ObjectiveFor prepubertal and urgently treated malignant tumor patients, ovarian tissue cryopreservation and transplantation represent more appropriate fertility preservation methods. Current clinical practices often involve freezing ovarian tissue with high concentrations of cryoprotectants (CPAs) and thawing with water baths. These processes lead to varying degrees of toxicity and devitrification damage to ovarian tissue. Therefore, this paper proposes optimized methods for vitrification of ovarian tissues based on sodium alginate hydrogel encapsulation and magnetic induction nanowarming technology. MethodsFirstly, the study investigated the effects of sodium alginate concentration, the sequence of hydrogel encapsulation and CPAs loading on vitrification efficiency of encapsulated ovarian tissue. Additionally, the capability of sodium alginate hydrogel encapsulation to reduce the required concentration of CPAs was validated. Secondly, a platform combining water bath and magnetic induction nanowarming was established to rewarm ovarian tissue under various concentrations of magnetic nanoparticles and magnetic field strengths. The post-warming follicle survival rate, antioxidant capacity, and ovarian tissue integrity were evaluated to assess the efficacy of the method. ResultsThe study found that ovarian tissue encapsulated with 2% sodium alginate hydrogel exhibited the highest follicle survival rate after vitrification. The method of loading CPAs prior to encapsulation proved more suitable for ovarian tissue cryopreservation, effectively reducing the required concentration of CPAs by 50%. A combination of 8 g/L Fe₃O₄ nanoparticles and an alternating magnetic field of 300 Gs showed optimal warming effectiveness for ovarian tissue. Combining water bath rewarming with magnetic induction nanowarming yielded the highest follicle survival rate, enhanced antioxidant capacity, and preserved tissue morphology. ConclusionSodium alginate hydrogel encapsulation of ovarian tissue reduces the concentration of CPAs required during the freezing process. The combination of magnetic induction nanowarming with water bath provides an efficient method ovarian tissue rewarming. This study offers novel approaches to optimize ovarian tissues vitrification.

[Objective] To analyze the effects of different factors and red blood cell transfusion thresholds on the efficacy of neonatal red blood cell (RBC) transfusion, in order to provide more references for neonatal transfusions to better achieve rational and effective blood use. [Methods] A retrospective collection of data from 282 neonates who received RBC transfusions at our hospital from 2022 to 2023 was conducted, including birth weight, gestational age, number of blood transfusions, length of hospital stay, assisted ventilation during RBC transfusion, and laboratory test results before and after transfusion. SPSS software was used for statistical analysis to comprehensively analyze the impact of different factors on the efficacy of RBC transfusion in neonates. [Results] The results showed that the gestational age and weight of newborns at birth were negatively correlated with their length of hospital stay and the number of RBC transfusions during hospitalization. Newborns with younger gestational age and lower weight had longer hospital stays and more RBC transfusions during hospitalization. After administering RBCs according to the standard of 15 mL/kg, there was a statistically significant difference in the efficacy of RBC transfusion at different transfusion thresholds. In non-critical situations, RBC transfusions were ineffective when the pre-transfusion hemoglobin (Hb) level was >120 g/L. When the pre-transfusion Hb level was ≤70 g/L, RBC transfusions achieved higher efficacy in both critical and non-critical situations. [Conclusion] In critical situations, the group with pre-transfusion Hb values ≤ 70 g/L has the best RBC transfusion effect, while in non-critical situations, the group with pre-transfusion Hb levels between 81 and 90 g/L has the best RBC transfusion effect. Overall, the efficacy of RBC transfusion in non-critical situations is higher than that in critical situations.

