Reactive oxygen species (ROS) are major contributors to radiation therapy-induced side effects in cancer patients. A fusion antioxidant enzyme comprising glutathione S-transferase (GST), superoxide dismutase 1 (SOD1), and a transmembrane peptide has been shown to effectively mitigate ROS-induced damage. To enhance its targeting capability, the fusion protein was further modified by incorporating a matrix metalloproteinase-2/9 substrate peptide (X) and the transmembrane peptide R9, yielding the antioxidant enzyme GST-SOD1-X-R9 (GS1XR). This modification reduced its transmembrane ability in tumor cells, thereby selectively protecting normal cells from oxidative stress. However, the use of non-human GST poses potential immunogenicity risks. In this study, we employed seamless cloning technology to construct an expression vector containing the human GST gene to replace the non-human GST gene, and then expressed and purified novel fusion antioxidant enzymes GS1R and GS1XR. The protective effects of newly constructed GS1R and GS1XR against hydrogen peroxide (H₂O₂)-induced oxidative damage in L-02 cells were then evaluated using GS1 as a control. Enzymatic activity assays revealed that the specific activity of GST in GS1XR remained unchanged compared to the unmodified protein, while SOD activity was enhanced. Exposure to 200 μmol/L H₂O₂ transiently activated the nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway; however, this activation diminished after 24 h, reducing cell viability to 48.4%. Both GS1R and GS1XR effectively scavenged intracellular ROS, directly counteracting oxidative stress and promoting Nrf2 nuclear translocation, thereby activating antioxidant pathways and restoring cell viability to normal levels. The two enzymes showed comparable efficacy. In contrast, GS1, lacking transmembrane capability, was restricted to scavenging extracellular ROS and provided only limited protection. In conclusion, both novel fusion antioxidant enzymes demonstrated significant potential in safeguarding normal cells from ROS-mediated oxidative damage. The findings provide a foundation for further investigation in related field.
Humans ; Oxidative Stress/drug effects* ; Hydrogen Peroxide ; Antioxidants/metabolism* ; Glutathione Transferase/metabolism* ; Recombinant Fusion Proteins/pharmacology* ; Superoxide Dismutase-1 ; Reactive Oxygen Species/metabolism* ; Superoxide Dismutase/biosynthesis*

Vitiligo is an immune memory skin disease. T-cell factor 1 (TCF1) is essential for maintaining the memory T-cell pool. There is an urgent need to investigate the characteristics of peripheral memory T-cell profile and TCF1⁺ T-cell frequencies in patients with vitiligo. In this study, 31 patients with active vitiligo (AV), 22 with stable vitiligo (SV), and 30 healthy controls (HCs) were included. We measured circulating memory and TCF1⁺ T-cell frequencies using flow cytometry. The Spearman's rank test was used to evaluate the correlation between cell frequencies and disease characteristics. Receiver operating characteristic curves (ROC) were constructed to investigate the discriminative power of the cell subpopulations. Circulating CD4⁺ and CD8⁺ terminally differentiated effector memory T-cell (T_EMRA) frequencies were significantly higher in the AV group than in HCs (P < 0.05). TCF1⁺ T-cell subpopulations were widespread increased in patients with vitiligo (P < 0.05). After adjusting for potential confounders, CD8⁺ and CD4⁺ central memory (T_CM) cells, and CD8⁺ T_EMRA were correlated with disease activity (P < 0.05). The combined diagnostic value of the four (naïve, effector memory, T_CM, and T_EMRA) CD8⁺TCF1⁺ T-cell subsets was relatively high (area under the ROC curve (AUC) = 0.804, sensitivity = 71.70%, specificity = 83.34%), and the CD8⁺ T-cell subsets combination performed well in discriminating disease activity (AUC = 0.849, sensitivity = 70.97%, specificity = 90.91%). We demonstrated an altered circulating memory T-cell profile and increased TCF1⁺ T-cell percentage in patients with vitiligo. T-cell subpopulations had a strong value for vitiligo diagnosis and activity evaluation. This evidence presents a potential new pharmacological target for inhibiting autoimmunity that leads to vitiligo.

