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.

OBJECTIVE To investigate the improvement effect and mechanism of processed Oxytropis falcata on renal fibrosis （RF） in rats. METHODS RF model was induced by adenine. After modeling， the rats were divided into the model group， positive control group （colchicine， 0.45 mg/kg）， and processed O. falcata low-， medium- and high-dose groups （0.5， 1， 2 g/kg）， respectively. Additionally， a blank group without modeling was set up， with 8 rats in each group. The positive control group and the various dosage groups of processed O. falcata were given the corresponding medicinal solutions intragastrically， while the blank group and model group were given equal volume of normal saline intragastrically， once daily for 28 consecutive days. The appearance and histopathological morphology of the rats’ kidneys were observed. Serum levels of renal function indexes [bl ood urea nitrogen （BUN）， creatinine （Cr） ] and inflammatory factors [interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） ] in rats were detected. Protein expressions of fibronectin （FN）， α -smooth muscle actin （ α -SMA） and collagen type Ⅰ （Col-Ⅰ） in renal tissue of rats were determined. mRNA expressions of transforming growth factor-β 1 （TGF-β 1 ）， Smad3 and extracellular signal-regulated kinase 1/2 （ERK1/2） in renal tissues were measured. Protein expression of TGF-β 1 and phosphorylation levels of Smad3 and ERK1/2 in renal tissues were detected. RESULTS Compared with the blank group， the rats in the model group exhibited enlarged kidneys with pale color， rough and uneven surface. There was a significant infiltration of inflammatory cells and vacuolated cells in the renal tubules， along with marked proliferation of collagen fibers. Serum levels of BUN， Cr， IL-6 and TNF-α， protein expressions of α -SMA， Col-Ⅰ and FN in renal tissues， mRNA expressions of TGF-β 1 ， Smad3， ERK1 and ERK2 and protein expression of TGF-β 1 as well as phosphorylation levels of Smad3 and ERK1/2 in renal tissues were increased significantly （ P ＜0.05）. Compared with the model group， renal pathological changes of rats were alleviated in processed O. falcata groups， with reduced infiltration of inflammatory cells and proliferation of collagen fibers. The levels of the aforementioned quantitative indicators were all significantly reversed （ P ＜0.05）. CONCLUSIONS Processed O. falcata can improve renal function in RF rats， alleviate inflammatory responses， and reduce abnormal collagen fiber deposition. Its mechanism of action may be related to the inhibition of the activity of the TGF-β 1 /Smad signaling pathway.

BackgroundResident physicians represent a high-risk group for sleep disorders， exhibiting a significantly higher prevalence of such conditions compared with the general population， which severely impairs their physical and mental health. It is hypothesized that perceived stress negatively impacts sleep quality through psychological mechanisms， such as depleting self-control resources and triggering anxiety. However， this pathway warrants empirical validation. ObjectiveTo explore the mediating role of self-control and anxiety emotions in the association between perceived stress and sleep quality among resident physicians， and to elucidate the underlying psychological mechanisms， aiming at providing theoretical basis for developing targeted psychological interventions. MethodsA cross-sectional survey was conducted in April 2025. First- to third- year resident physicians at a hospital in Fuyang City were recruited as participants （n=372）. The Chinese Perceived Stress Scales （CPSS）， the Chinese version of the Dual-Mode of Self-Control Scale （DMSC-S）， the Pittsburgh Sleep Quality Index （PSQI）， and the Generalized Anxiety Disorder Scale-7 item （GAD-7） were used for group testing. The model 6 of the Process macro version 4.1 was ultilized to examine the mediating pathway of self-control and anxiety emotions between perceived stress and sleep quality. ResultsA total of 322 valid questionnaires were collected， yielding an effective responsive rate of 86.56%. Among the respondents， 146 （45.34%） reported poor sleep quality. The CPSS score and GAD-7 score of resident physicians were positively correlated with the PSQI score （r=0.727， 0.784， P<0.01）， while the DMSC-S score was negatively correlated with the PSQI score （r=-0.615， P<0.01）. Perceived stress directly and positively predicted poor sleep quality （B=0.124， P<0.01）， with the direct effect accounting for 31.39% of the total effect. Furthermore， perceived stress indirectly affected sleep quality through the independent mediating effects of self-control and anxiety emotions. The indirect effect values of 0.053 （95% CI： 0.019 - 0.091） and 0.192 （95% CI： 0.141 - 0.249）， accounting for 13.42% and 48.61% of the total effect， respectively. Perceived stress also impact sleep quality through the serial mediating effect of self-control and anxiety， with the indirect effect value of 0.026 （95% CI： 0.005 - 0.049）， accounting for 6.58% of the total effect. ConclusionThe perceived stress of resident physicians can influence sleep quality by impairing self-control， exacerbating anxiety， and through the serial mediation of both factors.

