OBJECTIVE: To prepare decellularized nerve grafts from alpha-1, 3-galactosyltransferase (GGTA1) gene-edited pigs and explore their biocompatibility for xenotransplantation. METHODS: The sciatic nerves from wild-type pigs and GGTA1 gene-edited pigs were obtained and underwent decellularization. The alpha-galactosidase (α-gal) content in the sciatic nerves of GGTA1 gene-edited pigs was detected by using IB4 fluorescence staining and ELISA method to verify the knockout status of the GGTA1 gene, and using human sciatic nerve as a control. HE staining and scanning electron microscopy observation were used to observe the structure of the nerve samples. Immunofluorescence staining and DNA content determination were used to evaluate the degree of decellularization of the nerve samples. Fourteen nude mice were taken, and subcutaneous capsules were prepared on both sides of the spine. Decellularized nerve samples of wild-type pigs ( n=7) and GGTA1 gene-edited pigs ( n=7) were randomly implanted in the subcutaneous capsules. Blood was drawn at 1, 3, 5, and 7 days after implantation to detect neutrophil counting. RESULTS: IB4 fluorescence staining and ELISA detection showed that GGTA1 gene was successfully knocked out in the nerves of GGTA1 gene-edited pigs. HE staining showed that the structure of the decellularized nerve from GGTA1 gene-edited pigs was well preserved; the nerve basement membrane tube structure was visible under scanning electron microscopy; no cell nuclei was observed, and the extracellular matrix components was retained in the nerve grafts by immunofluorescence staining; and the DNA content was significantly reduced when compared with the normal nerves ( P<0.05). In vivo experiments showed that the number of neutrophils in the two groups were similar at 1, 3, and 7 days after implantation, with no significant difference ( P>0.05); only at 5 days, the number of neutrophils was significantly lower in the GGTA1 gene-edited pigs than in the wild-type pigs ( P<0.05). CONCLUSION The decellularized nerve grafts from GGTA1 gene-edited pigs have well-preserved nerve structure, complete decellularization, retain the natural nerve basement membrane tube structure and components, and low immune response after xenotransplantation through in vitro experiments.
Animals ; Transplantation, Heterologous ; Galactosyltransferases/genetics* ; Sciatic Nerve/immunology* ; Swine ; Tissue Engineering/methods* ; Humans ; Graft Rejection/prevention & control* ; Gene Editing ; Mice ; Mice, Nude ; Heterografts/immunology* ; Animals, Genetically Modified ; Tissue Scaffolds ; Decellularized Extracellular Matrix

Purpose: The risk stratification of pediatric anaplastic large cell lymphoma (ALCL) has not been standardized. In this study, new risk factors were included to establish a new risk stratification system for ALCL, and its feasibility in clinical practice was explored. Materials and Methods: On the basis of the non-Hodgkin’s lymphoma Berlin–Frankfurt–Munster 95 (NHL-BFM-95) protocol, patients with minimal disseminated disease (MDD), high-risk tumor site (multiple bone, skin, liver, and lung involvement), and small cell/lymphohistiocytic (SC/LH) pathological subtype were enrolled in risk stratification. Patients were treated with a modified NHL-BFM-95 protocol combined with an anaplastic lymphoma kinase inhibitor or vinblastine (VBL). Results: A total of 136 patients were enrolled in this study. The median age was 8.8 years. The 3-year event-free survival (EFS) and overall survival of the entire cohort were 77.7% (95% confidence interval [CI], 69.0% to 83.9%) and 92.3% (95% CI, 86.1% to 95.8%), respectively. The 3-year EFS rates of low-risk group (R1), intermediate-risk group (R2), and high-risk group (R3) patients were 100%, 89.5% (95% CI, 76.5% to 95.5%), and 67.9% (95% CI, 55.4% to 77.6%), respectively. The prognosis of patients with MDD (+), stage IV cancer, SC/LH lymphoma, and high-risk sites was poor, and the 3-year EFS rates were 45.3% (95% CI, 68.6% to 19.0%), 65.7% (95% CI, 47.6% to 78.9%), 55.7% (95% CI, 26.2% to 77.5%), and 70.7% (95% CI, 48.6% to 84.6%), respectively. At the end of follow-up, one of the five patients who received maintenance therapy with VBL relapsed, and seven patients receiving anaplastic lymphoma kinase inhibitor maintenance therapy did not experience relapse. Conclusion This study has confirmed the poor prognostic of MDD (+), high-risk site and SC/LH, but patients with SC/LH lymphoma and MDD (+) at diagnosis still need to receive better treatment (ClinicalTrials.gov number, NCT03971305).

