搜索结果：Biomedical physics & engineering express

Acoustic reporter genes (ARGs) enable genetically engineered bacteria to express gas vesicles (GVs), which function as intracellular acoustic scatterers for noninvasive ultrasound imaging. While second-generation ARGs (bARGSer) exhibit enhanced nonlinear acoustic properties, their signal-level acoustic characteristics remain incompletely understood. Moreover, how basic nonlinear pulse schemes and imaging parameters should be selected based on these acoustic characteristics for bARGSer-expressing bacteria has not been systematically evaluated. In this study, we investigated the pressure- and frequency-dependent acoustic characteristics of Escherichia coli BL21 expressing bARGSer, and compared with purified gas vesicles (GVSer) isolated from bacteria under identical conditions. Collapse behavior and nonlinear acoustic responses were characterized at selected imaging frequencies (5, 8, and 10 MHz) under varying acoustic pressures, while attenuation properties were measured over a broader frequency range (4-20 MHz). Several basic nonlinear pulse schemes were evaluated to assess their contrast-to-tissue ratio (CTR) performance for bARGSer-expressing bacteria under acoustically characterized pressure conditions. Engineered bacteria exhibited progressive vesicle collapse at acoustic pressures exceeding 1.5 MPa across all tested frequencies, indicating minimal frequency dependence of the collapse threshold within the investigated range. Below the collapse threshold, pressure-dependent nonlinear scattering was observed above 0.7 MPa, with harmonic amplitudes exceeding those of linear scatterers. Higher transmission frequencies resulted in increased harmonic-to-fundamental ratios, although detection bandwidth limitations influenced spectral observations. Attenuation measurements revealed frequency- and concentration-dependent increases in acoustic attenuation, while pressure-dependent attenuation exhibited a non-monotonic trend, increasing prior to collapse and decreasing thereafter due to vesicle destruction. In addition, compared with intracellular GVs, purified GVSer demonstrated slightly lower collapse thresholds and stronger nonlinear responses, and lower acoustic attenuation. Phantom experiments comparing multi-pulse imaging schemes revealed that, among the evaluated schemes, triple-pulse amplitude modulation (AM3) at 1.81 MPa provided the highest CTR under the tested conditions, where partial GV collapse began to occur. In vivo tumor imaging further confirmed enhanced visibility of the bacterial region using this optimized AM3 condition. These findings provide a basis for optimizing imaging frequency, pressure, and basic pulse-scheme selection in ultrasound imaging of second-generation ARGs-expressing bacteria, and may inform future development of GV-specific nonlinear imaging methods.

In the radiation biodosimetry field, transcriptomic studies seek to determine whether the expression of a small set of genes can be used to estimate the radiation dose that a person was exposed to. Most such studies consider only photon exposure, but in the detonation of an improvised nuclear device, neutrons would likely comprise a significant proportion of the dose. In this study, we compare the effects on gene expression in human blood between two neutron-producing radiation sources: a source designed to mimic the neutron energy spectrum of a nuclear weapon (CINF; the Columbia IND Neutron Facility at the Radiological Research Accelerator Facility) and a source from a nuclear reactor (RINSC; the Rhode Island Nuclear Science Center). The radiation sources have very different neutron energy spectra: mainly fast neutrons (CINF) versus thermal neutrons (RINSC). We have compared differential gene expression at 24 h after exposure to 0, 0.5, 1, 2, and 4 Gy total doses delivered to human blood samples from the same donors in parallel at the two sites. Our analysis suggests that there is a core response to radiation that is conserved at both sites, with larger magnitude and additional components in the CINF response. These results suggest that neutron energy does affect gene expression, and that this must be accounted for in the development of biodosimetry approaches.

With this status report, we aim to provide a timely snapshot of the protein engineering field as a broad and rapidly advancing discipline that integrates computational, molecular biology, structure-guided, evolutionary, and synthetic approaches to create new and improved proteins with tailored structures and useful functions. The report is organized into eight thematic areas spanning core methodologies and major application domains, including enzymes, therapeutics, detection, synthetic biology, and materials. Contributions from experts across these areas highlight both the historical foundations and recent advances in their respective fields, with particular emphasis on the growing influence of machine learning and artificial intelligence-based methods. Emerging from this broad overview is a central message: protein engineering appears to be entering a golden age, defined by a rapidly accelerating pace of progress, even as significant challenges in design, screening, and real-world application remain. Looking ahead, the continued integration of computational and experimental strategies is poised to further accelerate the impact of protein engineering across an expanding range of economically and societally important sectors, from therapeutics and molecular imaging to diagnostics, plastic recycling, and industrial chemistry.