S-methyl-L-cysteine sulfoxide (SMCO) is a non-protein sulfur-containing amino acid with a variety of functions. There are few reports on the enzymes catalyzing the biosynthesis of SMCO from S-methyl-L-cysteine (SMC). In this study, the flavin-containing monooxygenase gene derived from Schizosaccharomyces pombe (spfmo) was heterologously expressed in Escherichia coli BL21(DE3) and the enzymatic properties of the expressed protein were analyzed. The optimum catalytic conditions of the recombinant SpFMO were 30 ℃ and pH 8.0, under which the enzyme activity reached 72.77 U/g. An appropriate amount of Mg²⁺ improved the enzyme activity. The enzyme kinetic analysis showed that the K_m and k_cat/K_m of SpFMO on the substrate SMC were 23.89 μmol/L and 61.71 L/(min·mmol), respectively. Under the optimal reaction conditions, the yield of SMCO synthesized from SMC catalyzed by SpFMO was 12.31% within 9 h. This study provides reference for the enzymatic synthesis of SMCO.
Schizosaccharomyces/genetics* ; Escherichia coli/metabolism* ; Recombinant Proteins/metabolism* ; Cysteine/biosynthesis* ; Mixed Function Oxygenases/metabolism* ; Schizosaccharomyces pombe Proteins/metabolism* ; Oxygenases/metabolism* ; Kinetics

BACKGROUND:The development of calcium phosphate bone cement is limited due to its poor mechanical properties and weak osteogenic ability.Silicate bioactive glass is highly favored due to its excellent biological activity and osteogenic ability.Simultaneously,fiber structures can enhance the mechanical strength of materials.OBJECTIVE:To investigate the mechanical properties,biocompatibility,and osteogenic effect of silicate bioactive glass fiber composite calcium phosphate bone cement.METHODS:Different mass percentages(0％,10％,and 20％)of silicate bioactive glass fiber were added to the solid phase of calcium phosphate bone cement,mixed with the liquid phase and cured for 48 hours to obtain silicate bioactive glass fiber composite calcium phosphate bone cement.The mechanical properties,setting time,and ion precipitation of the cement were characterized.The three groups of bone cement extracts were co-cultured with MC3T3-E1 cells.The cell compatibility of the materials was evaluated by CCK-8 assay,live/dead staining,and phalloidin staining.After osteogenic induction,the osteogenic induction ability of the materials was evaluated by alkaline phosphatase staining,alizarin red staining,RUNX2 immunofluorescence staining,and RT-PCR.RESULTS AND CONCLUSION:(1)With the increase of silicate bioactive glass fiber content,the compressive strength and flexural strength of bone cement increased,and the setting time was prolonged.When bone cement was immersed in simulated body fluid,the precipitation of silicon ions,calcium ions,and phosphorus ions could be detected.Moreover,with the increase of silicate bioactive glass fiber content,the mass concentration of silicon ions and phosphorus ions released by bone cement increased,and the mass concentration of calcium ions decreased.(2)Live/dead staining and phalloidin staining results exhibited that silicate bioactive glass fiber composite calcium phosphate bone cement had no toxic effect on MC3T3-E1 cells.CCK-8 assay results showed that silicate bioactive glass fiber composite calcium phosphate bone cement could promote the proliferation of MC3T3-E1 cells.(3)With the increase of silicate bioactive glass fiber content in bone cement,the alkaline phosphatase activity and extracellular calcium deposition of MC3T3-E1 cells increased,the expression of RUNX2 protein increased,and the expression of alkaline phosphatase,osteocalcin,osteopontin,and RUNX2 mRNA expression increased.(4)The results indicate that silicate bioactive glass fibers can enhance the mechanical properties and osteogenic induction ability of calcium phosphate bone cement,among which 20％silicate bioactive glass fibers have a more obvious effect.

