Objective To evaluate the safety and efficacy of a self-developed micro-normothermic machine perfusion (NMP) system (micro-perfusion device) for preserving isolated porcine limbs. Methods Five healthy Landrace pigs were selected, and their left and right forelimbs were randomly divided into the NMP group and static cold storage (SCS) group. The NMP group was perfused with the self-developed micro-perfusion device and polymerized hemoglobin perfusate for 32 hours at normothermia, while the SCS group was preserved at 4 ℃. Hemodynamic parameters such as perfusion pressure and flow were monitored. The pH value, partial pressure of oxygen (PO₂), lactic acid (Lac), creatine kinase (CK) and lactate dehydrogenase (LDH) in the perfusate were measured. Hematoxylin-eosin staining was used to assess the muscle tissue structure, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling was employed to evaluate muscle cell apoptosis, and immunohistochemistry staining was applied to detect the expressions of tumor necrosis factor (TNF)-α and interleukin (IL)-6. A mixed-effects model was used to analyze the effects of time and treatment methods on tissue structure, cell apoptosis and inflammatory factors. Results The device could stably maintain a perfusion pressure of (69±15) mmHg and a flow rate of (117±42) mL/min. The pH value and electrolytes of the perfusate were generally stable, with PO₂ maintained at a high level. Lac was maintained at 5.38(3.81, 6.45) mmol/L, while CK and LDH increased over time. After 32 hours of perfusion in the NMP group, both the myocyte spacing and apoptosis rate were better than those in the SCS group. Mixed-effects model analysis showed that there were statistically significant differences in the effects of NMP treatment and SCS treatment on myocyte spacing and apoptosis rate per unit time (both P < 0.05). There were no statistically significant differences in TNF-α and IL-6 between the two groups, and mixed-effects model analysis showed no statistically significant differences in the effects of NMP treatment and SCS treatment on TNF-α and IL-6 per unit time (both P > 0.05). Conclusions The micro-perfusion device used in this study may achieve 32-hour normothermic preservation in a porcine limb amputation model, maintain basic metabolism and ionic homeostasis, reduce muscle structural damage and cell apoptosis without inducing additional inflammatory responses. This technology is expected to significantly extend the time window for replantation of amputated limbs in disaster rescue and long-distance transportation, providing an important technical basis for clinical translation and subsequent replantation research.

BACKGROUND: Temozolomide (TMZ) resistance is a significant challenge in treating glioblastoma (GBM). Collagen remodeling has been shown to be a critical factor for therapy resistance in other cancers. This study aimed to investigate the mechanism of TMZ chemoresistance by GBM cells reprogramming collagens. METHODS: Key extracellular matrix components, including collagens, were examined in paired primary and recurrent GBM samples as well as in TMZ-treated spontaneous and grafted GBM murine models. Human GBM cell lines (U251, TS667) and mouse primary GBM cells were used for in vitro studies. RNA-sequencing analysis, chromatin immunoprecipitation, immunoprecipitation-mass spectrometry, and co-immunoprecipitation assays were conducted to explore the mechanisms involved in collagen accumulation. A series of in vitro and in vivo experiments were designed to assess the role of the collagen regulators prolyl 4-hydroxylase subunit alpha 1 (P4HA1) and yes-associated protein (YAP) in sensitizing GBM cells to TMZ. RESULTS: This study revealed that TMZ exposure significantly elevated collagen type I (COL I) expression in both GBM patients and murine models. Collagen accumulation sustained GBM cell survival under TMZ-induced stress, contributing to enhanced TMZ resistance. Mechanistically, P4HA1 directly binded to and hydroxylated YAP, preventing ubiquitination-mediated YAP degradation. Stabilized YAP robustly drove collagen type I alpha 1 ( COL1A1) transcription, leading to increased collagen deposition. Disruption of the P4HA1-YAP axis effectively reduced COL I deposition, sensitized GBM cells to TMZ, and significantly improved mouse survival. CONCLUSION P4HA1 maintained YAP-mediated COL1A1 transcription, leading to collagen accumulation and promoting chemoresistance in GBM.
Temozolomide ; Humans ; Glioblastoma/drug therapy* ; Animals ; Mice ; Cell Line, Tumor ; Drug Resistance, Neoplasm/genetics* ; YAP-Signaling Proteins ; Hydroxylation ; Dacarbazine/pharmacology* ; Adaptor Proteins, Signal Transducing/metabolism* ; Transcription Factors/metabolism* ; Collagen/biosynthesis* ; Collagen Type I/metabolism* ; Prolyl Hydroxylases/metabolism* ; Antineoplastic Agents, Alkylating/therapeutic use*

