Pharmacological USP2 targeting suppresses ovarian cancer growth by potentiating apoptosis and ferroptosis.

Ovarian cancer is a frequently observed type of gynaecologic malignancy generally associated with poor prognosis around the world. Ubiquitin-specific proteases (USPs) form the largest subfamily of deubiquitylating enzymes and have emerged as potential therapeutic targets against human cancers. Through a systematic analysis of the prognostic significance of USP expression, USP2 was found to be inversely correlated with patient survival in ovarian cancer. Accordingly, we investigated the effects of pharmacological inhibition of USP2 on ovarian cancer by exploiting its small molecule inhibitor ML364. Our findings show that ML364 effectively hindered ovarian cancer growth and migration using a series of in vitro assays. In addition to apoptosis induction, ML364 also sensitized ovarian cancer cells to ferroptosis. Mechanistically, ML364 treatment resulted in cyclin D1 downregulation, increased poly (ADP-ribose) polymerase (PARP) cleavage, and elevated ROS levels in ovarian cancer cells. Collectively, our findings suggest USP2 as a potential therapeutic target in ovarian cancer, and hence, its pharmacological inhibition warrants further investigation.

Distinct Olfactory Bulb-Cortex Neural Circuits Coordinate Cognitive Function in Parkinson's Disease.

Cognitive dysfunction stands as a prevalent and consequential non-motor manifestation in Parkinson's disease (PD). Although dysfunction of the olfactory system has been recognized as an important predictor of cognitive decline, the exact mechanism by which aberrant olfactory circuits contribute to cognitive dysfunction in PD is unclear. Here, we provide the first evidence for abnormal functional connectivity across olfactory bulb (OB) and piriform cortex (PC) or entorhinal cortex (EC) by clinical fMRI, and dysfunction of neural coherence in the olfactory system in PD mice. Moreover, we discovered that 2 subpopulations of mitral/tufted (M/T) cells in OB projecting to anterior PC (aPC) and EC precisely mediated the process of cognitive memory respectively by neural coherence at specific frequencies in mice. In addition, the transcriptomic profiling analysis and functional genetic regulation analysis further revealed that biorientation defective 1 (Bod1) may play a pivotal role in encoding OBM/T-mediated cognitive function. We also verified that a new deep brain stimulation protocol in OB ameliorated the cognitive function of Bod1-deficient mice and PD mice. Together, aberrant coherent activity in the olfactory system can serve as a biomarker for assessing cognitive function and provide a candidate therapeutic target for the treatment of PD.

Identifying specific functional roles for senescence across cell types.

Cellular senescence plays critical roles in aging, regeneration, and disease; yet, the ability to discern its contributions across various cell types to these biological processes remains limited. In this study, we generated an in vivo genetic toolbox consisting of three p16Ink4a-related intersectional genetic systems, enabling pulse-chase tracing (Sn-pTracer), Cre-based tracing and ablation (Sn-cTracer), and gene manipulation combined with tracing (Sn-gTracer) of defined p16Ink4a+ cell types. Using liver injury and repair as an example, we found that macrophages and endothelial cells (ECs) represent distinct senescent cell populations with different fates and functions during liver fibrosis and repair. Notably, clearance of p16Ink4a+ macrophages significantly mitigates hepatocellular damage, whereas eliminating p16Ink4a+ ECs aggravates liver injury. Additionally, targeted reprogramming of p16Ink4a+ ECs through Kdr overexpression markedly reduces liver fibrosis. This study illuminates the functional diversity of p16Ink4a+ cells and offers insights for developing cell-type-specific senolytic therapies in the future.

Establishment of a prediction model of pulmonary artery hypertension in patients with hyperthyroidism.