Transcranial ultrasound stimulation (TUS) is an emerging non-invasive neuromodulation modality that offers deep brain access with high spatial precision. However, its broader application is limited by the difficulty of reliably generating complex transcranial acoustic fields, particularly for multi-target stimulation through the skull. These limitations can lead to focal distortion, off-target exposure, and reduced reliability of neuromodulation outcomes. Here, we introduce a physics-aware thickness-only acoustic hologram (TOAH) technique for precise transcranial ultrasound neuromodulation. Unlike conventional approaches that rely on simplified phase-based approximations, TOAH directly generates fabrication-ready holographic implementations while preserving consistency between numerical field synthesis and physical acoustic realization. This enables accurate formation of single-, dual-, and tri-focal stimulation patterns under transcranial conditions. We validated TOAH through in silico simulations, ex vivo acoustic measurements through skulls, and in vivo experiments. Compared with state-of-the-art methods, TOAH improved focal reconstruction, energy confinement, and multi-focal balance while reducing off-target acoustic leakage. Human-skull simulations further supported robust multi-focal reconstruction under clinically relevant transcranial conditions. In a neuropathic pain mouse model, bilateral thalamic stimulation induced measurable changes in neuronal activity, reflected by reduced c-Fos expression, together with preliminary improvements in pain-related behavioral responses. These findings support the capability of the proposed technique to enable spatially localized and reproducible neuromodulation in vivo. Collectively, this work provides a practical proof-of-concept strategy for achieving high-precision, multi-target transcranial neuromodulation and supports further investigation for neuroscience research and future therapeutic applications.

DNA-protein interactions underlie genome activity, governing gene expression as well as the physical organisation of DNA. Until recently, DNA-protein complexes were predominantly described at atomic resolution using short DNA fragments, concealing how proteins recognise and manipulate the long, supercoiled DNA present in cells. Now single-molecule imaging and cryo-electron microscopy (cryo-EM) are showing how longer DNA sequences are recognised by proteins and computations are predicting how these elements influence larger-scale genomic structures. Here we discuss how the polymeric nature of DNA influences its atomic-level structure and dynamics, as well as the implications for DNA recognition and ultimately biological function. We emphasise how theory and simulation help interpret these effects, which are difficult to replicate using conventional experimental settings.

Transcriptional condensates concentrate the machinery required for RNA polymerase II mediated transcription. These structures range from numerous small, short-lived species, to a handful of larger, stable assemblages. Large condensates have been implicated in driving potent transcription of several super-enhancer regulated genes, yet the underlying mechanisms and the range of their client genes remain unclear. Here, we developed a biochemical approach which combines density gradient centrifugation and affinity purification to partially purify large transcriptional condensates from nuclei, allowing systematic characterization of their nucleic acid components. We find that transcriptional condensate isolates engage thousands of gene promoters and harbor the nascent transcriptome, but do not stably co-purify with distal enhancers. Binding patterns of RNA polymerase II within condensates suggest these structures could facilitate promoter escape and promoter-proximal pause release. Together, our work supports a promoter-centric condensate organization and paves the way towards understanding the functional link between condensate architecture and nascent transcription.

Ephrin type-A receptor 2 (EphA2) is a promising target in pancreatic ductal adenocarcinoma (PDAC). However, the clinical potential of targeting EphA2 requires further imaging evaluation. This study developed and preliminarily evaluated a novel 68Ga-labeled radiotracer for PET imaging of EphA2 expression in PDAC patients. 68Ga-FZEAR-1, 68Ga-FZEAR-2, and 68Ga-FZEAR-3 were synthesized, and their stability, affinity, pharmacokinetics were evaluated in vitro and in vivo. Further biological evaluation was performed in EphA2-positive and EphA2-negative tumor xenografts. A pilot first-in-human PET/CT study of the lead candidate, 68Ga-FZEAR-2, was subsequently conducted in two PDAC patients. The three 68Ga-radiotracers were synthesized with high radiochemical purity (> 99%) and demonstrated high stability and nanomolar affinity for EphA2. Cellular uptake was consistent with EphA2 expression and blocking assays confirmed specificity. In vivo, all tracers exhibited high tumor accumulation with low off-target uptake. 68Ga-FZEAR-2 showed favorable tumor-targeting ability, pharmacokinetics, and safety profile. In the pilot clinical study (n = 2), 68Ga-FZEAR-2 PET/CT visualized primary and metastatic lesions clearly without adverse effects. Immunohistochemical analysis confirmed EphA2 expression in lesions exhibiting high tracer uptake. A series of 68Ga-labeled tracers for EphA2 imaging was successfully developed and evaluated. The lead candidate, 68Ga-FZEAR-2, showed promising targeting specificity and favorable pharmacokinetics. A pilot study indicated that 68Ga-FZEAR-2 provided preliminary evidence of noninvasive visualization of EphA2 expression in PDAC patients, thereby suggesting its potential as a tool for precision diagnosis in PDAC.

