BACKGROUND: T cell dysfunction, which includes exhaustion, anergy, and senescence, is a distinct T cell differentiation state that occurs after antigen exposure. Although T cell dysfunction has been a cornerstone of cancer immunotherapy, its potential in transplant research, while not yet as extensively explored, is attracting growing interest. Interferon regulatory factor 4 (IRF4) has been shown to play a pivotal role in inducing T cell dysfunction. METHODS: A novel ultra-low-dose combination of Trametinib and Rapamycin, targeting IRF4 inhibition, was employed to investigate T cell proliferation, apoptosis, cytokine secretion, expression of T-cell dysfunction-associated molecules, effects of mitogen-activated protein kinase (MAPK) and mammalian target of rapamycin (mTOR) signaling pathways, and allograft survival in both in vitro and BALB/c to C57BL/6 mouse cardiac transplantation models. RESULTS: In vitro , blockade of IRF4 in T cells effectively inhibited T cell proliferation, increased apoptosis, and significantly upregulated the expression of programmed cell death protein 1 (PD-1), Helios, CD160, and cytotoxic T lymphocyte-associated antigen (CTLA-4), markers of T cell dysfunction. Furthermore, it suppressed the secretion of pro-inflammatory cytokines interferon (IFN)-γ and interleukin (IL)-17. Combining ultra-low-dose Trametinib (0.1 mg·kg -1 ·day -1 ) and Rapamycin (0.1 mg·kg -1 ·day -1 ) demonstrably extended graft survival, with 4 out of 5 mice exceeding 100 days post-transplantation. Moreover, analysis of grafts at day 7 confirmed sustained IFN regulatory factor 4 (IRF4) inhibition, enhanced PD-1 expression, and suppressed IFN-γ secretion, reinforcing the in vivo efficacy of this IRF4-targeting approach. The combination of Trametinib and Rapamycin synergistically inhibited the MAPK and mTOR signaling network, leading to a more pronounced suppression of IRF4 expression. CONCLUSIONS Targeting IRF4, a key regulator of T cell dysfunction, presents a promising avenue for inducing transplant immune tolerance. In this study, we demonstrate that a novel ultra-low-dose combination of Trametinib and Rapamycin synergistically suppresses the MAPK and mTOR signaling network, leading to profound IRF4 inhibition, promoting allograft acceptance, and offering a potential new therapeutic strategy for improved transplant outcomes. However, further research is necessary to elucidate the underlying pharmacological mechanisms and facilitate translation to clinical practice.
Animals ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Interferon Regulatory Factors/metabolism* ; Heart Transplantation/methods* ; T-Lymphocytes/immunology* ; Sirolimus/therapeutic use* ; Pyridones/therapeutic use* ; Graft Survival/drug effects* ; Pyrimidinones/therapeutic use* ; Cell Proliferation/drug effects* ; Apoptosis/drug effects* ; Male ; Signal Transduction/drug effects*