OBJECTIVE: To analyze the efficacy and access safety of extracorporeal membrane oxygenation (ECMO) in series with continuous renal replacement therapy (CRRT) access for critically ill patients using propensity score matching analysis, and to explore the potential influencing factors of infection. METHODS: A total of 200 critically ill patients who received both ECMO and CRRT treatment in the intensive care unit (ICU) of Huai'an Second People's Hospital from December 2020 to December 2024 were retrospectively selected as the research subjects. They were divided into the independent operation group (72 cases) and the series system group (128 cases) according to the access connection mode of ECMO and CRRT. Propensity score matching analysis was used to perform 1 : 1 matching for patients of the two groups. The general data [age, gender, body mass index (BMI), clinical diagnosis, underlying disease, intubation method, intubation position, disease severity, ECMO support duration, catheter indwelling duration, oxygenation index (PaO₂/FiO₂) at 48 hours after ECMO initiation, serum creatinine (SCr), procalcitonin (PCT), hemoglobin (Hb), white blood cell count (WBC), platelet count (PLT)], treatment status [ECMO initiation duration, ECMO operation duration, ECMO flow, left ventricular ejection fraction (LVEF), CRRT initiation duration, CRRT catheter indwelling duration, inflow and outflow volume of replacement fluid], clinical outcome indicators (28-day survival rate, length of ICU stay, renal function recovery, fluid balance compliance rate), and access safety indicators (incidence of ECMO access thrombosis, incidence of infection, and incidence of bleeding events) of all the patients were collected. Subgroup analysis was conducted based on the occurrence of infection, and multivariate Logistic regression analysis was used to screen the potential risk factors for infection in critically ill patients receiving both ECMO and CRRT treatment. RESULTS: Finally, a total of 120 patients were successfully matched, with 60 patients in both the independent operation group and the series system group. No statistically significant differences were observed in the general data between the two groups, indicating comparability. Compared with the independent operation group, the ECMO flow at 48 hours after ECMO initiation, SCr, and alanine transaminase (ALT) of the patients in the series system group were significantly decreased, while the LVEF at 48 hours after ECMO initiation was significantly increased, additionally, the CRRT initiation duration, CRRT catheter indwelling duration, and the length of ICU stay were significantly shortened, and the inflow and outflow volume of replacement fluid were significantly increased. The incidence of infection and bleeding events in the series system group was significantly lower than that in the independent operation group [infection incidence: 11.67% (7/60) vs. 36.67% (22/60), bleeding event incidence: 8.33% (5/60) vs. 48.33% (29/60), both P < 0.05]. No significant difference was found in the other general data, treatment status, clinical outcome indicators, or access safety indicators between the two groups. Among the 120 patients, 29 cases developed infection (accounting for 24.17%), and 91 cases had no infection (accounting for 75.83%). Compared with the non-infection group, the catheter indwelling duration was significantly prolonged and PCT was significantly increased in the infection group, while the PLT and the proportion of patients with ECMO and CRRT access connected via the series system were significantly decreased. Multivariate Logistic regression analysis showed that catheter indwelling duration [odds ratio (OR) = 1.277, 95% confidence interval (95%CI) was 1.001-1.629, P = 0.049], PCT (OR = 1.529, 95%CI was 1.222-1.914, P < 0.001], PLT (OR = 0.953, 95%CI was 0.926-0.981, P = 0.001), and access connection mode (OR = 0.289, 95%CI was 0.090-0.930, P = 0.037) were potential risk factors for infection in critically ill patients. CONCLUSIONS The ECMO-in-series CRRT access can accelerate the initiation of CRRT, avoid local bleeding, stabilize patients' cardiac, hepatic and renal functions, reduce potential infection risks, and improve the prognosis of patients.
Humans ; Extracorporeal Membrane Oxygenation/adverse effects* ; Critical Illness/therapy* ; Retrospective Studies ; Continuous Renal Replacement Therapy ; Male ; Female ; Intensive Care Units ; Propensity Score ; Middle Aged ; Renal Replacement Therapy ; Adult ; Aged ; Risk Factors