OBJECTIVES: Multiple myeloma (MM) is a plasma cell malignancy occurring in middle and old age. MM is still an incurable disease due to its frequent recurrence and drug resistance. However, its pathogenesis is still unclear. Abnormal amino acid metabolism is one of the important characteristics of MM, and the important metabolic pathway of amino acids participates in protein synthesis as basic raw materials. Aminoacyl transfer ribonucleic acid synthetase (ARS) gene is a key regulatory gene in protein synthesis. This study aims to explore the molecular mechanism for ARS, a key factor of amino acid metabolism, in regulating amino acid metabolism in MM and affecting MM growth. METHODS: The corresponding gene number was combined with the gene expression profile GSE5900 dataset and GSE2658 dataset in Gene Expression Omnibus (GEO) database to standardize the gene expression data of ARS. GSEA_4.2.0 software was used to analyze the difference of gene enrichment between healthy donors (HD) and MM patients in GEO database. GraphPad Prism 7 was used to draw heat maps and perform data analysis. Kaplan-Meier and Cox regression model were used to analyze the expression of ARS gene and the prognosis of MM patients, respectively. Bone marrow samples from 7 newly diagnosed MM patients were collected, CD138⁺ and CD138^- cells were obtained by using CD138 antibody magnetic beads, and the expression of ARS in MM clinical samples was analyzed by real-time RT-PCR. Human B lymphocyte GM12878 cells and human MM cell lines ARP1, NCI-H929, OCI-MY5, U266, RPMI 8266, OPM-2, JJN-3, KMS11, MM1.s cells were selected as the study objects. The expression of ARS in MM cell lines was analyzed by real-time RT-PCR and Western blotting. Short hairpin RNA (shRNA) lentiviruses were used to construct gene knock-out plasmids (VARS-sh group). No-load plasmids (scramble group) and gene knock-out plasmids (VARS-sh group) were transfected into HEK 293T cells with for virus packaging, respectively. Stable expression cell lines were established by infecting ARP1 and OCI-MY5 cells, and the effects of knockout valyl-tRNA synthetase (VARS) gene on proliferation and apoptosis of MM cells were detected by cell counting and flow cytometry, respectively. GEO data were divided into a high expression group and a low expression group according to the expression of VARS. Bioinformatics analysis was performed to explore the downstream pathways affected by VARS. Gas chromatography time-of-flight mass spectrometry (GC-TOF/MS) and high performance liquid chromatography (HPLC) were used to detect the valine content in CD138⁺ cells and ARP1, OCI-MY5 cells and supernatant of knockdown VARS gene in bone marrow samples from patients, respectively. RESULTS: Gene enrichment analysis showed that tRNA processing related genes were significantly enriched in MM compared with HD (P<0.0001). Further screening of tRNA processing-pathway related subsets revealed that cytoplasmic aminoacyl tRNA synthetase family genes were significantly enriched in MM (P<0.0001). The results of gene expression heat map showed that the ARS family genes except alanyl-tRNA synthetase (AARS), arginyl-tRNA synthetase (RARS), seryl-tRNA synthetase (SARS) in GEO data were highly expressed in MM (all P<0.01). With the development of monoclonal gammopathy of undetermined significance (MGUS) to MM, the gene expression level was increased gradually. Kaplan-Meier univariate analysis of survival results showed that there were significant differences in the prognosis of MM patients in methionyl-tRNA synthetase (MARS), asparaginyl-tRNA synthetase (NARS) and VARS between the high expression group and the low expression group (all P<0.05). Cox regression model multivariate analysis showed that the high expression of VARS was associated with abnormal overall survival time of MM (HR=1.83, 95% CI 1.10 to 3.06, P=0.021). The high expression of NARS (HR=0.90, 95% CI 0.34 to 2.38) and MARS (HR=1.59, 95% CI 0.73 to 3.50) had no effect on the overall survival time of MM patients (both P>0.05). Real-time RT-PCR and Western blotting showed that VARS, MARS and NARS were highly expressed in CD138⁺ MM cells and MM cell lines of clinical patients (all P<0.05). Cell counting and flow cytometry results showed that the proliferation of MM cells by knockout VARS was significantly inhibited (P<0.01), the proportion of apoptosis was significantly increased (P<0.05). Bioinformatics analysis showed that in addition to several pathways including the cell cycle regulated by VARS, the valine, leucine and isoleucine catabolic pathways were upregulated. Non-targeted metabolomics data showed reduced valine content in CD138⁺ tumor cells in MM patients compared to HD (P<0.05). HPLC results showed that compared with the scramble group, the intracellular and medium supernatant content of ARP1 cells and the medium supernatant of OCI-MY5 in the VARS-shRNA group was increased (all P<0.05). CONCLUSIONS MM patients with abnormal high expression of VARS have a poor prognosis. VARS promotes the malignant growth of MM cells by affecting the regulation of valine metabolism.
Humans ; Valine-tRNA Ligase ; Multiple Myeloma/genetics* ; Metabolomics ; Amino Acids ; RNA, Transfer