Oral squamous cell carcinoma (OSCC) accounts for over 90% of oral malignancies, with more than 370 000 new cases and approximately 188 000 deaths annually worldwide. In China, there are roughly 65 000 new cases and 35 000 deaths each year, showing a significant upward trend compared with 2015 statistics. Despite continuous advancements in treatment modalities, the 5-year survival rate remains stagnant at 50%-60%, where tumor heterogeneity and therapy resistance persist as fundamental barriers to precision oncology. To address these critical challenges, this study established a standardized bioban-king protocol for OSCC patient-derived organoids (PDOs) (Patent: Method for constructing an oral squamous cell carcinoma organoid bank, ZL202311378598.3). Through groundbreaking optimization of culture media, enzymatic digestion kinetics, and stepwise cryopreservation, we achieved a biobanking success rate exceeding 95% and pioneered synchronous cultivation of matched primary tumors, lymph node metastases, and adjacent normal mucosa from individual patients, preserving spatial heterogeneity and stromal interactions. Leveraging this platform, we developed high-throughput drug screening: Quantified heterogeneity-driven differential chemoresponse using adenosine triphosphate (ATP)-based viability assays; We discovered resistance mechanisms: Identified sialylated cancer IgG (SIA-cIgG)-mediated cis-platin resistance (primary/secondary) through PTPN13 suppression, with anti-SIA-cIgG combination therapy demonstrating synergistic efficacy. Besides, we elucidated metastatic drivers: CRISPR-Cas9-edited organoids revealed WDR54 promoted metastasis via H3K4me3/H4K16ac epigenetic reprogramming, activating epithelial-mesenchymal plasticity (EMP) and inducing partial epithelial-mesenchymal transition (pEMT). This "holographic patient-mirroring" platform provided unprecedented resolution for OSCC precision therapy and had been formally incorporated into the Chinese Stomatological Association Technical Guidelines (Technical guideline for establishing patient-derived oral squamous cell carcinoma organoid banks, CHSA 2024-08). Future integration of immune-competent organoids, 3D-bioprinted vasculature, and multi-omics-AI systems will accelerate personalized oncology. These innovations will accelerate clinical translation of personalized therapeutic regimens, ultimately bridging the gap between bench research and bedside application.
Humans ; Organoids/pathology* ; Mouth Neoplasms/genetics* ; Carcinoma, Squamous Cell/pathology* ; Tissue Banks ; Biological Specimen Banks

Objective To explore the underlying mechanisms by which time-restricted feeding(TRF)attenuates dextran sodium sulfate(DSS)-induced colitis in mice via modulation of regenerating islet-derived protein 3 gamma(Reg3γ)expression and gut microbiota.Methods Six-week-old C57BL/6 mice were stratified by body weight and randomized into ad libitum feeding(AL)and TRF groups(n=7).The AL mice were given unrestricted food access,whereas the TRF mice were allowed feeding only during 00:00 and 08:00 daily,for totally 4 weeks.Mouse colitis model was induced at the fourth week by adding 2.5%dextran sodium sulfate(DSS)in drinking water for 6 d.Disease severity and the effects of TRF were assessed with disease activity index(DAI)scoring,colon length measurement,HE staining and histopathological scoring,and mRNA expression levels of regenerating islet-derived 3 gamma(Reg3g)and inflammatory cytokines in colonic tissues.Another 14 mice were randomized into AL plus antibiotic cocktail(AL+ABX)and TRF plus antibiotic cocktail(TRF+ABX)groups,with 7 animals in each group.ABX was administered to deplete gut microbiota and evaluate the microbiota dependence of TRF in attenuating colitis.Fecal samples from AL and TRF mice were analyzed by 16S ribosomal RNA sequencing(16S rRNA-seq),and serum lipopolysaccharide(LPS)level was measured.The colonic epithelial cells were collected for RNA-seq.Results After modeling,the AL mice exhibited typical colitis symptoms,such as weight loss,diarrhea,and hematochezia.TRF intervention significantly attenuated these above symptoms,with lower DAI scores from day 4 post-modeling(P<0.001),reduced colon shortening(P<0.01),preserved tissue architecture,and decreased inflammation.RT-qPCR analysis showed that TRF down-regulated colonic mRNA expression levels of Reg3g and pro-inflammatory cytokines(IL-1 β,IL-6,TNF-α)(P<0.05)while up-regulated that of anti-inflammatory factor IL-10(P<0.000 1)when compared with the corresponding levels in AL mice.ABX treatment led no significant differences between the AL+ABX and TRF+ABX groups in term of DAI score,colon length,or histopathology.Obviously down-regulated Reg3g was observed in the TRF+ABX group than the AL+ABX group(P<0.05),whereas L-1β,IL-6,TNF-α and IL-10 showed no notable changes.16S rRNA-seq revealed that TRF markedly reshaped gut microbiota composition,with increased Gram-positive bacterial abundance,reduced Gram-negative bacteria,with concomitant lower serum LPS level(P<0.001).RNA-seq also indicated significant suppression of NF-κB and other inflammation-related signaling pathways in the TRF group.Conclusion TRF attenuates DSS-induced colitis in mice by downr-egulating Reg3γ expression,reshaping gut microbiota,and reducing serum LPS level,and thereby suppressing NF-κB-mediated inflammatory signaling pathways.