OBJECTIVE: This study aims to assess the tricuspid annular plane systolic excursion (TAPSE)/PASP ratio as a potential indicator for predicting the probability of developing pulmonary arterial hypertension (PAH) in hyperthyroidism patients. A nomogram model will be developed based on our findings, as well as the receiver operating characteristic (ROC) curve. METHODS: The study involved 166 hyperthyroid patients treated at Yijishan Hospital, and the period covered August 2021 to August 2022. Patients were divided into two groups according to pulmonary artery systolic pressure ≥35 mmHg. Univariate and multivariate logistic analyses were performed on the two groups' demographic and laboratory data to identify potential diagnostic markers. These parameters were evaluated using ROC curves to determine their precision in forecasting PAH. The findings were validated by plotting a calibration curve based on a line chart model. RESULTS: In the study, eventually, 80 patients were enrolled: 30 in the PAH group and 50 in the No PAH group. Multipleistic regression analysis predicted the occurrence risk of developing PAH. When paired with other conventional echocardiographic parameters (such as TAPSE, MPI, and SV) and serological markers (such as FT3 and FT4), the developed model demonstrated outstanding predictive performance with an area under the ROC curve of 0.985, a Youden index of 0.971, a sensitivity of 100%, and a specificity of 97.1%. CONCLUSIONS: The nomogram model constructed by combining the TAPSE/PASP ratio with FT3 and FT4 serum markers, as well as conventional ultrasound parameters SV and MPI in hyperthyroidism patients, demonstrates robust discriminatory ability and consistency.

Effects and mechanisms of using "clean" smoke particles in the smoking process on the formation of ß-carboline heterocyclic amines (ß-CHAs) in smoked meat patties.

ß-Carboline heterocyclic amines (ß-CHAs), known for their synergistic neurotoxic and carcinogenic effects, are predominantly produced by humans through cigarette smoke and food and are found particularly in meats cooked at high temperatures. Few studies have explored the differences in the mechanisms of accumulation of ß-CHAs in smoked meat and meat processed at high temperatures. In this research, the concentration of ß-CHAs in smoked meats prepared using a variety of wood materials was measured using LCMS/MS. Additionally, key volatile organic compound markers associated with ß-CHAs accumulation in smoke were identified through GCMS and multivariate statistical analysis and subsequently confirmed in a chemical simulation system. Three types of strainers, each with a distinct aperture size, were used to assess the efficacy of particle filtration in reducing ß-CHAs levels in smoked meat. The findings indicated that smoke exposure indeed increases the ß-CHAs content of meat. However, only the strainer capable of filtering PM2.5-sized particles reduced the amount of ß-CHAs present compared to the control group. In contrast, strainers with larger pore sizes facilitated excessive accumulation of ß-CHAs. The presence of aldehydes such as 1 H-pyrrole-2-carboxaldehyde, 5-methylfurfural, benzaldehyde, furfural, and nonanal exhibited a positive correlation with the accumulation of ß-CHAs. Conversely, phenolic compounds, including 2-methoxy-4-vinylphenol, 2-methoxy-5-methylphenol, p-cresol, phenol, 2-methoxy-4-(1-propenyl)-, (Z)-, phenol, 3-ethyl-, and phenol, 4-ethyl-2-methoxy-, showed a negative correlation. Thus, filters made from chelated carbonyl trap materials both chemically and physically disrupt the buildup of ß-CHAs in smoked meats. The use of this approach will not only improve the quality of these products but will also contribute to decreasing the amount of inhalation pollutants released into the environment.

Construction of ROS-Responsive Hyaluronic Acid Modified Paclitaxel and Diosgenin Liposomes and Study on Synergistic Enhancement of Anti-Ovarian Cancer Efficacy.

Purpose: Ovarian cancer is a fatal gynecologic malignancy with a high rate of abdominal metastasis. Chemotherapy still has a poor clinical prognosis for ovarian cancer patients, with cell proliferation and angiogenesis leading to invasion, migration, and recurrence. To overcome these obstacles, we constructed a novel HA-modified paclitaxel and diosgenin liposome (PEG-TK-HA-PDLPs) using two novel functional materials, DSPE-PEG2000-HA and DSPE-PEG2000-TK-PEG5000, to specifically deliver the drugs to the tumor site in order to reduce OC cell proliferation and anti-angiogenic generation, thereby inhibiting invasion and migration. Methods and Results: PEG-TK-HA-PDLPs were prepared by film dispersion, with ideal physicochemical properties and exhibits active targeting for enhanced cellular uptake. The ZIP synergy score for PTX and Dios was calculated using the online SynergyFinder software to be 3.15, indicating synergy. In vitro results showed that PEG-TK-HA-PDLPs were highly cytotoxic to ID8 cells, induced ID8 cell apoptosis, and inhibited ID8 cell migration and invasion. In vivo studies showed that PEG-TK-HA-PDLPs could prolong the circulation time in the blood, accumulate significantly in the tumor site, and effectively fight against angiogenesis with significant anti-tumor effects. Conclusion: The production of PEG-TK-HA-PDLPs is an effective strategy for the treatment of OC.