The feature issue "Multimodal Optical Biosensing for Precision Medicine and Healthcare," published in Biomedical Optics Express, is a curated selection of advances in optical biosensing technologies and their applications in healthcare and precision medicine. The demand for precise, real-time, non-invasive diagnostic and therapeutic tools has led to multimodal optical biosensing as the recent direction by merging complementary methodologies (e.g., optical biosensors, optical coherence tomography, optical biophysics and photobiology, optical diagnostics, microscopy, and terahertz spectroscopy and imaging). This feature issue contains a collection of articles that showcase advances in sensor design and validation, signal processing and imaging methodologies, as well as clinical translation. The contributions published showcase tremendous advances in the microscale and nanoscale development of biosensing platforms and are directed toward uses including disease diagnosis, monitoring, and therapeutic guidance via improved sensitivity, specificity, accuracy, robustness, reliability. This collection will provide an avenue for publishing new research, fostering interdisciplinary collaboration between technology and medicine development, and advancing the field of next-generation optical biosensors towards precision healthcare.

Substantial progress has been made in developing artificial small-diameter vascular grafts (SDVGs), yet the contribution of perivascular adipose tissue (PVAT) to vascular graft healing remains poorly understood. This study investigated the interaction of biodegradable thermoplastic polyurethane (TPU) SDVGs with PVAT using in vitro and in vivo models. Vascular and perivascular cells were cultured on TPU and analyzed by viability assays, immunofluorescence staining, scanning electron microscopy, quantitative polymerase chain reaction, and secretome and cell lysate analysis. TPU grafts were implanted into male Sprague Dawley rats for 1 week or 3 months (control = sham; n = 6/group) and assessed for cellularization, expression of inflammation-related genes, and adipocyte subtype markers. TPU supported viability, attachment, and phenotype preservation of in vitro-seeded cells. Immunofluorescence staining revealed increased adiponectin expression, and adipokine profiling identified 14 substantially up-regulated adipokines in TPU-seeded PVAT-derived cells. Histology showed rapid cellular coverage of grafts within 1 week of implantation, including CD34+ endothelial progenitor cells on the lumen. Quantitative polymerase chain reaction demonstrated compartment-specific dynamics: Inflammatory markers within the grafts changed predominantly after 3 months, whereas adjacent PVAT showed an early response with Tnfa induction already at 1 week. PVAT further displayed evidence of desirable immune modulation, with altered expression of innate (Cd11c and Arg1) and adaptive (Foxp3 and Tbet) immune response markers, alongside partial white-to-brown adipocyte remodeling. These findings highlight PVAT's central role-through adipokine secretion, adipocyte browning, and inflammation regulation-in vascular graft healing and underscore the importance of considering PVAT-driven adipokine dynamics in SDVG design to ensure long-term patency.

B7-H3 (CD276) is an emerging target for cancer theranostics, highlighting the need for imaging probes capable of noninvasively quantifying B7-H3 expression in tumors. Here, we developed three 68Ga-labeled B7-H3-targeting bicyclic peptide tracers with different PEG linker lengths. All tracers showed high radiochemical purity (>96%), favorable in vitro stability, and rapid blood clearance. Among them, [68Ga]Ga-B7H3-FZ1 exhibited the highest affinity (KD = 83.22 nM). Micro-PET/CT imaging demonstrated that tumor uptake of [68Ga]Ga-B7H3-FZ1 correlated positively with B7-H3 expression across multiple tumor models. In H1299 tumors. B7-H3 overexpression increased uptake from 1.09 ± 0.18 to 3.50 ± 0.97%ID/g at 30 min postinjection, confirming target specificity. Biosafety studies indicated no obvious toxicity. These results support [68Ga]Ga-B7H3-FZ1 as a promising PET tracer for noninvasive B7-H3 imaging.