Objective:To evaluate the clinical efficacy of multi-disciplinary single center's CCCG-HB-2016 regimen in the treatment of hepatoblastoma (HB) in children.Methods:Clinical data of 36 HB patients treated with CCCG-HB-2016 program from Aug 2016 to March 2020 were analyzed.Results:These 36 patients included 20 boys and 16 girls. The serum AFP was all higher than 2 792 ng/ml,there was a correlation between AFP and tumor risk stratification ( H=14.973, P<0.05). Twenty eight cases (77.78%) were epithelial type and 8 cases (22.22%) were mixed epithelial mesenchymal type.All children were treated by tumor resection combined with chemotherapy, and there was a correlation between tumor risk stratification and surgical resection of liver lobe ( H=8.847, P<0.05). The probability of bone marrow suppression in the low-risk group was 58.33% (35/60),that in the intermediate-risk group was 73.49% (61/83) and in the high-risk group was 80.23% (69/86).All 36 cases were followed up to March 31, 2020,with an average follow-up of 21.9 months and the median survival was 22.5 months.The overall survival rate (OS) and event-free survival rate (EFS) were 97.2% and 83.3% respectively. Conclusions:The multidisciplinary CCCG-HB-2016 regimen was with a high success rate and along with a high incidence of bone marrow suppression.

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

Objective To investigate the application value of ultrasound-guided fine-needle aspiration cytology of lymph nodes in the pre-radiotherapy evaluation of superficial lymph nodes metastases in patients with lung cancer and esophageal cancer. Methods A total of 115 patients with lung cancer and esophageal cancer prepared for thoracic radiotherapy from February 2017 to September 2018 in Cancer Hospital of Huanxing Chaoyang District Beijing were retrospectively analyzed. Ultrasound-guided fine-needle aspiration cytology examination was performed in 166 lymph nodes. Puncture tissues were prepared for cytology production. Then cytological specialist read the film to evaluate if the cancer cells metastasis were present after conventional papanicolaou staining. The effects of short diameter of lymph nodes, blood flow signal and tumor characteristics of patients on the results of cytological examination were analyzed. Results The ultrasound results showed the median short diameter of lymph nodes was 0.6 cm (0.2-3.5 cm), and 25 (15.1％) lymph nodes had blood flow signals, 50 (30.1％) lymph nodes had positive results of puncture tissues cytology. The positive rate of puncture tissues cytology was 10.0％(4/40), 21.6％(21/97) and 86.2％(25/29) in the shortdiameter of lymph nodes <0.5 cm group, ≥0.5 cm and <1 cm group, and ≥1 cm group, respectively (P<0.01). Cytology positive rate of lymph nodes with or without blood flow signal detected by ultrasound was 48.0％ (12/25) and 27.0％ (38/141), and there was a statistical difference (P = 0.034). Location of primary tumor, T stage and chemotherapy history had no significant influence on the cytological results in patients with lung cancer (all P> 0.05). Pathological types had a significant influence on cytological results (χ2= 8.050, P= 0.045). Lymph node metastasis of the upper mediastinum was a risk factor for lymph node metastasis in lower neck and supraclavicular region (χ2= 9.699, P= 0.002). Location of primary tumor, T stage and chemotherapy history had no significant influence on cytological results in patients with esophageal cancer (all P> 0.05). Conclusions Ultrasound-guided fine-needle aspiration cytology is safe and efficient. It can be used to evaluate the metastasis status of superficial lymph nodes in pre-radiotherapy patients with lung cancer and esophageal cancer.

