As the major pathway mediating specific protein degradation in eukaryotes, ubiquitin-proteasome system (UPS) is involved in various physiological and pathological processes such as cell cycle regulation, immune response, signal transduction and DNA-repair. Deubiquitinases (DUB) maintain the balance of UPS and related physiological processes via reversibly removing ubiquitin from the covalently modified protein substrates, which have been implicated in various disease processes in case of their imbalance expression. Because DUB plays critical regulating roles in the UPS pathway, they may be also the ideal drug targets for severe and intractable human diseases, such as cancer and neurodegenerative disease. With the rapid development of proteomic technology, systematical investigation of specific substrates and interacting proteins of varied DUB via mass spectrometry approach may shed light on these DUB's biological function and regulating roles in the physiological and pathogenic states. In this review, we briefly introduce the characteristics of DUB and summarize the recent application and progresses of proteomics in DUB research.
Humans ; Mass Spectrometry ; Proteasome Endopeptidase Complex ; metabolism ; Proteomics ; Signal Transduction ; Ubiquitin ; metabolism ; Ubiquitin-Specific Proteases ; metabolism

Protein ubiquitination is one of the most important and widely exist protein post-translational modifications in eukaryotic cells, which takes the ubiquitin and ubiquitin chains as signal molecules to covalently modify other protein substrates. It plays an important roles in the control of almost all of the life processes, including gene transcription and translation, signal transduction and cell-cycle progression, besides classical 26S protesome degradation pathway. Varied modification sites in the same substrates as well as different types of ubiquitin linkages in the same modification sites contain different structural information, which conduct different signal or even determine the fate of the protein substrates in the cell. Any abnormalities in ubiquitin chain formation or its modification process may cause severe problem in maintaining the balance of intracellular environment and finally result in serious health problem of human being. In this review, we discussed the discovery, genetic characteristics and the crystal structure of the ubiquitin. We also emphasized the recent progresses of the assembly processes, structure and their biological function of ubiquitin chains. The relationship between the disregulation and related human diseases has also been discussed. These progress will shed light on the complexity of proteome, which may also provide tools in the new drug research and development processes.
Humans ; Proteome ; Ubiquitin ; chemistry ; Ubiquitination

Objective We increase the soluble expression of artificial tandem hybrid ubiquitin binding domains ( ThUBD) in prokaryotic cytoplasm of Escherichia coli BL21 ( DE3 ) , which offer an effective and special profiling for ubiquitin conjugates( UbC) .Methods Codon optimization of the ThUBD was performed, followed by analysis of codon relative adaptiveness based on relative frequency of synonymous codon ( RFSC) of E.coli.Further induced expression and yeast ubiquitin conjugate enrichment quantified the soluble ThUBD-S and tested the ability to bind UbC.Results The statistical result showed that the percentage of codon of the highest usage frequency was increased from 48%to 75%, and codon adaptation index( CAI) was increased from 0.63 to 0.88 after codon optimization, which might suggest a higher expression of the ThUBD in E.coli BL21 (DE3).The subsequent SDS-PAGE indicated that the soluble target protein was increased four times, which accounted for 13.06%of total cell lysis.Further ubiquitinated proteome of yeast demonstrated that the ability to bind and enrich UbC of optimized ThUBD-S did not change compared with original ThUBD.Conclusion The expression of ThUBD-S can quadruple after codon optimization.At the same time, codon optimization does not impact its soluble expression and the ability to bind UbC.