Organophosphorus nerve agents are the most threatening chemical warfare agents and terrorist agents.The number of nerve agents and their related chemicals involved in the verification of Chemical Weapon Convention(CWC)exceeds ten million,with the majority being isomers.Accurate structural identification of these chemicals has always been one of the challenges in CWC related verification analysis.In this work,a total of 17 kinds of alkyl phosphonate isomers and structural analogs from 5 groups were designed and synthesized,and then analyzed by gas chromatography-mass spectrometry(GC-MS)and gas chromatography-infrared spectroscopy(GC-FTIR).The spectra of isomers or structural analogs obtained from two techniques as well as the structural information provided therein were compared and analyzed.The results showed that for isomers or structural analogs with similar MS spectra,FTIR spectra could provided more structural fingerprint information of compounds and had advantages in confirming structures.Combined with the excellent separation ability of GC,GC-FTIR can be used to assist GC-MS in the structural confirmation of alkyl phosphates,achieving rapid and accurate identification of isomers or structural analogues.

Objective To explore the effects of different glucose concentrations on the synthesis and secretion of bone collagen in osteoblasts and the impact of diabetes on bone quality in mice.Methods(1)Primary osteoblasts were extracted from the skulls of neonatal mice via collagenase digestion and cultured in four groups under different glucose concentrations:normal glucose(5.5 mmol/L),moderate glucose(11.5 mmol/L),moderate-high glucose(16.5 mmol/L),and high glucose(25 mmol/L).EdU staining was performed to evaluate cell proliferation,while the Transwell assay was used to assess cell migration.Immunofluorescence and Western blotting were performed to detect and quantitatively analyze the content of type Ⅰ collagen(Col-1).Alizarin red S(ARS)staining and alkaline phosphatase(ALP)staining were applied to assess the effects of different glucose concentrations on osteogenic differentiation.(2)Six-week-old male C57BL/6 mice were randomly divided into control group and model group(5 in each group).The model group was fed a high-fat diet for 4 weeks followed by streptozotocin(STZ)injection to establish a diabetic mouse model.The osteogenic differentiation capacity of primary osteoblasts from both groups was assessed.(3)Micro-computed tomography(Micro-CT)was employed to analyze femoral bone mineral density(BMD),bone volume/tissue volume(BV/TV),trabecular number(Tb.N),and trabecular separation(Tb.Sp).Three-point bending test was conducted to evaluate mechanical parameters including maximum load,Young's modulus,fracture energy,and stiffness.RT-qPCR was employed to assess the expression of osteogenic differentiation genes(Alp,Opn,Col1a1,and Lox).Masson staining and Mallory staining were used to evaluate Col-1 content in trabecular bone.Results(1)EdU and Transwell assay results demonstrated that with the gradual increase in glucose concentration,the proliferation and migration abilities of osteoblasts were significantly decreased(P<0.001),and the protein expression levels of Col-1 and lysyl oxidase(LOX)were significantly reduced(P<0.01 or P<0.001).ARS and ALP staining revealed that calcium salt deposition and ALP activity in osteoblasts were significantly decreased with increasing glucose concentration(P<0.05 or P<0.001).(2)Compared with control group,mice in model group exhibited typical"three polies and one weight loss"symptoms(polyuria,polydipsia,polyphagia,and weight loss)of diabetes,and ARS and ALP staining showed a significant reduction in osteoblasts(P<0.001).(3)Micro-CT and three-point bending test results indicated that,compared with control group,mice in model group showed microarchitectural deterioration of bone,decreased Tb.N,increased Tb.Sp,and significantly reduced maximum load,Young's modulus,fracture energy,and stiffness(P<0.05).RT-qPCR results showed that the relative mRNA expression levels of osteogenic differentiation genes(Alp,Opn,Col1a1,and Lox)were significantly decreased in model group compared with control group(P<0.01 or P<0.001).Masson and Mallory staining indicated a significant reduction in collagen content in model group compared with control group(P<0.01).Conclusions High-glucose environment inhibits osteoblast proliferation,differentiation,and migration.Diabetic mice exhibit reduced bone quality and increased bone fragility,potentially mediated by decreased lysyl oxidase and collagen levels.