Objective:To investigate the potential mechanism of cellular senescence-related mitochondrial autophagy genes in diabetic retinopathy (DR).Methods:The DR gene datasets GSE53257 and GSE60436 from the GEO database and screened the differentially expressed genes (DEG) were downloaded. Cellular senescence-related genes and mitochondrial autophagy-related genes from the GeneCards database, and the intersection of the two to obtain the DR-related differentially expressed genes (CSRMRDEG) were collected. The obtained CSRMRDEG was subjected to Gene Ontology (GO) functional enrichment analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis, protein-protein interaction network (PPI) analysis, and hub gene identification using Maximal Clique Centrality (MCC), Degree, Maximum Neighborhood Component (MNC)、Edge Percolated Component (EPC) and Closeness algorithms. Gene Set Enrichment Analysis (GSEA) was conducted to obtain the enriched pathways of DEG, and ssGSEA immune infiltration analysis was performed to screen the correlation between immune cells and DR. The diagnostic efficacy of hub genes for DR was evaluated by drawing the receiver operating characteristic (ROC) curve and calculating the area under the curve (AUC). Meanwhile, the Wilcoxon rank sum test was used to compare the differences in the infiltration level of immune cells between the DR Group and the control group.Results:23 DR-related CSRMRDEG were obtained. GO analysis showed that they were mainly enriched in the pathways of dicarboxylic acid, biosynthetic process of folate-containing compounds, tetrahydrofolate conversion, mitochondrial matrix, mitochondrial endomembrane, structural components of ribosomes, and glutamate transmembrane transporter protein activity. The results of KEGG pathway enrichment analysis showed that CSRMRDEG was highly enriched in pathways such as the folate carbon pool, biosynthesis of cofactors, and pyruvate metabolism. The PPI analysis results show that there are 16 related CSRMRDEG. Five algorithms (MCC, Degree, MNC, EPC, Closeness) obtained the nine Hub genes. The results of ROC curve analysis showed that the AUC of the expression levels of 9 hub genes for diagnosing DR ranged from 0.7-0.9. The ssGSEA results showed that there were statistically significant differences in Wilcoxon of central memory CD4 + T cells, macrophages, natural killer cells, and helper T cell 1 between the DR group and the control group ( Z=-2.85, -2.23, -2.10, -2.52; P<0.05). Conclusion:Mitochondrial autophagy genes related to cellular senescence are potential diagnostic targets for DR.