Recently, photodynamic therapy (PDT) has gained considerable attention as a promising therapeutic approach for the treatment of psoriasis. Unfortunately, the activation of high mobility group box 1 protein (HMGB1) by PDT triggers innate and adaptive immune responses, which exacerbate skin inflammation. Herein, we combined glycyrrhizic acid (GA), a natural anti-inflammatory compound and immunomodulator derived from the herb Glycyrrhiza uralensis Fisch., with PDT actuated by the photosensitizer chlorin e6 (Ce6) by co-loading them in GA-based lipid nanoparticles coated with a catechol-modified quaternary chitosan salt (GC NPs/QCS-C). GC NPs/QCS-C exhibited high drug loading efficacy, uniform size distribution, an ideal topical adhesive property, enhanced skin retention and penetration in psoriasis-like lesions, and high intracellular uptake in epidermal cells compared with the counterparts. Subsequently, the transdermal administration of GC NPs/QCS-C followed by near-infrared laser radiation in an imiquimod-induced psoriasis-like mouse model significantly ameliorated psoriasis symptoms, promoted the apoptosis of hyperproliferative epidermal cells, and alleviated the inflammatory cascade. The significant therapeutic outcomes of GC NPs/QCS-C were attributed to the synergistic effects of GA and PDT on modulating immune cell recruitment and inhibiting dendritic cell maturation. Our results demonstrated that the topical bio-adhesive nanosystem that combines GA and Ce6 offers a synergistic chemo-phototherapeutic strategy for psoriasis treatment.

OBJECTIVE: Since Omicron will likely persist, this trial evaluates the safety and efficacy of Shufeng Jiedu Granule (SFJDG) for mild Omicron infection, aims at finding new therapies especially for home-treated patients. METHODS: This randomized, double-blind, placebo-controlled, multi-center phase III trial involves 844 patients, divided into a treatment group (422) and control group (422). Participants will receive SFJDG or placebo for 7 d (1.2 g/bag, 2 bags, 3 times/d). Hospital evaluations will be done on days 1 and 8, with telephone assessments on days 3 and 5. Follow-up continues on days 10 and 14. Diary cards will track symptom scores and safety data. The primary outcome is the time to sustained clinical recovery from corona virus disease 2019 (COVID-19) symptoms. An interim analysis will occur after 70 % of patients complete follow-up, with Type I error correction (α1 = 0.015) at interim analysis based on O'Brien-Fleming-type cumulative error spending function. RESULTS: This phase III trial evaluates the efficacy and safety of SFJDG for mild COVID-19, focusing on real-world applicability for home-managed patients. The study's randomized, double-blind, placebo-controlled design ensures methodological rigor, while its comprehensive outcome measures address both symptom recovery and treatment safety. By emphasizing symptom resolution and recovery time, the trial aligns with the clinical priorities for managing mild cases of COVID-19. The findings could offer valuable insights into SFJDG's role in improving patient outcomes and addressing gaps left by existing antiviral therapies, particularly in symptom management. CONCLUSION The global risk assessment remains high due to the ongoing virulence of SARS-CoV-2 Omicron sub-lineages. This Phase III study adopts a robust methodology to investigate SFJDG as a treatment for mild COVID-19 as well as it's effectiveness and safety. Furthermore, this study aim to provide sufficient scientific evidence for the market registration of SFJDG especially for home-treated patients. If successful, SFJDG could be a meaningful addition to therapeutic options for mild infections, supporting public health strategies in managing the ongoing impact of SARS-CoV-2.

Objective To investigate the susceptibility of Cryptococcus neoformans strains to antifungal drugs and examine the relevant clinical manifestations and laboratory test results in a tertiary hospital in Shanghai during the period from 2019 to 2023.Methods The isolates were identified by MALDI-TOF and biochemical identification cards.The minimum inhibitory concentration(MIC)values of 5-fluorocytosine,amphotericin B,fluconazole,voriconazole,and itraconazole against C.neoformans strains were measured using broth microdilution method.The corresponding clinical data were reviewed and compared.Results Majority(78.7％)of the 75 strains of C.neoformans were isolated from cerebrospinal fluid(CSF).The prevalence of wild type(WT)strains was the lowest(36.0％)for itraconazole and the highest(94.7％)for voriconazole.Cryptococcus capsular antigen test was positive in 62 strains.The results of Cryptococcus capsular antigen test was consistent with fungal culture in 96.9％of the cases.Conclusions Most of the C.neoformans strains were isolated from CSF.The prevalence of non-WT C.neoformans strains was the highest for itraconazole.The prevalence of WT C.neoformans strains was the highest for voriconazole.