Acute hypobaric hypoxic brain damage is a potentially fatal high-altitude sickness. Autophagy plays a critical role in ischemic brain injury, but its role in hypobaric hypoxia (HH) remains unknown. Here we used an HH chamber to demonstrate that acute HH exposure impairs autophagic activity in both the early and late stages of the mouse brain, and is partially responsible for HH-induced oxidative stress, neuronal loss, and brain damage. The autophagic agonist rapamycin only promotes the initiation of autophagy. By proteome analysis, a screen showed that protein dynamin2 (DNM2) potentially regulates autophagic flux. Overexpression of DNM2 significantly increased the formation of autolysosomes, thus maintaining autophagic flux in combination with rapamycin. Furthermore, the enhancement of autophagic activity attenuated oxidative stress and neurological deficits after HH exposure. These results contribute to evidence supporting the conclusion that DNM2-mediated autophagic flux represents a new therapeutic target in HH-induced brain damage.
Mice ; Animals ; Hypoxia ; Oxidative Stress ; Autophagy ; Cognition ; Sirolimus/therapeutic use*

Objective:To understand the epidemiological characteristics of human respiratory syncytial virus (HRSV) among acute respiratory infection (ARI) cases in 16 provinces of China from 2009 to 2023.Methods:The data of this study were collected from the ARI surveillance data from 16 provinces in China from 2009 to 2023, with a total of 28 278 ARI cases included in the study. The clinical specimens from ARI cases were screened for HRSV nucleic acid from 2009 to 2023, and differences in virus detection rates among cases of different age groups, regions, and months were analyzed.Results:A total of 28 278 ARI cases were enrolled from January 2009 to September 2023. The age of the cases ranged from<1 month to 112 years, and the age M ( Q1, Q3) was 3 years (1 year, 9 years). Among them, 3 062 cases were positive for HRSV nucleic acid, with a total detection rate of 10.83%. From 2009 to 2019, the detection rate of HRSV was 9.33%, and the virus was mainly prevalent in winter and spring. During the Corona Virus Disease 2019 (COVID-19) pandemic, the detection rate of HRSV fluctuated between 6.32% and 18.67%. There was no traditional winter epidemic peak of HRSV from the end of 2022 to the beginning of 2023, and an anti-seasonal epidemic of HRSV occurred from April to May 2023. About 87.95% (2 693/3 062) of positive cases were children under 5 years old, and the difference in the detection rate of HRSV among different age groups was statistically significant ( P<0.001), showing a decreasing trend of HRSV detection rate with the increase of age ( P<0.001). Among them, the HRSV detection rate (25.69%) was highest in children under 6 months. Compared with 2009-2019, the ranking of HRSV detection rates in different age groups changed from high to low between 2020 and 2023, with the age M (Q1, Q3) of HRSV positive cases increasing from 1 year (6 months, 3 years) to 2 years (11 months, 3 years). Conclusion:Through 15 years of continuous HRSV surveillance analysis, children under 5 years old, especially infants under 6 months old, are the main high-risk population for HRSV infection. During the COVID-19 pandemic, the prevalence and patterns of HRSV in China have changed.