Objective To investigate the effect of the fusion of leader peptide on the structure of human manganese superoxide dismutase (SOD2) and anti-cisplatin (DDP)-induced renal injury. Methods The effect of mitochondrion targeting sequence (MTS) on the structure and activity of SOD2 was analyzed by structure prediction and superoxide dismutase (SOD) specific-activity determination. The DDP injury model of Kunming (KM) mice was established, and amifostine (AMFT) was set as a positive control. Indicators such as kidney index, renal function, kidney antioxidant capacity, and appearance and pathology changes of mice kidney were used to evaluate the effect of MTS-SOD2 against DDP-induced kidney injury. Results The MTS leader peptide seemed to change the secondary and tertiary structures of SOD2 to some extent, but it also increased the specific activity of the MTS-SOD2 protein. Pre-administration of a medium dose of MTS-SOD2 (0.84 mg/kg) before the use of DDP significantly reduced the level of renal malondialdehyde and increased the SOD activity and total antioxidant capacity (T-AOC) in the kidney, thereby reducing the renal pathological damage and consequently maintaining renal function. The overall protective effect of MTS-SOD2 was comparable to or even better than that of 200 mg/kg AMFT. Conclusion The MTS leader peptide enhances the activity of SOD2 and confers it with an excellent anti-DDP-induced renal-injury effect because of its transmembrane function.

Antioxidant enzymes fused with cell-penetrating peptides could enter cells and protect cells from irradiation damage. However, the unselective transmembrane ability of cell-penetrating peptide may also bring antioxidant enzymes into tumor cells, thus protecting tumor cells and consequently reducing the efficacy of radiotherapy. There are active matrix metalloproteinase (MMP)-2 or MMP-9 in most tumor cellular microenvironments. Therefore, a fusion protein containing an MMP-2/9 cleavable substrate peptide X, a cell-penetrating peptide R9, a glutathione S-transferase (GST), and a human Cu, Zn superoxide dismutase (SOD1), was designed and named GST-SOD1-X-R9. In the tumor microenvironment, GST-SOD1-X-R9 would lose its cell-penetrating peptide and could not enter tumor cells due to the cleavage of substrate X by active MMP-2/9, thereby achieving selected entering normal cells. The complete nucleotide sequence of SOD1-X-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1. The pGEX4T-1-SOD1-X-R9 recombinant plasmid was obtained, and soluble expression of the fusion protein was achieved. GST-SOD1-X-R9 was purified by ammonium sulfate precipitation and GST affinity chromatography. The molecular weight of the fusion protein was approximately 47 kDa, consistent with the theoretical value. The SOD and GST activities were 2 954 U/mg and 328 U/mg, respectively. Stability test suggested that almost no change in either SOD activity or GST activity of GST-SOD1-X-R9 was observed under physiological conditions. The fusion protein could be partially digested by collagenase Ⅳ in solution. Subsequently, the effect of MMP-2/9 activity on transmembrane ability of the fusion protein was tested using 2D and 3D cultured HepG2 cells. Little extracellular MMP-2 activity of HepG2 cells was observed under 2D culture condition. While under the 3D culture model, the size and the MMP-2 activity of the HepG2 tumor spheroid increased daily. GST-SOD1-R9 proteins showed the same transmembrane efficiency in 2D cultured HepG2 cells, but the transmembrane efficiency of GST-SOD1-X-R9 in 3D cultured HepG2 spheres was reduced remarkably. This study provided a basis for further investigating the selectively protective effect of GST-SOD1-X-R9 against oxidative damage in normal cells.
Ammonium Sulfate ; Antioxidants ; Cell-Penetrating Peptides/pharmacology* ; Endopeptidases ; Glutathione Transferase/metabolism* ; Humans ; Matrix Metalloproteinase 2/genetics* ; Matrix Metalloproteinase 9/genetics* ; Recombinant Fusion Proteins ; Recombinant Proteins ; Superoxide Dismutase/metabolism* ; Superoxide Dismutase-1