Spatial transcriptomic techniques provide a wealth of information useful in guiding drug development, while three-dimensional (3D) cell cultures have demonstrated power in accelerating drug approvals. However, techniques for robust spatial analysis of 3D cultures are limited. Here, we present a transfection-based method for constructing cellular spheroids through a layer-by-layer approach, in which DNA barcodes encode the spatial positioning of cells. Our technique facilitates multiplex single-cell RNA sequencing, providing spatial maps of gene expression and drug response, while correlative imaging reveals the locations of barcoded cell populations and quantifies local tissue elasticity. We show that model HeLa 3D spheroids display heterogeneous responses to drugs, which may arise through diffusion gradients of the drug, or from differences in metabolism, nutrient supply, and cellular stressors. The ability to create spatially encoded cellular assemblies may help to reveal spatial variation in gene expression within 3D culture models.

Objective.Glioblastoma exhibits highly heterogeneous growth and infiltration patterns, limiting the predictive value of routine imaging and population-averaged tumor growth models. This study aimed to develop a clinically feasible, biomarker-informed reaction-diffusion framework that integrates standard magnetic resonance imaging with routinely available immunohistochemical (IHC) markers to support patient-specific modeling of glioblastoma growth.Approach.A reaction-diffusion model was formulated using standard clinical MRI sequences, including contrast-enhanced T1-weighted and T2-weighted images, together with IHC biomarkers, particularly the antigen protein (Ki-67) and Isocitrate dehydrogenase 1 (IDH-1) status. Baseline diffusion and proliferation parameters were estimated from MRI-derived growth metrics. A pathological coefficient was introduced as a biologically interpretable modifier of the proliferation term to incorporate microscopic tumor aggressiveness into the macroscopic growth model. The coefficient was identified through constrained inverse parameter estimation and subsequently interpreted using subgroup-specific representative values. Short-term agreement between simulated tumor extent and postoperative follow-up MRI was assessed within the available cohort.Main results.The proposed framework improved agreement between simulated and observed tumor extent within the studied cohort and demonstrated biologically coherent behavior across molecular subgroups. The pathology-informed proliferation term allowed the model to reflect differences in tumor aggressiveness associated with Ki-67 expression and IDH-1 status. In addition, the model generated spatial representations of tumor infiltration extending beyond the visible margins detected by routine MRI, highlighting regions of potential microscopic spread that are not directly observable on standard imaging.Significance.This study presents an exploratory step toward linking routinely acquired pathological biomarkers with physics-based glioblastoma growth modeling, by using standard-of-care MRI and IHC data, the proposed framework provides a clinically accessible tumor-growth prior that may support future treatment-response modeling, survival analysis, and individualized planning. Further validation in larger independent cohorts and extension to three-dimensional modeling are required before prospective clinical application.

Fluoride (F) exposure is widely recognized for its role in systemic fluorosis; however, its effects on alveolar bone, a structurally and functionally complex tissue, remain poorly understood. This study investigated the molecular, physicochemical, and morphological alterations in alveolar bone following prolonged, dose-dependent F exposure. Thirty male Swiss mice were assigned to three groups receiving deionized water containing 0, 10, or 50 mg F/L for 60 days. Plasma F levels were measured, and hemimandibles were analyzed using proteomics, gene expression, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, micro-computed tomography, as well as osteocyte density and collagen content assessments. F exposure increased plasma levels in a dose-dependent manner and promoted molecular dysregulation involving proteins associated with DNA organization, cytoskeleton, and energy metabolism, alongside altered expression of genes related to BMP, TGFB1, IL, CCL, MMP, and RANKL pathways. These changes were accompanied by modifications in the mineral and physicochemical profile, including reduced crystallinity and alterations in phosphate, carbonate, and amide composition. Structural impairments were evidenced by reduced osteocyte density, decreased collagen content, and compromised alveolar bone architecture, particularly at higher exposure levels. Collectively, these findings demonstrate that prolonged fluoride exposure induces dose-dependent alterations linking molecular dysregulation to structural impairment in alveolar bone, providing mechanistic insight into how environmental exposure may impact oral tissue integrity and function.

Ribosomes are central to protein synthesis and a frequent target for antibiotics. In fast-growing bacteria, the ribosome content is proportional to the growth rate; how ribosomes and protein synthesis are regulated during nutrient starvation remains poorly understood, particularly in single cells. To address this, we fluorescently labeled ribosomal proteins (RPs) in Salmonella and explored their variations and regulation in single cells. We show that the RP levels become heterogeneous during the transition to the stationary phase. Unexpectedly, cells with higher RP levels responded less to the induction of gene expression but accumulated more virulence gene products. Our work further reveals that adenosine 3',5'-monophosphate (cAMP) signaling increases the heterogeneity of the levels of RPs and other gene products. Fluorescence dilution assay and proteomic analysis indicate that cAMP signaling promotes gene expression heterogeneity by directing proteome-wide adaptation, which enables growth heterogeneity, hence differential dilution of gene products during nutrient depletion.