Extensive epigenetic reprogramming involves in memory CD8+ T-cell differentiation. The elaborate epigenetic rewiring underlying the heterogeneous functional states of CD8+ T cells remains hidden. Here, we profile single-cell chromatin accessibility and map enhancer-promoter interactomes to characterize the differentiation trajectory of memory CD8+ T cells. We reveal that under distinct epigenetic regulations, the early activated CD8+ T cells divergently originated for short-lived effector and memory precursor effector cells. We also uncover a defined epigenetic rewiring leading to the conversion from effector memory to central memory cells during memory formation. Additionally, we illustrate chromatin regulatory mechanisms underlying long-lasting versus transient transcription regulation during memory differentiation. Finally, we confirm the essential roles of Sox4 and Nrf2 in developing memory precursor effector and effector memory cells, respectively, and validate cell state-specific enhancers in regulating Il7r using CRISPR-Cas9. Our data pave the way for understanding the mechanism underlying epigenetic memory formation in CD8+ T-cell differentiation.
CD8-Positive T-Lymphocytes/metabolism* ; Cell Differentiation ; Chromatin/immunology* ; Animals ; Mice ; Immunologic Memory ; Epigenesis, Genetic ; SOXC Transcription Factors/immunology* ; NF-E2-Related Factor 2/immunology* ; Mice, Inbred C57BL ; Gene Regulatory Networks ; Enhancer Elements, Genetic

Naringenin (4,5,7-trihydroxyflavonoid) is a naturally occurring bioflavonoid found in citrus fruits, which plays an important role in metabolic syndrome, neurological disorders, and cardiovascular diseases. However, the pharmacological mechanism and biological function of naringenin on anti-angiogenesis and anti-tumor immunity have not yet been elucidated. Our study firstly demonstrates that naringenin inhibits the growth of hepatocellular carcinoma (HCC) cells both in vivo and in vitro. Naringenin diminishes the ability of HCC cells to induce tube formation and migration of human umbilical vein endothelial cells (HUVECs) and suppresses neovascularization in chicken chorioallantoic membrane (CAM) assays. Meanwhile, in vivo results demonstrate that naringenin can significantly upregulate level of CD8⁺ T cells, subsequently increasing the level of immune-related cytokines in the tumor immune microenvironment. Mechanistically, we found that naringenin facilitate the K48-linked ubiquitination and subsequent protein degradation of vascular endothelial growth factor A (VEGFA) and mesenchymal-epithelial transition factor (c-Met), which reduces the expression of programmed death ligand 1 (PD-L1). Importantly, combination therapy naringenin with PD-L1 antibody or bevacizumab provided better therapeutic effects in liver cancer. Our study reveals that naringenin can effectively inhibit angiogenesis and anti-tumor immunity in liver cancer by degradation of VEGFA and c-Met in a K48-linked ubiquitination manner. This work enlightens the potential effect of naringenin as a promising therapeutic strategy against anti-angiogenesis and anti-tumor immunity in HCC.