Objective:To propose and verify a surgical classification system for the axial primary malignant and aggressive benign tumor.Methods:The CZH surgical classification system was originally developed for the axial primary malignant and aggressive benign tumor. The CZH surgical classification system includes seven types, according to the anatomic features and the extension of tumor violation. A total of 136 patients (79 males and 57 females) with axial primary malignant and aggressive benign tumor from multiple tertiary centers who received surgery from July 2006 to July 2019 were included. The average age was 44.40±17.55 years (8-83 years) old. There were 99 malignant tumors and 37 aggressive benign tumors included. The number of patients with each classification was presented as followed, Type I 13, Type II 15, Type IIIa 3, Type IIIb 20, Type IVa 43, Type IVb 12, Type Va 21, Type Vb 3, Type VI 2, Type VIIa 3 and Type VIIb 1. Surgical procedures were selected according to different types in classification. The inter- and intra-observer consistencies were evaluated by the Kendall's W test. The VAS, Frankel score, overall survival and recurrence free survival were recorded during the follow-up. Results:The inter- and intra-observer consistent coefficient was 0.973 and 0.996, respectively ( P<0.05). The single posterior approach was adopted for the Type II tumors. Other patients underwent surgery by the combined antero-posterior approach. The majority in anterior approach (113 cases) was the modified submandibular approach. The reconstruction modes included anterior "T" shape titanium mesh (112 cases) or the 3D printed prothesis (7 cases) combined with the posterior occipto-cervical fusion (92 cases) or the pedicle screw system (44 cases). The average surgical duration and the volume of intraoperative bleeding was 348.40±136.14 min (60-760 min) and 1 225.69±859.40 ml (80-4 000 ml), respectively. The operation duration and volume of intraoperative bleeding among each type were with statistical difference. The patients with Type IV, V tumors had longer operation duration than those with Type II tumors. Those with Type V and VII tumors had longer operation duration than those with Type I tumors. The patients with Type V tumors had more intraoperative bleeding than those with Type I-IV tumors. The average preoperative VAS score was 4.15±2.25 and then was reduced significantly to 0.62±0.71 and 0.38±0.59 at one and three months after operation, respectively. The Frankel score was also significantly ameliorated at one and three months postoperatively. There were 22 postoperative complications (16.2%). The complications included cerebral spinal fluid leak (12.5%), dysphagia and/or dysphonia (7.4%), dyspnea (5.1%), wound infection (3.7%), wound hemorrhage (2.2%) and pharyngeal dehiscence (1.5%). The incidence of postoperative complication was 25.9% in Type IV-VII tumors, while 11.8% in Type I-III tumors. Conclusion:CZH surgical classification system was verified with high observer consistency. This classification system could assist surgeons to select proper surgical approaches, resection modes and reconstruction modes, and thus ensure the safety of surgery and reduce the recurrence. The tumors in Type IV, V and VII may be with more challenging for surgeons. The incidence of postoperative complication in Type IV-VII tumors may be higher than that in Type I-III tumors.

Retinoic acid, an active metabolite of vitamin A, exerts multiple effects on regulating embryonic development and inducing differentiation, proliferation, apoptosis as well as resistance in various cancer cells. Apart from the classic genomic action (binding to the nuclear receptors to regulate the expression of its downstream target genes), retinoic acids also play important roles in anti-cancer effect through non-genomic pathways (via extranuclear and non-transcriptional effects).
Apoptosis ; Cell Differentiation ; Genomics ; Humans ; Neoplasms ; drug therapy ; Receptors, Retinoic Acid ; Tretinoin

Two proteins of similar molecular weight (named as ASPR-C-1 and ASPR-C-2) from the crude drug of Angelica sinensis were purified and characterized by 80% ammonium sulfate precipitation, Sephadex G-50 gel filtration chromatography, and DEAE-Sepharose anion exchange chromatography. The molecular weight of ASPR-C-1 and ASPR-C-2 on SDS-PAGE was 17.33 kDa and 17.18 kDa, respectively. They were mainly monomeric in solution, but partially formed dimers and they were glycoproteins with glycosyl content of 2.6% and 8.2%, respectively. Both ASPR-C-1 and ASPR-C-2 were identified to be members of pathogenesis-related 10 family of proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and have ribonuclease activities with the specific activity of 73.60 U/mg and 146.76 U/mg, respectively. The optimum pH of the two isoforms was similar, at about 5.6, while their optimum temperatures were different. The optimum temperature of ASPR-C-1 was 50 ℃, and that of ASPR-C-2 was 60 ℃. Both isoforms presented highest thermal stability at 60 ℃. However, ASPR-C-2 was more thermotolerant than ASPR-C-1. The latter was rapidly inactivated and retained only about 20% residual activity while the former still maintained about 80% of its original activity at a higher treatment temperature (80 to 100 ℃). In addition, Fe²⁺ had an activating effect on the ribonuclease activities of two isoforms while Ca²⁺, Mg²⁺, Zn²⁺, Mn²⁺, Ag⁺, Cu²⁺, EDTA (Elhylene diamine tetraacetic acid), dithiothreitol and sodium dodecylsulphate showed different degrees of inhibition of the enzyme activities. Our findings provide a foundation for further research on the biological function of PR-10 protein from Angelica sinensis.
Angelica sinensis ; Chromatography, Gel ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Enzyme Stability ; Hydrogen-Ion Concentration ; Kinetics ; Molecular Weight ; Protein Isoforms ; Temperature