BACKGROUND: Wnt signaling pathway plays an important regulative role in the embryonic development processes. Accordingly, it is of great significance to establish the cell model of Wnt signaling pathway so as to conduct study on it. OBJECTIVE: To establish Wnt signaling pathway cell model by transfecting L-M (TK-) cells with Wnt3a eukaryotic expression plasmid, and to investigate the effect of canonical Wnt signal pathway on the β-catenin subcellular distribution. METHODS: The eukaryotic expression plasmid pgk-Wnt3a-pcDNA3.0 after amplification was digested by restriction endonuclease first. Then it was transfected together with the control plasmid pgk-neo-pcDNA3.0 into L-M (TK-) cells via lipofection, after which the cell colony was screened by G418 for amplification. RT-PCR was used for detecting the expression products and the indirect immunofluorescence assay for observing the effect of Wnt3a on the β-catenin subcellular localization of L-M (TK-) cells. RESULTS AND CONCLUSION: The Wnt3a plasmid was verified by endonuclease digestion to have produced the expected plasmids after amplification. According to the RT-PCR detection to the 10 stably-transfected cell colonies achieved by 3 weeks of G418 screening, it was seen, on the L-Wnt3a cDNA, a strip of bright band of 320 bp in length, which showed that the products of amplification were exactly the expected fragments and that the Wnt3a plasmid was expressed on mRNA transcriptional level after being transfected with L-M (TK-) cells. In contrast, no expected band was found on the cDNA of L-M (TK-) calls transfecting the control plasmid. In addition, the immunofiuorescence assay detection showed that the protein expression of Wnt3a was found in the cytoplasm of the L-M(TK-) cells tranfecting Wnt3a plasmid, while for those transfecting the control plasmid, it was opposite. β-catenin, as showing by bright red fluorescence, was found to concentrate and enter into the nucleus of the L-M (TK-) cells transfecting Wnt3a plasmid, while for those transfecting the control plasmid, it was opposite. Cell model with continually activated Wnt signaling pathway is established. The stable expression of Wnt3a in L-M (TK-) cells transfected with pgk-Wnt3a-pcDNA3.0 is obtained. The expression of Wnt3a is able to promote the transfer of β-catenin from cytoplasms into nucleus in L-M (TK-) cells.

Objective To evaluate the safety,tumor radical and early postoperative efficacy through comparison of laparoscopic gastric D2 radical surgery with traditional open gastric D2 radical surgery.Methods 254 patients with upper stomach cancer underwent surgery were selected,132 cases using conventional open gastrectomy (the traditional laparotomy group),122 patients underwent laparoscopic radical gastrectomy (laparoscopic surgery group).Laparoscopic surgery group with traditional open surgery group had no statistically significant differences in gender,age,tumor location,histological type and TNM staging.Results Open surgery group and laparoscopic surgery group had statistically significant differences in operative time [(235.78±31.56) min,(256.43±54.08) min,P ＜ 0.001],blood loss [(326.69±89.73) ml,(158.31±62.98) ml,P ＜ 0.001],incision length [(16.53±2.34) cm,(5.51±1.15) cm,P ＜ 0.001],gastrointestinal recovery time [(4.22±0.91) d,(3.31±0.83) d,P ＜ 0.001],first time eating liquid [(5.78±0.95) d,(5.56±0.78) d,P ＜ 0.001] and postoperative hospital stay [(12.62±2.89) d,(11.18±1.78) d,P ＜ 0.001].The total number of lymph node dissection and complications was not statistically significant.Conclusions Laparoscopic gastric D2 radical surgery is a safe,minimally invasive surgical method.Laparoscopic gastric D2 radical surgery has the same lymph node dissection and good early outcome compared with the traditional gastric D2 radical surgery,but postoperative recovery fast and less invasive.

OBJECTIVETo evaluate the clinical feasibility of laparoscope-assisted D2 radical total gastrectomy for gastric cancer.

METHODSFrom March 2011 to March 2013, 216 gastric cancer patients underwent D2 radical total gastrectomy in our hospital, including 118 laparoscopic and 98 open procedures. The intraoperative and postoperative outcomes between the two groups were compared.

RESULTSCompared with open surgery group, laparoscopic group had longer operation time [(253.9 ± 26.1) min vs. (206.2 ± 23.9) min, P<0.01], less intraoperative blood loss [(138.4 ± 34.0) ml vs. (266.3 ± 58.7) ml, P<0.01], shorter postoperative hospital stay [(10.3 ± 1.4) d vs. (13.9 ± 2.0) d, P<0.01], and lower morbidity of postoperative complication [16.1% (19/118) vs. 29.6% (29/98), P<0.05]. There were no significant differences in the number of lymph node removed [(26.2 ± 6.2 vs. 27.6 ± 5.6)], postoperative morbidity of complication [1.7% (2/118) vs. 2.0% (2/98)], and 2-year survival rate(99.0% vs. 98.6%) between the two groups (all P>0.05).

CONCLUSIONAs compared to open surgery, laparoscope-assisted D2 radical total gastrectomy is safe, feasible with the advantage of rapid postoperative recovery.