Objective To compare the effect of nutritional support with and without tube feeding on the incidence of adverse reactions in radiotherapy for esophageal cancer.Methods A total of 120 esophageal cancer patients with high-risk factors receiving radiotherapy were selected and randomly assigned into the tube feeding (TF,n=60) and non-tube feeding groups (NTF,n=60) according to the random number method.Nutritional interventions were performed during radiotherapy in both groups.The incidence of esophagitis and myelosuppressioa,aulmonary infectioa,autritional status and the completion of radiotherapy were observed and statistically compared between TF and NTF groups.Results In the TF groua,ahe incidence of ≥ grade 2 esophagitis was lower than that in the NTF group.The incidence of grade 3 esophagitis significantly differed between two groups (P＜ 0.05).The incidence of grade 1-2 myelosuppression did not differ between two groupa,ahereas the incidence of grade 3 myelosuppression in the TF group was significantly lower than that in the NTF group (P＜0.05).In the TF groua,ahe incidence of pulmonary infection was remarkably lower than that in the NTF group (P＜0.05).The changes of BMI and prealbumin in the TF group were better than those in the NTF group (both P＜0.05).One patient in the NTF group failed to complete the radiotherapy due to grade Ⅳ esophagitia,and 5 cases in the NTF group (P＜ 0.05).In the TF groua,ahe length of hospital stay was significantly shortened by 6.2 d on average (P＜ 0.05).Conclusions During radiotherapy for esophageal cancer patients with high-risk factora,autritional support with tube feeding can effectively reduce the incidence of adverse reactiona,amprove the completion rate of treatmena,and shorten the average length of hospital stay.

Objective: To explore the new gene mutation and clinical manifestations of Caroli syndrome in 2 blood brother cases. Methods: The clinical data of the 2 brothers with clinical diagnosis of Caroli syndrome were analyzed, and the related genes were detected by second-generation sequencing technology. Results: The PKHD1 gene of both brothers had the mutation of c. 5059A>T(exon32). c. 4358(exon 32)delC shift mutation, neither of which has been reported in OMIM database. Conclusion After analysis, c. 4358(exon32) delC and c. 5059A>T(exon32) may be the new pathogenic mutation of this disease, with the same mutant genotype but completely different phenotype.

Objective To explore the effect of jejunum tube implantation on the reduction of adverse reactions,outcomes improvement and cost reduction in esophageal cancer patients with radiotherapy. Methods Eighty-two esophageal cancer patients with nutritional risk in Beijing Chaoyang District Huanxing Cancer Hospital from August 2014 to December 2016 were analyzed. Forty-one cases received jejunum tube implantation (implantation group) and 41 cases received non-nutrient tube implantation (control group), and data on their nutritional status,clinical efficacy,adverse reactions,and hospitalization costs were collected and compared. Results Compared with the control group, the implantation group has higher body mass index [(21.7±0.5)kg/m2vs. (19.5±0.3) kg/m2,t =2.12,P =0.031], hemoglobin[(120.0±2.1) g/L vs. (115.0±6.3) g/L, t= 2.12, P= 0.031] and clinical efficacy [95.1 % (39/41) vs. 87.8 % (36/41), χ2= 6.11, P= 0.022]. The incidence of adverse reactions in implantation group was significantly lower than that in control group[29.3 % (12/41) vs. 51.2 %(21/41),χ 2=8.12,P=0.002). The total cost in implantation group was lower than that in control group (138 000 yuan vs. 147 000 yuan, t= 2.09, P= 0.037), and the average hospitalization day was significantly reduced (42.1 d vs. 48.4 d, t = 2.27, P = 0.029). Conclusion Enteral nutrition support with jejunal feeding tube can reduce the incidence of adverse reactions, improve the curative effect, shorten hospitalization time and save medical cost for patients with esophageal cancer.

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.