The Neurovascular Unit Dysfunction in the Molecular Mechanisms of Epileptogenesis and Targeted Therapy.

Epilepsy is a multifaceted neurological syndrome characterized by recurrent, spontaneous, and synchronous seizures. The pathogenesis of epilepsy, known as epileptogenesis, involves intricate changes in neurons, neuroglia, and endothelium, leading to structural and functional disorders within neurovascular units and culminating in the development of spontaneous epilepsy. Although current research on epilepsy treatments primarily centers around anti-seizure drugs, it is imperative to seek effective interventions capable of disrupting epileptogenesis. To this end, a comprehensive exploration of the changes and the molecular mechanisms underlying epileptogenesis holds the promise of identifying vital biomarkers for accurate diagnosis and potential therapeutic targets. Emphasizing early diagnosis and timely intervention is paramount, as it stands to significantly improve patient prognosis and alleviate the socioeconomic burden. In this review, we highlight the changes and molecular mechanisms of the neurovascular unit in epileptogenesis and provide a theoretical basis for identifying biomarkers and drug targets.

Tracing the origin of alveolar stem cells in lung repair and regeneration.

Alveolar type 2 (AT2) cells are stem cells of the alveolar epithelia. Previous genetic lineage tracing studies reported multiple cellular origins for AT2 cells after injury. However, conventional lineage tracing based on Cre-loxP has the limitation of non-specific labeling. Here, we introduced a dual recombinase-mediated intersectional genetic lineage tracing approach, enabling precise investigation of AT2 cellular origins during lung homeostasis, injury, and repair. We found AT1 cells, being terminally differentiated, did not contribute to AT2 cells after lung injury and repair. Distinctive yet simultaneous labeling of club cells, bronchioalveolar stem cells (BASCs), and existing AT2 cells revealed the exact contribution of each to AT2 cells post-injury. Mechanistically, Notch signaling inhibition promotes BASCs but impairs club cells' ability to generate AT2 cells during lung repair. This intersectional genetic lineage tracing strategy with enhanced precision allowed us to elucidate the physiological role of various epithelial cell types in alveolar regeneration following injury.

A rat model of adenoid hypertrophy constructed by using ovalbumin and lipopolysaccharides to induce allergy, chronic inflammation, and chronic intermittent hypoxia.

BACKGROUND: Adenoid hypertrophy (AH) is a common pediatric disease that significantly impacts the growth and quality of life of children. However, there is no replicable and valid model for AH. METHODS: An AH rat model was developed via comprehensive allergic sensitization, chronic inflammation induction, and chronic intermittent hypoxia (CIH). The modeling process involved three steps: female Sprague-Dawley rats (aged 4-5 weeks) were used for modeling. Allergen sensitization was induced via intraperitoneal administration and intranasal provocation using ovalbumin (OVA); chronic nasal inflammation was induced through intranasal lipopolysaccharide (LPS) administration for sustained nasal irritation; CIH akin to obstructive sleep apnea/hypopnea syndrome was induced using an animal hypoxia chamber. Postmodel establishment, behaviors, and histological changes in nasopharynx-associated lymphoid tissue (NALT) and nasal mucosa were assessed. Arterial blood gas analysis and quantification of serum and tissue levels of (interleukin) IL-4 and IL-13, OVA-specific immunoglobulin E (sIgE), eosinophil cationic protein (ECP), tumor necrosis factor (TNF-α), IL-17, and transforming growth factor (TGF)-ß were conducted for assessment. The treatment group received a combination of mometasone furoate and montelukast sodium for a week and then was evaluated. RESULTS: Rats exhibited notable nasal symptoms and hypoxia after modeling. Histopathological analysis revealed NALT follicle hypertrophy and nasal mucosa inflammatory cell infiltration. Elevated IL-4, IL-13, IL-17, OVA-sIgE, ECP, and TNF-α levels and reduced TGF-ß levels were observed in the serum and tissue of model-group rats. After a week of treatment, the treatment group exhibited symptom and inflammatory factor improvement. CONCLUSION: The model effectively simulates AH symptoms and pathological changes. But it should be further validated for genetic, immunological, and hormonal backgrounds in the currently used and other strains and species.

FLOWERING LOCUS T1 and TERMINAL FLOWER1 regulatory networks mediate flowering initiation in apple.