Objective:To compare the protective effects of the static cold storage (SCS) and normothermic machine perfusion (NMP) on the skeletal muscle of the amputated limbs of pigs.Methods:Four Landrace pigs were selected, from which eight limbs were amputated and divided into SCS group ( n=5) and NMP group ( n=3) according to the random number table method. After blood collection from the carotid artery, an amputated limb model was established by amputating the limbs at the scapulohumeral joints. The limbs in the SCS group were wrapped in sterile cloth and stored at 4 ℃ for 24 hours. In the NMP group, the limbs were mechanically perfused with a red blood cell-containing perfusion fluid at 37 ℃ for 24 hours, with 70% of the perfusion fluid replaced every 6 hours. Before the experiment, cross-matching tests with the saline medium were conducted between donor and recipient pigs to evaluate blood coagulation and blood safety in the NMP group. An allogeneic red blood cell perfusion fluid was prepared and the levels of pH, Na +, K +, Cl -, Ca 2+, glucose (Glu), hematocrit (Hct), lactic acid (Lac) and osmotic pressure of the perfusion fluid were measured. At 0, 6, 12, 18, and 24 hours after perfusion, the skin temperature and oxyhemoglobin saturation (SaO 2) levels in the NMP group were monitored and the levels of pH, Glu, creatine kinase (Ck), K +, Ca 2+, and Na +levels of the perfusion fluid were analyzed to evaluate the metabolism of the skeletal muscle in the amputated limbs. The mean intercellular distance and apoptosis index of the myocytes were quantitatively analyzed and histopathological changes were observed by performing HE staining and TUNEL staining on the skeletal muscle of the amputated limbs in both groups at 0 and 24 hours after perfusion. After perfusion was ended, the weight gain rate and swelling degree of the amputated limbs were compared between the two groups and the overall state of the amputated limbs was evaluated. Results:The result of the cross-matching test between donor and recipient pig blood was negative. The parameters in the prepared red blood cell-containing perfusion fluid generally maintained within a normal range: pH 7.38±0.04, Na + concentration (138.30±4.48)mmol/L, K + concentration (3.50±0.26)mmol/L, Glu concentration (6.11±2.08)mmol/L, and osmotic pressure (305.67±3.79)mmol/L. However, slightly higher Cl - and Ca 2+ concentrations [(118.34±12.00)mmol/L and (2.00±0.15)mmol/L] and lower Hct and lactate concentrations [0.30±0.03 and (1.54±0.38)mmol/L] were detected when compared with the reference range. During the perfusion, the average skin temperature of the amputated limbs in the NMP group was (36.13±0.98)℃, with the skin temperatures at 6, 12, 18, and 24 hours after perfusion being significantly higher than that at 0 hour ( P<0.01), while no significant difference among the skin temperatures at 6, 12, 18, and 24 hours after perfusion was observed ( P>0.05). The SaO 2 levels in the skin of the amputated limbs in the NMP group averaged over 95%, which showed no significant difference at 0, 12, 18, and 24 hours after perfusion ( P>0.05), while a significant elevation was observed at 6 hours compared with that at 0 hour ( P<0.05). There were no significant differences in pH, Glu, Na +, and Ca 2+ levels in the NMP group at 0, 6, 12, 18, and 24 hours after perfusion ( P>0.05), while the Ck levels at 18 and 24 hours were both significantly higher than that at 6 hours after perfusion ( P<0.05), and the Ck levels at 6, 12, 18, and 24 hours were all significantly higher than that at 0 hour ( P<0.05). The K + level progressively increased with the perfusion time, with significant elevations at 18 and 24 hours after perfusion compared with that at 0 hour ( P<0.05). HE staining revealed well-preserved muscle fiber continuity and regular arrangement in the NMP group and the SCS group at 0 hour, with an intercellular distance of (8.95±0.60)μm. At 24 hours, the NMP group exhibited slight skeletal muscle fiber rupture and swelling, with a slightly increased intercellular distance of (14.75±0.90)μm, significantly greater than that at 0 hour ( P<0.01). At 24 hours, the SCS group showed marked skeletal muscle fiber rupture and swelling, with a significantly increased intercellular distance of (23.51±1.49)μm, significantly larger than those at 0 hour in the same group and at 24 hours in the NMP group ( P<0.01). TUNEL immunofluorescence staining indicated a tiny amount of apoptotic cells in the skeletal muscle in both groups at 0 hour, with an apoptotic index of (4.26±1.62)%. There was a small number of apoptotic cells in the skeletal muscle in the NMP group at 24 hours, with an apoptotic index of (25.94±2.69)%, significantly larger than that in the same group at 0 hour ( P<0.01). The SCS group exhibited a large number of apoptotic cells at 24 hours, with an apoptotic index of (62.97±3.22)%, significantly larger than those at 0 hour in the same group and at 24 hours in the NMP group ( P<0.01). In comparison with the SCS group at 24 hours, the amputated limbs in the NMP group showed red color in the appearance, no symptoms of ischemic muscle contracture and good joint movement despite slight edema in the subcutaneous layer. At 24 hours, the weight gain rate of the amputated limbs was (15.82±0.89)% in the NMP group, significantly higher than (0.97±0.28)% in the SCS group ( P<0.01). Conclusion:Compared with SCS, NMP with the red blood cell-containing perfusion fluid prepared with the allogeneic blood for the amputated limbs of pigs can alleviate the ischemic injury of the muscle fibers and inhibit the apoptosis of the muscle cells by sustaining stable energy and oxygen supply and balancing ion homeostasis and pH of the perfusion fluid.