Objective: To investigate the correlation among α2, 6-sialyltransferase (ST6Gal-Ⅰ), CD36 desialylation, and caveolin-1 (Cav-1) in phorbol ester (PMA)-induced MEG-01 cell model, as well as their potential mechanism in immune thrombocytopenia (ITP). Methods: MEG-01 cells were treated with 10 ng/mL PMA for 48 hours (control group: 0.1% DMSO). Flow cytometry was used to detect cell surface markers: desialylation (CD41 RCA ) and α2, 6-sialylation (CD41 SNA ). Western blot was performed to analyze the protein expressions of ST6Gal-Ⅰ, CD36, and Cav-1. Results: Flow cytometry analysis revealed that, compared with the control group (set as 100%), the proportion of CD41 RCA positive cells in the MEG-01 cells after PMA intervention significantly increased to (127.79±2.01)%, while the proportion of CD41 SNA positive cells significantly decreased to (78.09±1.76)% (both P<0.05). Western blot analysis results showed that, compared with the control group, PMA intervention significantly downregulated the expression of ST6Gal-Ⅰ protein (0.602±0.023 vs 0.768±0.068) and Cav-1 protein (1.012±0.028 vs 1.253±0.068) (both P<0.05), while significantly upregulating the expression of CD36 protein (0.936±0.033 vs 0.694±0.070, P<0.05). Conclusion: PMA can significantly inhibit the expression of ST6Gal-Ⅰ, accompanied by increased desialylation (β-galactose exposure), elevated CD36, and downregulated Cav-1. These changes suggest that the increased exposure of CD36 antigen and the disorder of membrane microenvironment may be involved in the pathological process of ITP, providing a new direction for mechanism research.

Based on whole-genome identification of the TPS gene family in Perilla frutescens and screening, cloning, bioinformatics, and expression analysis of the synthetic enzyme for the insect-resistant component germacrene D, this study lays the foundation for understanding the biological function of the TPS gene family and the insect resistance mechanism in P. frutescens. This study used bioinformatics tools to identify the TPS gene family of P. frutescens based on its whole genome and predicted the physicochemical properties, systematic classification, and promoter cis-elements of the proteins. The relative content of germacrene D was detected in both normal and insect-infested leaves of P. frutescens, and the germacrene D synthase was screened and isolated. Gene cloning, bioinformatics analysis, and expression profiling were then performed. The results showed that a total of 99 TPS genes were identified in the genome, which were classified into the TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g subfamilies. Conserved motif analysis showed that the TPS in P. frutescens has conserved structural characteristics within the same subfamily. Promoter cis-element analysis predicted the presence of light-responsive elements, multiple hormone-responsive elements, and stress-responsive elements in the TPS family of P. frutescens. Transcriptome data revealed that most of the TPS genes in P. frutescens were highly expressed in the leaves. GC-MS analysis showed that the relative content of germacrene D significantly increased in insect-damaged leaves, suggesting that it may act as an insect-resistant component. The germacrene D synthase gene was screened through homologous protein binding gene expression and was found to belong to the TPS-a subfamily, encoding a 64.89 kDa protein. This protein was hydrophilic, lacked a transmembrane structure and signal peptide, and was predominantly expressed in leaves, with significantly higher expression in insect-damaged leaves compared to normal leaves. In vitro expression results showed that germacrene D synthase tended to form inclusion bodies. Molecular docking showed that farnesyl pyrophosphate(FPP) fell into the active pocket of the protein and interacted strongly with six active sites. This study provides a foundation for further research on the biological functions of the TPS gene family in P. frutescens and the molecular mechanisms underlying its insect resistance.
Perilla frutescens/chemistry* ; Plant Proteins/chemistry* ; Multigene Family ; Sesquiterpenes, Germacrane/metabolism* ; Alkyl and Aryl Transferases/chemistry* ; Phylogeny ; Gene Expression Regulation, Plant