Flower bud formation is a critical process that directly determines yield and fruit quality in fruit crops. Floral induction is modulated by the balance between 2 flowering-related proteins, FLOWERING LOCUS T (FT) and TERMINAL FLOWER1 (TFL1); however, the mechanisms underlying the establishment and maintenance of this dynamic balance remain largely elusive. Here, we showed that in apple (Malus × domestica Borkh.), MdFT1 is predominantly expressed in spur buds and exhibits an increase in expression coinciding with flower induction; in contrast, MdTFL1 exhibited downregulation in apices during flower induction, suggesting that MdTFL1 has a role in floral repression. Interestingly, both the MdFT1 and MdTFL1 transcripts are directly regulated by transcription factor basic HELIX-LOOP-HELIX48 (MdbHLH48), and overexpression of MdbHLH48 in Arabidopsis (Arabidopsis thaliana) and tomato (Solanum lycopersicum) results in accelerated flowering. Binding and activation analyses revealed that MdbHLH48 functions as a positive regulator of MdFT1 and a negative regulator of MdTFL1. Further studies established that both MdFT1 and MdTFL1 interact competitively with MdWRKY6 protein to facilitate and inhibit, respectively, MdWRKY6-mediated transcriptional activation of target gene APPLE FLORICAULA/LFY (AFL1, an apple LEAFY-like gene), ultimately regulating apple flower bud formation. These findings illustrate the fine-tuned regulation of flowering by the MdbHLH48-MdFT1/MdTFL1-MdWRKY6 module and provide insights into flower bud formation in apples.

Dynamics of Endothelial Cell Generation and Turnover in Arteries During Homeostasis and Diseases.

BACKGROUND: Endothelial cell (EC) generation and turnover by self-proliferation contributes to vascular repair and regeneration. The ability to accurately measure the dynamics of EC generation would advance our understanding of cellular mechanisms of vascular homeostasis and diseases. However, it is currently challenging to evaluate the dynamics of EC generation in large vessels such as arteries because of their infrequent proliferation. METHODS: By using dual recombination systems based on Cre-loxP and Dre-rox, we developed a genetic system for temporally seamless recording of EC proliferation in vivo. We combined genetic recording of EC proliferation with single-cell RNA sequencing and gene knockout to uncover cellular and molecular mechanisms underlying EC generation in arteries during homeostasis and disease. RESULTS: Genetic proliferation tracing reveals that ≈3% of aortic ECs undergo proliferation per month in adult mice during homeostasis. The orientation of aortic EC division is generally parallel to blood flow in the aorta, which is regulated by the mechanosensing protein Piezo1. Single-cell RNA sequencing analysis reveals 4 heterogeneous aortic EC subpopulations with distinct proliferative activity. EC cluster 1 exhibits transit-amplifying cell features with preferential proliferative capacity and enriched expression of stem cell markers such as Sca1 and Sox18. EC proliferation increases in hypertension but decreases in type 2 diabetes, coinciding with changes in the extent of EC cluster 1 proliferation. Combined gene knockout and proliferation tracing reveals that Hippo/vascular endothelial growth factor receptor 2 signaling pathways regulate EC proliferation in large vessels. CONCLUSIONS: Genetic proliferation tracing quantitatively delineates the dynamics of EC generation and turnover, as well as EC division orientation, in large vessels during homeostasis and disease. An EC subpopulation in the aorta exhibits more robust cell proliferation during homeostasis and type 2 diabetes, identifying it as a potential therapeutic target for vascular repair and regeneration.

Effect of Angelica dahurica, Angelica dahurica polysaccharides, and imperatorin on free and bound heterocyclic amine generation in roasted beef patties and release profiles of bound heterocyclic amines during in vitro digestion.

This study explored the suppressive activity of Angelica dahurica (AD), AD polysaccharides, and imperatorin on free and bound heterocyclic amine (HA) formation in roast beef patties and release profiles of bound HAs during in vitro digestion. The suppressive effects and potential mechanisms associated with free radical quenching were explored using UPLC-MS/MS, multivariate statistical analysis, and electron paramagnetic resonance (EPR). AD (0.5%, 1.0%, and 1.5%) and imperatorin (0.005%, 0.010%, and 0.015%) showed a dose-dependent inhibition for both free and bound HAs, with AD polysaccharides showing a slight inhibitory capacity. The maximum inhibition of free and bound HAs was 36.31% (1.5% AD) and 35.68% (0.015% imperatorin). The EPR results demonstrated that alkyl radicals and 1O2 were the pivotal free radicals for HAs. Furthermore, AD and imperatorin dose-dependently decreased the level of these radicals. Intriguingly, after in vitro digestion, only AD polysaccharides significantly inhibited the release of bound HAs, with imperatorin even facilitating the release process. In this study, the capacity of the AD polysaccharide to suppress the release of bound HAs and the ability of AD and imperatorin to inhibit free and bound HAs in beef patties were identified for the first time.

