclusionsOur results provide methodological optimizations that enable trial-by-trial decreases in photostimulation response to be mitigated in all-optical experiments. This will reduce an external source of trial-by-trial variability in future all-optical experiments.

High-affinity poly-cytosine DNA modulated metal-organic framework-DNA interactions and their application in detection of hepatocellular carcinoma biomarker.

CD200, an immunoregulatory glycoprotein of the immunoglobulin superfamily, suppresses inflammatory signaling by engaging its receptor CD200R, which is predominantly expressed on myeloid cells. To enhance the immune-evading properties of viral vectors, we engineered lentiviral particles displaying the CD200 ectodomain (CD200ED) to exploit anti-inflammatory response and phagocytosis resistance. A fusion gene encoding the mouse CD200 ectodomain and core streptavidin (CD200ED-coreSA) was cloned into the pET-30a(+) plasmid, expressed in E. coli Lemo21(DE3), and purified via immobilized metal affinity chromatography (IMAC). Successful protein assembly was confirmed by SDS-PAGE and western blot. Biotinylated VSV-G pseudotyped lentiviral vectors, encoding a green fluorescent protein reporter, were functionalized with CD200ED-coreSA. When exposed to murine J774A.1 macrophages, CD200ED-modified lentiviruses significantly reduced pro-inflammatory cytokine production - evidenced by 47.1% decrease in TNF-α and 55% decrease in IL-6 - compared to unmodified controls. Additionally, CD200ED anchoring reduced macrophage phagocytosis of lentiviral particles by 25%. These findings demonstrate that CD200-tethering confers dual anti-inflammatory and phagocytosis resistance capabilities to viral vectors, offering a promising strategy to improve gene delivery efficiency in inflammatory environments.

The DHHC palmitoyltransferase Zdhhc22 is known to play a role in neuronal differentiation, synaptic regulation, and brain development. While transcriptomic data hint at region-specific expression, its exact spatiotemporal and cell-type distribution in the mammalian brain is unclear. For this purpose, we generated a bacterial artificial chromosome (BAC) transgenic mouse line that expresses the mCherry fluorescent reporter driven by the Zdhhc22 promoter. We then analyzed Zdhhc22 expression from embryonic day 13.5 (E13.5) through adulthood. mCherry fluorescence was detected in many brain regions, including the cortex, thalamus, midbrain, piriform cortex, and brainstem. Interestingly, a dynamic developmental gene expression pattern was observed: Zdhhc22 expression was initially restricted to the cortical marginal zone between E13.5 and E15.5, it then expanded into deeper cortical layers by E17.5, and at postnatal day 0 (P0), it persisted in deep layers while also appearing in a new subset of cortical plate neurons. Through co-immunostaining, mCherry expression was found to be predominantly neuronal, showing strong co-localization with NeuN and minimal overlap with glial cells. In the cortex, Zdhhc22 expression showed no co-localization with CUX1 or CTIP2 but did partially overlap with FOG2, a marker for layer VI pyramidal neurons. A particularly striking finding was that nearly all marginal zone mCherry-positive cells co-expressed RELN, identifying them as Cajal–Retzius cells. This neuronal specificity was maintained in the adult brain. Our findings validate the Zdhhc22-mCherry BAC transgenic line as a faithful model of endogenous Zdhhc22 expression, providing invaluable insight into its cellular specificity and a powerful new tool for future research.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00429-026-03075-y.