Objectives: Low-intensity pulsed ultrasound (LIPUS) has been widely utilized as a biophysical modality accelerating fracture healing, particularly in bones undergoing endochondral ossification. However, its efficacy in facilitating intramembranous ossification remains unclear. This study aimed to evaluate the effects of LIPUS and autologous bone (AB) on bone healing in a critical-size bone defect (CSBD) model of the rat calvaria. Methods: We performed micro-computed tomography (micro-CT) and immunohistochemical TNF-&#x3b1; analysis on bone specimens to assess osteogenesis. Results: Micro-CT demonstrated significant increases in newly formed bone on day 30 compared with days 7 and 15 across all groups (p < 0.001). The highest bone volume was observed in the AB group (26.83%), followed by the LIPUS group (23.74%), and the lowest in the control (15.85%). Immunohistochemical analysis revealed significantly higher TNF-&#x3b1; expression on day 7 in the control group (172.0 &#xb1; 1.1) than in the AB (133.8 &#xb1; 0.9) and LIPUS (125.2 &#xb1; 0.8) groups (p < 0.001). On day 15, TNF-&#x3b1; expression was significantly higher in the LIPUS group (137.7 &#xb1; 1.3) than in both the AB (134.2 &#xb1; 1.8) and control (126.6 &#xb1; 2.2) groups (p < 0.001). At day 30, TNF-&#x3b1; levels in the LIPUS group (147.6 &#xb1; 1.9) remained significantly higher than in the control group (115.8 &#xb1; 0.9) (p < 0.001), with no significant difference compared to the AB group (146.3 &#xb1; 0.8). Conclusions: Although AB grafting achieved the greatest bone volume, LIPUS demonstrated considerable regenerative potential and may represent a promising non-invasive therapeutic approach to enhance intramembranous bone regeneration.

To evaluate lesion detection patterns and the risk-stratification value of paired 68Ga-PSMA-11 and 18F-FDG PET/CT in patients with post-prostatectomy biochemical recurrence (BCR) and negative conventional imaging, and to explore the hypothesis-generating biological context of dual-tracer phenotypes using public single-cell transcriptomic data. This retrospective single-center study included patients with post-prostatectomy BCR who underwent paired 68Ga-PSMA-11 and 18F-FDG PET/CT within 14 days and had negative conventional imaging within 1 month before PET/CT. Overall dual-tracer PET positivity was defined as at least 1 positive lesion on either tracer. Among patients with PET-detectable disease on either tracer, phenotypes were classified as PSMA+/FDG-, PSMA+/FDG+, or PSMA-/FDG+. Public single-cell RNA sequencing data were analyzed to assess the relationship between FOLH1 expression and glycolytic activity. Among 58 included patients, 21 (36.2%) had at least one positive lesion on either tracer. Overall dual-tracer PET positivity increased across PSA strata and with a greater number of prespecified risk features (both P for trend < 0.001). Older age and higher PSA at PET were independently associated with overall dual-tracer PET positivity. Among patients with PET-detectable disease on either tracer, FDG-avid disease was associated with pathologic stage &#x2265;pT3, pathologic N1 disease, recurrence within 3 years after radical prostatectomy, and PSA doubling time&#x2009;&#x2264;&#x2009;6 months. In the public single-cell cohort, FOLH1 expression and glycolytic activity showed only a weak association. Paired 68Ga-PSMA-11 and 18F-FDG PET/CT revealed heterogeneity beyond lesion detection in patients with post-prostatectomy BCR and negative conventional imaging. Among patients with PET-detectable disease on either tracer, FDG-avid disease was enriched for adverse clinicopathologic features, and public single-cell transcriptomic analysis provided hypothesis-generating biological context for divergent dual-tracer phenotypes.

