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*

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.

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 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

Objective:To summarize the clinical characteristics and treatment experience of patients with botulism after cosmetic botulinum toxin injection.Methods:A retrospective study was conducted on patients admitted to the Department of Medical Aesthetic and Plastic Surgery of the Fifth Affiliated Hospital of Zhengzhou University for botulism after cosmetic botulinum toxin injection between January 1, 2023, and October 31, 2024. Clinical data and treatment regimens were collected. Patients received botulinum antitoxin injection, neurotrophic therapy, nutrition supplementation, modulation and enhancement of cellular immune function, and systemic supportive care based on their condition. Prior to antitoxin administration, a skin test was performed. Patients with a negative test received intramuscular injections of 10 000 U of antitoxin serum every 12 hours, while those with a positive result underwent a desensitization protocol. The cessation criterion was significant improvement of toxic symptoms. Data collected included age, gender, region, time of presentation, injection location, brand and type of toxin, injection time, sites and dose of injection, time to onset of initial symptoms, main symptoms, skin test result for antitoxin, dosage of antitoxin administered, length of hospital stay, adverse reactions and prognosis. Data analysis was performed using GraphPad Prism Version 10.2.0.Results:A total of 179 patients were included, with 8 cases in 2023 and 171 cases in 2024. The majority were female (97.2%, 174 cases). The age range was 18-62 years, with a median age of 35 years; the highest proportion was in the 20-40 age group (71.5%, 128 cases). Patients were from 23 different provinces, autonomous regions, and municipalities directly under the central government in China. The injected product was mostly an unspecified brand of botulinum toxin (57.5%, 103 cases). Injections were primarily administered in non-medical institutions, with beauty salons or private studios accounting for 88.8% (159 cases). Injection sites included the platysma (92 cases), masseter muscle (82 cases), orbicularis oculi muscle (82 cases), frontalis muscle (67 cases), among others, with some patients receiving injections at multiple sites. 69 cases (38.5%) of patients were unaware of the injected dose; for the remaining cases, based on information provided, the injected doses were all within the safe range. The incubation period was mostly 1-7 days. The main symptoms included fatigue (171 cases), dysphagia (137 cases), dizziness (101 cases), blurred vision (76 cases), and difficulty opening eyes (66 cases). 176 patients received botulinum antitoxin treatment; 82 cases (46.6%) had a positive skin test and received desensitization injections, while 94 cases (53.4%) had a negative test. The total dosage of antitoxin used ranged from 10 000 U to 240 000 U. Three patients received only adjuvant therapy such as neurotrophic support. Adverse reactions during treatment primarily included induration at the injection site and serum sickness, all of which resolved after symptomatic treatment with antihistamines, steroids, etc. The hospital stay ranged from 1 to 24 d, with an average of 4.6 d. Upon discharge, symptoms in all patients had alleviated or resolved. At the 6-month follow-up after discharge, 14 patients were lost to follow-up; the remaining patients recovered well with no other complications.Conclusion:Poisoning incidents due to the illegal or improper use of botulinum toxin are increasing. Administration of botulinum antitoxin is an effective means to ameliorate intoxicating symptoms. Patients should seek timely medical intervention and receive antitoxin treatment as early as possible. Desensitization administration does not affect the efficacy of the antitoxin.

Objective:To explore the efficacy of non-invasive prenatal testing (NIPT) of fetal free DNA in maternal peripheral blood in fetuses with increased nuchal translucency (NT).Methods:A retrospective analysis was conducted on 1 184 singleton pregnant women that underwent chromosomal microarray analysis (CMA) at Nanjing Drum Tower Hospital, Nanjing University Medical School from June 2014 to December 2022 due to fetal increased NT (≥3.0 mm). These subjects were categorized based on whether the increased NT was accompanied by other high-risk factors into isolated increased NT without advanced maternal age (further subdivided into 3.0 mm≤NT<3.5 mm, 3.5 mm≤NT<4.0 mm, and NT≥4.0 mm subgroups), isolated increased NT with advanced maternal age, increased NT with nasal bone abnormalities, increased NT with other soft markers, and increased NT with structural abnormalities groups. Assuming the sensitivity and specificity of NIPT and expanded NIPT at this center were both 100%, genomic abnormalities outside the detection range of NIPT or expanded NIPT were termed as residual risk of NIPT or expanded NIPT. Chi-square test and Bonferroni correction were used to compare the residual risks of NIPT and expanded NIPT among the three subgroups of isolated increased NT without advanced maternal age group. Results:(1) In the group of isolated increased NT without advanced maternal age: For the 3.0 mm≤NT<3.5 mm subgroup (329 cases), 19 abnormalities were detected by CMA [12 cases of chromosome aneuploidy, seven cases of pathogenic copy number variation (pCNV)], with residual risks of NIPT and expanded NIPT both at 2.1% (7/329). For the 3.5 mm≤NT<4.0 mm subgroup (173 cases), 29 abnormalities were detected by CMA (17 cases of chromosome aneuploidy, nine cases of pCNV, three cases of chromosome unbalanced translocation), with residual risks of NIPT at 8.1% (14/173) and expanded NIPT at 7.5% (13/173). For the NT≥4.0 mm subgroup (270 cases), CMA detected abnormalities in 70 cases (50 cases of chromosome aneuploidy, 16 cases of pCNV, three cases of unbalanced translocations, and one case of sex chromosome abnormality combined with pCNV). The residual risk of NIPT was 12.2% (33/270), and the residual risk of expanded NIPT was 7.0% (19/270). The residual risks of NIPT and expanded NIPT in the 3.0 mm≤NT<3.5 mm subgroup were lower than those in the 3.5 mm≤NT<4.0 mm and NT≥4.0 mm subgroups (Bonferroni correction, all P<0.017). (2) In the group of 92 cases with isolated increased NT and advanced maternal age, CMA detected abnormalities in 36 cases (29 cases of chromosome aneuploidy, five cases of pCNV, one case of trisomy 21 combined with sex chromosome abnormality, and one case of trisomy 18 combined with sex chromosome abnormality). The residual risk of NIPT was 7.6% (7/92), and that of expanded NIPT was 5.4% (5/92). (3) In the group of 49 cases with increased NT combined with nasal bone abnormalities, CMA detected abnormalities in 24 cases (23 cases of chromosome aneuploidy and one case of pCNV). The residual risks of NIPT and expanded NIPT were both 2.0% (1/49). (4) In the group of 26 cases with increased NT combined with other soft markers, CMA detected abnormalities in nine cases (six cases of chromosome aneuploidy, one case of pCNV, and two cases of chromosome unbalanced translocations). The residual risks of NIPT and expanded NIPT were both 11.5% (3/26). (5) In the group of 245 cases with increased NT combined with structural abnormalities, CMA detected abnormalities in 121 cases (107 cases of chromosome aneuploidy, seven cases of pCNV, four cases of chromosome unbalanced translocations, one case of trisomy 21 combined with trisomy 20, and two cases of trisomy 18 combined with sex chromosome abnormalities). The residual risk of NIPT was 16.7% (41/245), and that of expanded NIPT was 4.1% (10/245). Conclusions:For isolated NT≥3.5 mm or NT≥3.0 mm combined with other high-risk factors, chorionic villus sampling in early pregnancy can be recommended, advancing the timing of prenatal diagnosis from the second trimester to the first trimester. For fetuses with isolated 3.0 mm≤NT<3.5 mm, the 2.1% residual risk of chromosomal abnormalities should be fully informed during counseling, even if the risk of NIPT is low.

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.