Background::Homoharringtonine (HHT) is an effective anti-inflammatory, anti-viral, and anti-tumor protein synthesis inhibitor that has been applied clinically. Here, we explored the therapeutic effects of HHT in a mouse heart transplant model.Methods::Healthy C57BL/6 mice were used to observe the toxicity of HHT in the liver, kidney, and hematology. A mouse heart transplantation model was constructed, and the potential mechanism of HHT prolonging allograft survival was evaluated using Kaplan–Meier analysis, immunostaining, and bulk RNA sequencing analysis. The HHT-T cell crosstalk was modeled ex vivo to further verify the molecular mechanism of HHT-induced regulatory T cells (Tregs) differentiation. Results::HHT inhibited the activation and proliferation of T cells and promoted their apoptosis ex vivo. Treatment of 0.5 mg/kg HHT for 10 days significantly prolonged the mean graft survival time of the allografts from 7 days to 48 days ( P <0.001) without non-immune toxicity. The allografts had long-term survival after continuous HHT treatment for 28 days. HHT significantly reduced lymphocyte infiltration in the graft, and interferon-γ-secreting CD4 + and CD8 + T cells in the spleen ( P <0.01). HHT significantly increased the number of peripheral Tregs (about 20%, P <0.001) and serum interleukin (IL)-10 levels. HHT downregulated the expression of T cell receptor (TCR) signaling pathway-related genes ( CD4, H2-Eb1, TRAT1, and CD74) and upregulated the expression of IL-10 and transforming growth factor (TGF) -β pathway-related genes and Treg signature genes ( CTLA4, Foxp3, CD74, and ICOS). HHT increased CD4 + Foxp3 + cells and Foxp3 expression ex vivo, and it enhanced the inhibitory function of inducible Tregs. Conclusions::HHT promotes Treg cell differentiation and enhances Treg suppressive function by attenuating the TCR signaling pathway and upregulating the expression of Treg signature genes and IL-10 levels, thereby promoting mouse heart allograft acceptance. These findings may have therapeutic implications for organ transplant recipients, particularly those with viral infections and malignancies, which require a more suitable anti-rejection medication.

The NiFe2O4-graphene oxide nanocomposite modified with polyethylenimine(GO@PEI-NiFe2O4)was prepared to purify and enrich phosphopeptides from biosamples.The Ni2+and Fe3+ions on its surface could coordinate with phosphate groups and then selectively adsorb phosphopeptides.PEI was conducive to the above combination due to its high hydrophilicity.The material showed good magnetic response properties and could be rapidly separated from samples with the aid of magnet.With tryptic digest of β-casein as sample,the enrichment property of the material to phosphopeptides was studied,which was compared with the results of GO@NiFe2O4,revealing the adsorption mechanism of GO@PEI-NiFe2O4.The static and dynamic binding properties of GO@PEI-NiFe2O4 were investigated using pTyr as a representative phosphopeptide,and the adsorption capacity was 36.2 μg/mg.The results showed that the material could remove the interference of nonphosphopeptides and effectively enrich phosphopeptides in complex matrix.After enrichment by GO@PEI-NiFe2O4,1535 phosphopeptides were identified from the tryptic digest of rat liver by mass spectrum and the enrichment effect of GO@PEI-NiFe2O4 greatly outperformed commercial Fe3+-IMAC kits.This work provided an efficient material for the enrichment of phosphopeptides,showing potential applications in phosphoproteomics research.

An analytical method based on ultra high performance liquid chromatography-high resolution tandem mass spectrometry(UHPLC-HRMS/MS)and high performance liquid chromatography-triple quadrupole mass spectrometry(HPLC-TQ MS)was established to reveal the characteristics of various sulfur mustard analogs with different active thiol molecules in CWC Schedule 1.A.04.Firstly,the toxic agents were prepared by micro-directed synthesis,and then the differences of the reactivity and abundance of formed adducts between different sulfur mustards and glutathione(GSH),cysteine(Cys)and N-acetylcysteine(NAC)in incubation solution,plasma and cell were investigated,respectively.The results indicated that all target sulfur mustards could react with three kinds of thiol molecules.The content of Cys and sulfur mustard adducts in plasma was higher than that of GSH and sulfur mustard adducts,while NAC and sulfur mustard adducts might have fewer types of adducts due to low content or poor mass spectrometry response.Additionally,the content of GSH and sulfur mustard adducts in exposed cells was higher than that of Cys,which should be due to the significant difference in the content of thiol molecules in plasma and cells.