The medicinal materials of Bawei Chenxiang Pills(BCPs) were extracted via three methods: reflux extraction by water, reflux extraction by 70% ethanol, and extraction by pure water following reflux extraction by 70% ethanol, yielding three extracts of ST, CT, and CST. The efficacy of ST(760 mg·kg~(-1)), CT(620 mg·kg~(-1)), and CST(1 040 mg·kg~(-1)) were evaluated by acute myocardial ischemia(AMI) and p-chlorophenylalanine(PCPA)-induced insomnia in mice, respectively. Western blot was further utilized to investigate their hypnosis mechanisms. The main chemical components of different extracts were identified by the UPLC-Q-Exactive-MS technique. The results showed that CT and CST significantly increased the ejection fraction(EF) and fractional shortening(FS) of myocardial infarction mice, reduced left ventricular internal dimension at end-diastole(LVIDd) and left ventricular internal dimension at end-systole(LVIDs). In contrast, ST did not exhibit significant effects on these parameters. In the insomnia model, CT significantly reduced sleep latency and prolonged sleep duration, whereas ST only prolonged sleep duration without shortening sleep latency. CST showed no significant effects on either sleep latency or sleep duration. Additionally, both CT and ST upregulated glutamic acid decarboxylase 67(GAD67) protein expression in brain tissue. A total of 15 main chemical components were identified from CT, including 2-(2-phenylethyl) chromone and 6-methoxy-2-(2-phenylethyl) chromone. Six chemical components including chebulidic acid were identified from ST. The results suggested that chromones and terpenes were potential anti-myocardial ischemia drugs of BCPs, and tannin and phenolic acids were potential hypnosis drugs. This study enriches the pharmacological and chemical research of BCPs, providing a basis and reference for their secondary development, quality standard improvement, and clinical application.
Animals ; Drugs, Chinese Herbal/isolation & purification* ; Mice ; Male ; Sleep Initiation and Maintenance Disorders/physiopathology* ; Humans ; Myocardial Infarction/drug therapy* ; Myocardial Ischemia/drug therapy*

A targeted metabolomics study was conducted on the bile acid profiles in the liver and feces of rats induced by a high-fat diet and intervened by ginsenoside Rb_1, along with the detection of FXR pathway gene expression in the liver, to explore and clarify its mechanism of action. The content of biochemical indicators in the serum were detected using an automatic biochemical analyzer. Hematoxylin and eosin(HE) staining and oil red O staining were used to detect pathological changes and lipid deposition in the liver. RT-PCR was used to detect the mRNA expression of FXR, small heterodimer partner(SHP), cholesterol 7 alpha-hydroxylase(CYP7A1), and sterol regulatory element-binding protein-1c(SREBP-1c) in the liver. Targeted bile acid metabolomics technology was employed to analyze changes in bile acid profiles in liver tissue and feces, and a correlation analysis was performed between key genes such as FXR, SHP, CYP7A1, SREBP-1c and differential bile acid metabolites. The results showed that ginsenoside Rb_1 significantly reduced the levels of total cholesterol(TC), triglycerides(TG), low-density lipoprotein cholesterol(LDL-C), and high-density lipoprotein cholesterol(HDL-C) in the serum, alleviated the large fat vacuoles and lipid deposition in the liver, increased the expression of FXR mRNA in the liver, and decreased the expression of SREBP-1c mRNA. The expression of CYP7A1 and SHP mRNA was increased, but the differences were not statistically significant. Targeted bile acid metabolomics showed that ginsenoside Rb_1 could restore the levels of 9 bile acids in the liver and 8 bile acids in the feces. Ginsenoside Rb_1 also increased the percentage of taurocholic acid(TCA) in the liver(56.78%) and the percentage of 12-ketolithocholic acid(12-KLCA) in the feces(26.10%). Pathway enrichment analysis revealed two pathways involved in bile acid metabolism: primary bile acid biosynthesis and taurine and hypotaurine metabolism. Correlation analysis showed that FXR, SHP, CYP7A1, and SREBP-1c were positively correlated with multiple differential bile acids. These results suggest that ginsenoside Rb_1 may intervene in lipid metabolism disorders induced by a high-fat diet by regulating the FXR pathway and modulating bile acid profiles in the liver and feces.
Animals ; Bile Acids and Salts/metabolism* ; Rats ; Ginsenosides/pharmacology* ; Male ; Receptors, Cytoplasmic and Nuclear/genetics* ; Liver/drug effects* ; Diet, High-Fat/adverse effects* ; Metabolomics ; Rats, Sprague-Dawley ; Feces/chemistry* ; Cholesterol 7-alpha-Hydroxylase/metabolism* ; Sterol Regulatory Element Binding Protein 1/genetics* ; Humans