OBJECTIVE To study the anti-fatigue effects of differently-cultivated Astragalus extract in a hypoxic environment of the plateau and explore the related mechanisms.METHODS Fifty-six male KM mice were randomly divided into the hypoxic swimming control(HSC)group,imitation wild Astragalus extract(IWA)430,860 and 1 720 mg·kg-1 groups,and cultivated Astragalus extract(CA)463,925 and 1 850 mg·kg-1 groups.The drug was administered by gavage once daily for 15 days,while body mass was monitored every three days.After 15 days of gavage,the mice were subjected to load swimming(5%body weight)in a hypobaric chamber(simulating a 4 000 m altitude),with exhaus-tive swimming time measured to identify the optimal dosage.Following randomization,fifty male KM mice were assigned to five groups:normoxic control(NC),hypoxic control(HC),HSC,IWA 860 and CA 925 mg·kg-1.All groups underwent daily gavage for 15 d before 90 min non-weight-bearing swimming was conducted in the HSC,IWA 860 and CA 925 mg·kg-1 groups within a hypobaric chamber,followed by immediate measurement of muscle strength.Hematoxylin-eosin(HE)staining was used to observe the histopathological changes in liver and gastrocnemius muscle tissues.Blood urea nitrogen(BUN),blood glucose(BG)and serum lactic acid(LA),glutathione(GSH),glutathione peroxidase(GSH-Px),malondialdehyde(MDA),superoxide dismutase(SOD),liver glycogen(LG)and muscle glycogen(MG)in livers and muscles,and total antioxidant capacity(T-AOC)and reactive oxygen species(ROS)in muscles were measured by commercial kits.Taurine and hypotaurine were measured by HPLC.Enzyme-linked immunosorbent assay(ELISA)was used for cysteine sulfenic acid decarboxylase(CSAD)measure-ment.Western blotting was used to detect protein expressions of phosphatidylinositol 3-kinase(PI3K),protein kinase B(Akt),nuclear factor E2-related factor 2(Nrf2),and heme oxygenase-1(HO-1)in skeletal muscles.RESULTS Compared with the HSC group,the swimming time was prolonged in IWA 463,IWA 860,CA 925 and CA 1 850 mg·kg-1 groups.Compared with the HSC group,the muscle strength of mice in the IWA 860 mg·kg-1 group and the CA 925 mg·kg-1 group was significantly increased,histo-pathological damage in the liver and gastrocnemius muscle was reduced,serum levels of LA and BUN were significantly decreased,levels of BG,LG and MG were significantly increased,levels of GSH,GSH-Px and SOD were significantly increased,contents of MDA were significantly decreased,expressions of CSAD were significantly increased in liver tissue,contents of GSH,T-AOC,taurine and hypotaurine were significantly increased,levels of ROS were significantly decreased,and protein expressions of PI3K,Akt,Nrf2,HO-1 were significantly upregulated in muscle tissues.CONCLUSION Under simulated high-altitude hypoxic conditions,extracts of Astragalus membranaceus cultivated by two methods consis-tently exhibit anti-fatigue effects.Its mechanisms may be mitigating oxidative stress,augmenting taurine and hypotaurine metabolic regulation,and activating PI3K/Akt and Nrf2/HO-1 signaling pathways.IWA has a better anti-fatigue effect than CA.

Objective To explore the efficacy and safety of flexible ureteroscopic lithotripsy(FURL)combined with flexible negative-pressure sheath in treating patients with 3-4 cm renal calculi with CT value＜1100 Hu,and to identify a safe and effective treatment option for patients with such calculi.Methods A retrospective analysis was conducted on the clinical data of 95 patients undergoing surgical treatment at the Department of Urology,the Second People's Hospital of Anhui Province during Jun.2022 and May 2024.The patients were divided into two groups,including 42 in the FURL with flexible negative-pressure sheath group(FURL group),and 53 in the percutaneous nephrolithotomy(PCNL)group.General data,perioperative indicators,and complication rates were compared between the two groups.Results There were no statistically significant differences between the FURL group and PCNL group in stone-free rate(SFR)3 days postoperatively(83.3％vs.88.7％),1 month postoperatively(95.2％vs.92.5％),and 3 months postoperatively(97.6％vs.94.3％),as well as operation time[(77.65±9.05)min vs.(79.10±8.14)min](P＞0.05).The FURL group had shorter hospital stay[(5.98±1.12)days vs.(9.38±1.57)days],lower decrease in hemoglobin level[(3.17±0.85)g/L vs.(4.98±1.72)g/L],lower visual analog scale(V AS)score on postoperative day 1[(2.60±0.63)vs.(3.77±1.09)]and day 3[(2.29±0.99)vs.(2.70±0.89)],lower postoperative white blood cell count[(7.05±1.66)× 109 cells/L vs.(11.24±2.90)× 109 cells/L],lower C-reactive protein level[(25.73±7.57)ng/L vs.(31.14±5.53)ng/L],lower blood urea nitrogen level[(6.12±1.43)mmol/L vs.(9.85±3.07)mmol/L],and lower serum creatinine level[(84.48±11.57)μmol/L vs.(114.43±21.48)μmol/L](all P＜0.001).The total incidence of complications was also lower in the FURL group(4.8％vs.18.9％,P＜0.05).Conclusion FURL combined with flexible negative-pressure sheath can achieve comparable SFR as PCNL without extending operation time,and it can shorten hospital stay,reduce intraoperative blood loss,have little impact on renal function,reduce inflammatory response and decrease the incidence of complications.