STUDY DESIGN: A prospective cross-sectional study. PURPOSE: To evaluate the risk factors associated with the severity of pain intensity in patients with non-specific low back pain (NSLBP) in Southern China. OVERVIEW OF LITERATURE: Low back pain (LBP) is the leading cause of activity limitation and work absence throughout the world, so a firm understanding of the risk factor associated with NSLBP can provide early and prompt interventions that are aimed at attaining long-term results. METHODS: Participants were recruited from January 2014 to January 2016 and were surveyed using a self-designed questionnaire. Anonymous assessments included Short Form 36-Item Health Survey (SF-36) and Visual Analogue Scale (VAS). The association between the severity of NSLBP and these potential risk factors were evaluated. RESULTS: A total of 1,046 NSLBP patients were enrolled. The patients with primary school education, high body mass index (BMI), those exposed to sustained durations of driving and sitting, smoking, recurrent LBP had increased VAS and Oswestry Disability Index (ODI) scores with lower SF-36 scores (p<0.01). Workers and drivers compared with waiters and patients who lifted >10 kg objects in a quarter of their work time for >10 years had higher VAS and ODI scores with lower SF-36 scores (p<0.01). Multiple logistic regression showed lower levels of education, LBP for 1–7 days, long-lasting LBP in last year, smoking, long duration driving, and higher BMI were associated with more severe VAS score. CONCLUSIONS: The severity of NSLBP is associated with lower levels of education, poor standards of living, heavy physical labor, long duration driving, and sedentary lifestyle. Patients with recurrent NSLBP have more severe pain. Reducing rates of obesity, the duration of heavy physical work, driving or riding, and attenuating the prevalence of sedentary lifestyles and smoking may reduce the prevalence of NSLBP.
Anonyms and Pseudonyms ; Body Mass Index ; China ; Cross-Sectional Studies ; Education ; Health Surveys ; Humans ; Logistic Models ; Low Back Pain ; Obesity ; Prevalence ; Prospective Studies ; Risk Factors ; Sedentary Lifestyle ; Smoke ; Smoking

The fusion of cell permeable peptide TAT and bifunctional antioxidant enzymes, GST (Glutathione sulfur transferase)-TAT-SOD1 (Cu, Zn superoxide dismutase), is an intracellular superoxide scavenger. Compared with SOD1-TAT, GST-TAT-SOD1 has better protective effect on oxidative damage but less transduction efficiency. A novel cell permeable bifunctional antioxidant enzymes with the fusion of GST, SOD1 and polyarginine R9 was constructed for higher transduction efficiency. The full nucleotide sequence of SOD1-R9 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1 with the GST tag. After the successful construction of the prokaryotic expression vectors of GST-SOD1-R9, the recombinant vector was then transformed into Escherichia coli BL21 (DE3) and the GST-SOD1-R9 fusion protein was produced with the induction of IPTG. The soluble expression of GST-SOD1-R9 fusion protein was combining with the induction temperature and time. The best soluble expression was obtained with the induction temperature of 25 ℃ and the induction time of 11 h. The fusion protein was purified through the combination of 80% ammonium sulfate precipitation and affinity chromatography using glutathione agarose, and verified by SDS-PAGE and special enzymatic activity. The thermal and pH stability of GST-SOD1-R9 fusion protein were analyzed and the SOD and GST activity of fusion protein were proved to be well maintained under physiological conditions. Finally, the transduction efficiency of GST-SOD1-R9 fusion protein was proved to be better than GST-TAT-SOD1 fusion protein (P<0.05). These works establish a foundation for further study of the protective effect of GST-SOD1-R9 fusion protein against oxidative damage.

Superoxide dismutase (SOD) family is necessary to protect cells from the toxicity of reactive oxygen species produced during normal metabolism. Among SODs, manganese-containing superoxide dismutase (Mn-SOD, SOD2) is the most important one. The DNA fragment containing the full nucleotide of full-length human SOD2 was synthesized and inserted into the prokaryotic expression vector pGEX-4T-1 with tag GST. DNA construct was then transformed into Escherichia coli BL21 (DE3) and expression was induced with IPTG at 25 ℃. The recombinant fusion protein GST-SOD2 (46 kDa) was purified from the bacterial lysate by GST resin column affinity chromatography. GST tag was cleaved with thrombin, and a crude SOD2 recombinant protein (25 kDa) was obtained and further purified by heparin affinity chromatography. Activities of the two SOD2 proteins were 1 788 and 2 000 U/mg, respectively. Both SOD2 proteins were stable under physiological condition and cell-penetrating (P<0.05). Our findings open the possibility to study the structure and effects of two full-length recombinant SOD2 proteins.