Agricultural work involves exposure to airborne pollutants including dust and pesticides that can cause respiratory effects, yet little is known about these impacts in females. The inflammatory effects of inhaled glyphosate, alone or in combination with common agricultural exposures like lipopolysaccharide (LPS), remain unclear. The objective was to evaluate the inflammatory potential of single and combined exposures to glyphosate and LPS in female mice using physiological and structural measures, and synchrotron imaging. C57BL/6 female mice (n&#x2009;=&#x2009;20) were intranasally treated with glyphosate (GLY), LPS, LPS&#x2009;+&#x2009;glyphosate (LG), or HBSS (CTL) for 5 days. On day 5, an additional group of mice were transported to the Canadian Light Source synchrotron (CLS) for multiple image x-radiography (MIR) to assess lung injury. Following treatment, mice were euthanized and bronchoalveolar lavage fluid (BAL) and lung tissue were collected. Mice exposed to LG had significantly higher airway restriction; expression of pro-inflammatory cytokines/chemokines TNF-&#x3b1;, KC, IL-6, MCP-1, and MIP-2; levels of myeloperoxidase expression; greater recruitment of cells into the alveolar regions, disruption to the bronchial epithelium in the lungs, and compromised lung air-tissue interfaces in the MIR images compared to other treatment groups. It is likely that inflammatory adaptation is already occurring in the female mice by five days of exposure. These results reveal that female mice exposed to LG displayed physiological, structural, and lung injury effect that were different from mice exposed to LPS and GLY alone.

HDL biogenesis is mainly determined by intestinal and hepatic ABCA1. Inhibiting miR-10b increases macrophage ABCA1 but does not affect intestinal/hepatic ABCA1 or plasma HDL cholesterol in ApoE-/- mice. Given that miR-10a and miR-10b (with similar seed sequences) are comparably expressed in intestine/liver, we hypothesize they redundantly regulate intestinal/hepatic ABCA1 and HDL biogenesis. Primary mouse enterocytes and hepatocytes were treated with antagonist miR-10a, antagonist miR-10b, or their combination, followed by systemic inhibition studies in ApoE-/- and C57BL/6&#xa0;J mice. ABCA1 expression (qPCR/Western blot) and cholesterol efflux to ApoA-I/HDL were measured. Additionally, atherosclerotic lesions and HDL biogenesis in mice were assessed; ApoE-/- mice were further tested for intestinal barrier (intestinal permeability) and systemic inflammation (gut microbiota, inflammatory responses). Combined, not individual, miR-10a/b inhibition upregulated ABCA1 and increased cholesterol efflux in primary mouse enterocytes. Consistently, in ApoE-/- and wild-type C57BL/6J mice, combined miR-10 inhibition upregulated intestinal ABCA1, promoted HDL biogenesis, enhanced cholesterol efflux, and ultimately exerted anti-atherosclerotic effects. Concurrently, this dual inhibition reduced intestinal cholesterol accumulation, reshaped gut microbiota, and restored intestinal barrier integrity. Specifically, 16S rRNA sequencing shows that miR-10 inhibition decreased Proteobacteria (phylum level), while it increased Bacteroidetes (phylum level) and enriched Bifidobacterium and Lactobacillus (genus level). These effects collectively suppress systemic inflammation and exert anti-atherosclerotic effects in ApoE-/- mice. Additionally, miR-10 inhibition alleviated systemic inflammation in ApoE-/- mice by suppressing TLR4/NLRP3 inflammasome activation, with this suppression further verified in vitro. Inhibition of miR-10 attenuates atherosclerosis through enhanced intestinal ABCA1-mediated cholesterol efflux and barrier integrity, paralleling a favorable shift in gut microbiota composition particularly the enrichment of Bifidobacterium and Lactobacillus. This coordinated intestinal improvement reduces systemic inflammation and ultimately suppresses TLR4/NLRP3 inflammasome activation.

Real-time imaging of endogenous mRNA is essential for understanding the dynamics of gene regulation, yet appropriate animal models have been limited. We developed an Egr1-MPBS knock-in mouse line in which all endogenous Egr1 transcripts are tagged with 12 tandem MS2 and PP7 binding site (MPBS) pairs, enabling direct visualization of single mRNA molecules under physiological conditions. To establish proof of principle, we derived mouse embryonic fibroblast (MEF) lines from this model and performed live-cell imaging of fluorescently labeled Egr1 transcripts. We found that the insertion of the MPBS cassette did not alter endogenous mRNA stability or expression dynamics. This system allowed us to observe Egr1 transcriptional bursting and quantitatively track individual mRNA molecules as they diffused within the nucleus and cytoplasm. By integrating technological innovation with mechanistic insight, the Egr1-MPBS mouse establishes a broadly applicable platform for quantitative studies of gene expression and RNA trafficking at single-molecule resolution in living systems.