Nonobstructive azoospermia (NOA), one of the most severe types of male infertility, etiology often remains unclear in most cases. Therefore, this study aimed to detect four biallelic detrimental variants (0.5%) in the minichromosome maintenance domain containing 2 ( MCMDC2 ) genes in 768 NOA patients by whole-exome sequencing (WES). Hematoxylin and eosin (H&E) demonstrated that MCMDC2 deleterious variants caused meiotic arrest in three patients (c.1360G>T, c.1956G>T, and c.685C>T) and hypospermatogenesis in one patient (c.94G>T), as further confirmed through immunofluorescence (IF) staining. The single-cell RNA sequencing data indicated that MCMDC2 was substantially expressed during spermatogenesis. The variants were confirmed as deleterious and responsible for patient infertility through bioinformatics and in vitro experimental analyses. The results revealed four MCMDC2 variants related to NOA, which contributes to the current perception of the function of MCMDC2 in male fertility and presents new perspectives on the genetic etiology of NOA.
Humans ; Male ; Azoospermia/genetics* ; Meiosis/genetics* ; Spermatogenesis/genetics* ; Adult ; Exome Sequencing ; Microtubule-Associated Proteins/genetics* ; Alleles ; Infertility, Male/genetics*

The underlying cause of low response rates to existing immunotherapies is that tumor cells dominate tumor immune escape through surface antigen deficiency and inducing tumor immunosuppressive microenvironment (TIME). Here, we proposed an in situ tumor cell engineering strategy to disrupt tumor immune escape at the root by restoring tumor cell MHC-I/tumor-specific antigen complex (MHC-I/TSA) expression to promote T-cell recognition and by silencing tumor cell CD55 to increase the ICOSL⁺ B-cell proportion and reverse the TIME. A doxorubicin (DOX) and dual-gene plasmid (MAC pDNA, encoding both MHC-I/ASMTNMELM and CD55-shRNA) coloaded drug delivery system (LCPN@ACD) with tumor targeting and charge/size dual-conversion properties was prepared. LCPN@ACD-induced ICD promoted DC maturation and enhanced T-cell activation and infiltration. LCPN@ACD enabled effective expression of MHC-I/TSA on tumor cells, increasing the ability of tumor cell recognition and killing. LCPN@ACD downregulated tumor cell CD55 expression, increased the proportion of ICOSL⁺ B cells and CTLs, and reversed the TIME, thus greatly improving the efficacy of αPD-1 and CAR-T therapies. The application of this in situ tumor cell engineering strategy eliminated the source of tumor immune escape, providing new ideas for solving the challenges of clinical immunotherapy.

Neuropathic pain, a debilitating condition caused by dysfunction of the somatosensory nervous system, remains difficult to treat due to limited understanding of its molecular mechanisms. Bioinformatics analysis identified cerebellin 2 (CBLN2) as highly enriched in human and murine proprioceptive and nociceptive neurons. We found that CBLN2 expression is persistently upregulated in dorsal root ganglia (DRG) following spinal nerve ligation (SNL) in mice. In addition, transcription factor SOX11 binds to 12 cis-regulatory elements within the Cbln2 promoter to enhance its transcription. SNL also induced SOX11 upregulation, with SOX11 and CBLN2 co-localized in nociceptive neurons. The siRNA-mediated knockdown of Sox11 or Cbln2 attenuated SNL-induced mechanical allodynia and thermal hyperalgesia. High-throughput sequencing of DRG following intrathecal injection of CBLN2 revealed widespread gene expression changes, including upregulation of numerous NF-κB downstream targets. Consistently, CBLN2 activated NF-κB signaling, and inhibition with pyrrolidine dithiocarbamate reduced CBLN2-induced pain hypersensitivity, proinflammatory cytokines and chemokines production, and neuronal hyperexcitability. Together, these findings identified the SOX11/CBLN2/NF-κB axis as a critical mediator of neuropathic pain and a promising target for therapeutic intervention.
Animals ; Neuralgia/metabolism* ; Ganglia, Spinal/metabolism* ; Up-Regulation ; Mice ; NF-kappa B/metabolism* ; SOXC Transcription Factors/genetics* ; Male ; Neuroinflammatory Diseases/metabolism* ; Mice, Inbred C57BL ; Nerve Tissue Proteins/genetics* ; Hyperalgesia/metabolism* ; Signal Transduction ; Spinal Nerves