Aged ; Female ; Gastrectomy ; methods ; Humans ; Laparoscopy ; Laparotomy ; Male ; Middle Aged ; Retrospective Studies ; Stomach Neoplasms ; surgery ; Treatment Outcome

Objective : To investigate the effect and mechanism of exosome ( Exo) transported miR⁃223 on brain tissue injury and microglial activation in rats with traumatic brain injury ( TBI) . Methods : The miR⁃NC plasmid and miR⁃223 mimic plasmid were transfected into HEK293 cells by liposome method , and the expression level of miR⁃223 in the cells was determined by quantitative real⁃time PCR . Exo was extracted from transfected HEK293 cells and identified by transmission electron microscopy , nanoparticle tracking analysis and Western blot , the expression level of miR⁃223 in Exo was determined by quantitative real⁃time PCR . Forty SD rats were randomly divided into sham group , model group , NC⁃Exo group and miR⁃223 ⁃Exo group , with 10 rats in each group , TBI model was prepared by modified Feeney free fall method in all groups except sham group , rats in NC⁃Exo group and miR⁃223 ⁃Exo group were injected with cell⁃derived Exo transfected with miR⁃NC plasmid and cell⁃derived Exo transfected with miR⁃223 mimic plasmid via tail vein , respectively . Two weeks later , hematoxylin⁃eosin (HE) staining was used to observe the pathological changes of brain tissue in each group , Nissl staining was used to detect the changes and distribution of Nissl bodies in each group , enzyme⁃linked immunosorbent assay (ELISA) was used to measure the serum levels of tumor necrosis factor⁃α (TNF⁃α ) , interleukin⁃1β (IL⁃1β) and interleukin⁃6(IL⁃6) , immunofluorescence double staining was used to observe the expression of nod⁃like receptor family pyrin domain containing 3(NLRP3) and ionized calcium binding adaptor molecule 1 (Iba⁃1) , Western blot was used to detect the protein expression of NLRP3 , apoptosis⁃associated speck⁃like protein containing( ASC) and Caspase⁃1 . Results: After transfection , compared with control group and miR⁃NC group , the relative expression of miR⁃223 in miR⁃223 group significantly increased (P < 0. 05) . The isolated particles had typical Exo morphology , the peak particle size was about 120 nm , the Exo marker proteins CD9 , CD63 and CD81 were significantly overexpressed , and the relative expression of miR⁃223 significantly non of brain tissue in the miR⁃223 ⁃Exo group was improved , the morphology and number of Nissl bodies were re⁃increased (P < 0. 05) . Compared with the model group , the damage phenome stored , the levels of TNF⁃α , IL⁃1β and IL⁃6 in serum decreased ( P < 0. 05) , the intensity of NLRP3 and Iba⁃1 fluorescence staining in brain tissue decreased (P < 0. 05) , the relative protein expressions of NLRP3 , ASC and Caspase⁃1 in brain tissue were down⁃regulated (P < 0. 05) . Conclusion Exo operation of miR⁃223 can significant ly improve brain tissue injury and inhibit microglial activation in TBI rats , which may be related to the inhibition of NLRP3 .