Objective To investigate the relationship between daily intake of monounsaturated fatty acids(MUFAs)and polyunsaturated fatty acids(PUFAs)and non-alcoholic fatty liver disease(NAFLD),and to determine the threshold values of daily MUFAs and PUFAs intake for NAFLD risk.Methods Date were collected from the Dryad database.We enrolled a total of 1 068 healthy subjects aged 18 years and older(534 in the control group and 534 with NAFLD group)who had physical check-up in the Affiliated Nanping First Hospital of Fujian Medical University from April 2015 to August 2017.Comprehensive medical histories were obtained through questionnaires;information on dietary intake was collected using a semi-quantitative food frequency questionnaire and daily MUFAs and PUFAs intake were calculated.Baseline characteristics were compared between the two groups,and Logistic regression and restricted cubic spline(RCS)analyses were used to explore the relationship between daily MUFAs or PUFAs intake and NAFLD.Results Compared with the control group,the prevalence of hypertension,tea drinking,body mass index(BMI),daily energy intake,and daily MUFAs and PUFAs intakes were significant higher in patients with NAFLD(all P＜0.05),but the proportion of physical activities was significantly lower(P＜0.05).Logistic regression analysis revealed that after adjusting other confounding factors such as age,gender and BMI,for every 10 g increase in daily MUFAs or PUFAs intake,the risk of NAFLD increased by 53％(95％ CI:1.25-1.87,P＜0.001)and 3.30 times(95％ CI:2.98-6.20,P＜0.001),respectively.RCS indicated an approximately linear relationship between daily MUFAs intake and NAFLD(P for nonlinearity=0.064)and a nonlinear relationship between daily PUFAs intake and NAFLD(P for nonlinearity＜0.05).Subgroup analysis results were generally consistent,and there was statistical evidence of interactions between MUFAs and factors such as gender,hypertension and education level,with interaction between PUFAs and BMI observed(P＜0.05).Conclusion Increased daily intake of MUFAs or PUFAs is significantly associated with an increased risk of NAFLD,and further research is needed to clarify their specific roles in hepatic lipid accumulation.

Non-small cell lung cancer (NSCLC) is a malignant tumor with a high global incidence rate, accounting for about 10.54% of all new cancer cases and posing a serious threat to human health. Due to significant individual variations in the efficacy of immunotherapy among NSCLC patients, it is necessary to identify accurate detection indicators to screen appropriate populations, monitor treatment efficacy, and assist in prognosis assessment. Programmed death-ligand 1 (PD-L1), as an immunosuppressive molecule expressed on the surface of tumor cells and various immune cell membranes, can serve as a "companion diagnostic" or "supplementary diagnostic" tool to guide clinical treatment decisions for metastatic NSCLC patients. Given that tumor tissue PD-L1 testing is an invasive procedure and its reliability is still under debate, the assessment of PD-L1 expression via liquid biopsies, such as circulating tumor cells, will play a significant role in predicting treatment response and prognosis in NSCLC patients.

OBJECTIVES: To analyze MYO1B expression in gastric cancer, its association with long-term prognosis and its role in regulating biological behaviors of gastric cancer cells. METHODS: We analyzed MYO1B expression in gastric cancer and its correlation with tumor grade, tumor stage, and patient survival using the Cancer Public Database. We also examined MYO1B expression with immunohistochemistry in gastric cancer and paired adjacent tissues from 105 patients receiving radical surgery and analyzed its correlation with cancer progression and postoperative 5-year survival of the patients. GO and KEGG enrichment analyses were used to explore the biological functions of MYO1B and the key pathways. In cultured gastric cancer cells, we examined the changes in cell proliferation, migration and invasion following MYO1B overexpression and knockdown. RESULTS: Data from the Cancer Public Database showed that MYO1B expression was significantly higher in gastric cancer tissues than in normal tissues with strong correlations with tumor grade, stage and patient prognosis (P<0.05). In the clinical tissue samples, MYO1B was significantly overexpressed in gastric cancer tissues in positive correlation with Ki67 expression (r=0.689, P<0.05) and the parameters indicative of gastric cancer progression (CEA ≥5 μg/L, CA19-9 ≥37 kU/L, G3-4, T3-4, and N2-3) (P<0.05). Kaplan-Meier analysis and multivariate Cox regression analysis suggested that high MYO1B expression was associated with decreased postoperative 5-year survival and was an independent risk factor (HR: 3.522, 95%CI: 1.783-6.985, P<0.05). MYO1B expression level was a strong predictor of postoperative survival (cut-off value: 3.11, AUC: 0.753, P<0.05). GO and KEGG analyses suggested that MYO1B may regulate cell migration and the mTOR signaling pathway. In cultured gastric cancer cells, MYO1B overexpression significantly enhanced cell proliferation, migration, and invasion and promoted the phosphorylation of Akt and mTOR. CONCLUSIONS High MYO1B expression promotes proliferation, migration and invasion of gastric cancer cells and is correlated with poor patient prognosis.
Humans ; Stomach Neoplasms/metabolism* ; Cell Proliferation ; Prognosis ; Cell Movement ; Myosin Type I/genetics* ; Neoplasm Invasiveness ; Cell Line, Tumor ; Female ; Male