Synthesis and evaluation of butylphthalide-scutellarein hybrids as multifunctional agents for the treatment of Alzheimer's disease.

A series of butylphthalide and scutellarein hybrids 3-(alkyl/alkenyl) hydroxyphthalide derivatives were designed, synthesized and evaluated as multifunctional agents against Alzheimer's disease. In vitro bioactivity assays indicated that most of the compounds displayed excellent antioxidant activity and moderate to good inhibition activities of self-induced Aß1-42 aggregation. Among them, compound 7c was demonstrated as a potential and balanced multifunctional candidate displaying the best inhibitory effects on self- and Cu2+-induced Aß1-42 aggregation (90.2 % and 35.4 %, respectively) and moderate activity for disaggregation of Aß1-42 aggregation (42.5 %). In addition, 7c also displayed excellent antioxidant (2.42 Trolox equivalents), metal ions chelating, oxidative stress alleviation, neuroprotective and anti-neuroinflammatory activities. Furthermore, in vivo study demonstrated that 7c could ameliorate the learning and memory impairment induced by sodium nitrite and Aß1-42 in the step-down passive avoidance test. These balanced multifunctional profiles supporting compound 7c as a novel potential candidate for the treatment of AD.

Trust-Aware and Fuzzy Logic-Based Reliable Layering Routing Protocol for Underwater Acoustic Networks.

Routing protocols based on trust mechanisms have been widely investigated for wireless sensor networks, and the works have achieved good results, while there are few works on trusted routing for underwater acoustic networks (UANs). However, trust-aware routing is the key to improving the packet delivery rate and the energy efficiency of UANs. Therefore, inspired by the theory of trust evaluation, a trust-aware and fuzzy logic-based reliable layering routing protocol (TAFLRLR) is proposed. In the TAFLRLR protocol, to avoid the problem of the void area and improve the transmission reliability, the candidate nodes of the next-hop forwarding nodes are determined according to the layers of neighbor nodes. Moreover, a fuzzy logic-based trust evaluation mechanism (FLTEM) is provided, which employs the fuzzy comprehensive evaluation decision model to calculate the comprehensive trust value for underwater sensor nodes. Further, the node density of a candidate node and its comprehensive trust value are taken as the input of a fuzzy control system and the forwarding probability (FP) of the node is taken as the output, and the candidate node with the highest FP is selected as the best forwarding node. Simulation results illustrate the superiority and effectiveness of the TAFLRLR protocol in terms of energy efficiency, routing reliability, and transmission reliability.

RHBDF1 deficiency suppresses melanoma glycolysis and enhances efficacy of immunotherapy by facilitating glucose-6-phosphate isomerase degradation via TRIM32.

Melanoma is a dangerous form of skin cancer, making it important to investigate new mechanisms and approaches to enhance the effectiveness of treatment. Here, we establish a positive correlation between the human rhomboid family-1 (RHBDF1) protein and melanoma malignancy. We demonstrate that the melanoma RHBDF1 decrease dramatically inhibits tumor growth and the development of lung metastases, which may be related to the impaired glycolysis. We show that RHBDF1 function is essential to the maintenance of high levels of glycolytic enzymes, especially glucose-6-phosphate isomerase (GPI). Additionally, we discover that the E3 ubiquitin ligase tripartite motif-containing 32 (TRIM32) mediates the K27/K63-linked ubiquitination of GPI and the ensuing lysosomal degradation process. We prove that the multi-transmembrane domain of RHBDF1 is in competition with GPI, preventing the latter from interacting with NCL1-HT2A-LIN41 (NHL) domain of TRIM32. We also note that the mouse RHBDF1's R747 and Y799 are crucial for competitive binding and GPI protection. Artificially silencing the Rhbdf1 gene in a mouse melanoma model results in declined lactic acid levels, elevated cytotoxic lymphocyte infiltration, and improved tumor responsiveness to immunotherapy. These results provide credence to the hypothesis that RHBDF1 plays a significant role in melanoma regulation and suggest that blocking RHBDF1 may be an efficient technique for reestablishing the tumor immune microenvironment (TIME) in melanoma and halting its progression.

Mammalian mitochondrial translation infidelity leads to oxidative stress-induced cell cycle arrest and cardiomyopathy.

Proofreading (editing) of mischarged tRNAs by cytoplasmic aminoacyl-tRNA synthetases (aaRSs), whose impairment causes neurodegeneration and cardiac diseases, is of high significance for protein homeostasis. However, whether mitochondrial translation needs fidelity and the significance of editing by mitochondrial aaRSs have been unclear. Here, we show that mammalian cells critically depended on the editing of mitochondrial threonyl-tRNA synthetase (mtThrRS, encoded by Tars2), disruption of which accumulated Ser-tRNAThr and generated a large abundance of Thr-to-Ser misincorporated peptides in vivo. Such infidelity impaired mitochondrial translation and oxidative phosphorylation, causing oxidative stress and cell cycle arrest in the G0/G1 phase. Notably, reactive oxygen species (ROS) scavenging by N-acetylcysteine attenuated this abnormal cell proliferation. A mouse model of heart-specific defective mtThrRS editing was established. Increased ROS levels, blocked cardiomyocyte proliferation, contractile dysfunction, dilated cardiomyopathy, and cardiac fibrosis were observed. Our results elucidate that mitochondria critically require a high level of translational accuracy at Thr codons and highlight the cellular dysfunctions and imbalance in tissue homeostasis caused by mitochondrial mistranslation.

Polydatin alleviates mycoplasma pneumoniae-induced injury via inhibition of Caspase-1/GSDMD-dependent pyroptosis.

Mycoplasma pneumoniae (MP) is one of the main pathogens causing community acquired pneumonia (CAP) in children and adults. Previous pharmacological and clinical studies have shown that Polydatin (PD) exerts anti-inflammatory action by conferring protective benefit in MP pneumonia. However, the mechanism underlying the of PD on MP infection remains unclear. It was found that PD alleviated MP-induced injury by inhibiting caspase-1/gasdermin D (GSDMD)-mediated epithelial pyroptosis. The results demonstrated that PD inhibited the transformation of GSDMD to N-terminal gasdermin-N (GSDMD-N) by decreasing caspase-1 activation, as well as suppressed the formation and secretion of interleukin-1ß (IL-1ß) and interleukin-18 (IL-18), reversed Na, K-ATPase reduction, and suppressed LDH release both in vitro and vivo. Taken together, epithelial pyroptosis in BEAS-2B cells and lung injury in mice were prevented by PD. In conclusion, PD suppressed pulmonary injury triggered by MP infection, by inhibiting the caspase-1/GSDMD-mediated epithelial pyroptosis signaling pathway. Thus, PD may be regarded as a potential therapy for MP-induced inflammation.

Functional ProTracer identifies patterns of cell proliferation in tissues and underlying regulatory mechanisms.

A genetic system, ProTracer, has been recently developed to record cell proliferation in vivo. However, the ProTracer is initiated by an infrequently used recombinase Dre, which limits its broad application for functional studies employing floxed gene alleles. Here we generated Cre-activated functional ProTracer (fProTracer) mice, which enable simultaneous recording of cell proliferation and tissue-specific gene deletion, facilitating broad functional analysis of cell proliferation by any Cre driver.

Genetic recording of in vivo cell proliferation by ProTracer.

The ability to experimentally measure cell proliferation is the basis for understanding the sources of cells that drive organ development, tissue regeneration and repair. Recently, we generated a genetic approach to detect cell proliferation: we used genetic lineage-tracing technologies to achieve seamless recording of in vivo cell proliferation in a tissue-specific manner. We provide a detailed protocol (generation of mouse lines, characterization of mouse lines, mouse line crossing and cell-proliferation tracing) for using this genetic system to study cell proliferation. This cell-proliferation tracing system, which we term 'ProTracer' (Proliferation Tracer), permits lifelong noninvasive monitoring of cell proliferation of specific cell lineages in live animals. Compared with other short-term strategies that require execution of animals, ProTracer does not require sampling or animal sacrifice for tissue processing. To highlight these features, we used ProTracer to study the proliferation of hepatocytes during liver homeostasis and after tissue injury in mice. We show that the protocol is applicable to study any in vivo cell proliferation, which takes ~9 months to finish from mouse generation to data analysis. This protocol can easily be carried out by researchers skilled in mouse-related experiments.