Objective:To investigate the influences of age-adjusted Charlson comorbidity index (ACCI) on prognosis of patients undergoing laparoscopic radical gastrectomy.Methods:The retrospective cohort study was conducted. The clinicopathological data of 242 gastric cancer patients who underwent laparoscopic radical gastrectomy in 19 hospitals of the Chinese Laparoscopic Gastrointestinal Surgery Study Group-04 study, including 54 patients in Fujian Medical University Union Hospital, 32 patients in the First Hospital of Putian City, 32 patients in Renji Hospital Affiliated to Shanghai Jiaotong University School of Medicine, 31 patients in Zhangzhou Affiliated Hospital of Fujian Medical University, 17 patients in Nanfang Hospital of Southern Medical University, 11 patients in the First Affiliated Hospital with Nanjing Medical University, 8 patients in Qinghai University Affiliated Hospital, 8 patients in Meizhou People′s Hospital, 7 patients in Fujian Provincial Hospital, 6 patients in Zhongshan Hospital of Fudan University, 6 patients in Longyan First Hospital, 5 patients in the First Affiliated Hospital of Xinjiang Medical University, 5 patients in the First Hospital Affiliated to Army Medical University, 4 patients in the Second Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine, 4 patients in West China Hospital of Sichuan University, 4 patients in Beijing University Cancer Hospital, 3 patients in the First Affiliated Hospital of Xiamen University, 3 patients in Guangdong Provincial People′s Hospital, 2 patients in the First Affiliated Hospital of Xi′an Jiaotong University, from September 2016 to October 2017 were collected. There were 193 males and 49 females, aged 62(range, 23?74)years. Observation indicators: (1) age distribution, comorbidities and ACCI status of patients; (2) the grouping of ACCI and comparison of clinicopathological characteristics of patients in each group; (3) incidence of postoperative early complications and analysis of factors affecting postoperative early complications; (4) follow-up; (5) analysis of factors affecting the 3-year recurrence-free survival rate of patients. Follow-up was conducted using outpatient examination or telephone interview to detect postoperative survival of patients up to December 2020. Measurement data with normal distribution were represented as Mean± SD, and comparison between groups was conducted using the t test. Measurement data with skewed distribution were represented as M( Q1, Q3) or M(range), and comparison between groups was conducted using the Mann-Whitney U test. Count data were described as absolute numbers or percentages, and comparison between groups was conducted using the chi-square test or Fisher exact probability. Comparison of ordinal data was conducted using the nonparametric rank sum test. The X-Tile software (version 3.6.1) was used to analyze the best ACCI grouping threshold. The Kaplan-Meier method was used to calculate survival rates and draw survival curves. The Log-Rank test was used for survival analysis. The Logistic regression model was used to analyze the factors affecting postoperative early complications. The COX proportional hazard model was used for univariate and multivariate analyses of factors affecting the 3-year recurrence-free survival rate of patients. Multivariate analysis used stepwise regression to include variables with P<0.05 in univariate analysis and variables clinically closely related to prognosis. Results:(1) Age distribution, comor-bidities and ACCI status of patients. Of the 242 patients, there were 28 cases with age <50 years, 68 cases with age of 50 to 59 years, 113 cases with age of 60 to 69 years, 33 cases with age of 70 to 79 years. There was 1 patient combined with mild liver disease, 1 patient combined with diabetes of end-organ damage, 2 patients combined with peripheral vascular diseases, 2 patients combined with peptic ulcer, 6 patients combined with congestive heart failure, 8 patients combined with chronic pulmonary diseases, 9 patients with diabetes without end-organ damage. The ACCI of 242 patients was 2 (range, 0-4). (2) The grouping of ACCI and comparison of clinicopathological characteristics of patients in each group. Results of X-Tile software analysis showed that ACCI=3 was the best grouping threshold. Of the 242 patients, 194 cases with ACCI <3 were set as the low ACCI group and 48 cases with ACCI ≥3 were set as the high ACCI group, respectively. Age, body mass index, cases with preoperative comorbidities, cases of American Society of Anesthesiologists classification as stage Ⅰ, stage Ⅱ, stage Ⅲ, tumor diameter, cases with tumor histological type as signet ring cell or poorly differentiated adenocarcinoma and cases with tumor type as moderately or well differentiated adenocarcinoma, cases with tumor pathological T staging as stage T1, stage T2, stage T3, stage T4, chemotherapy cycles were (58±9)years, (22.6±2.9)kg/m 2, 31, 106, 85, 3, (4.0±1.9)cm, 104, 90, 16, 29, 72, 77, 6(4,6) in the low ACCI group, versus (70±4) years, (21.7±2.7)kg/m 2, 23, 14, 33, 1, (5.4±3.1)cm, 36, 12, 3, 4, 13, 28, 4(2,5) in the high ACCI group, showing significant differences in the above indicators between the two groups ( t=-14.37, 1.98, χ2=22.64, Z=-3.11, t=-2.91, χ2=7.22, Z=-2.21, -3.61, P<0.05). (3) Incidence of postoperative early complications and analysis of factors affecting postoperative early complications. Of the 242 patients, 33 cases had postoperative early complications, including 20 cases with local complications and 16 cases with systemic complica-tions. Some patients had multiple complications at the same time. Of the 20 patients with local complications, 12 cases had abdominal infection, 7 cases had anastomotic leakage, 2 cases had incision infection, 2 cases had abdominal hemorrhage, 2 cases had anastomotic hemorrhage and 1 case had lymphatic leakage. Of the 16 patients with systemic complications, 11 cases had pulmonary infection, 2 cases had arrhythmias, 2 cases had sepsis, 1 case had liver failure, 1 case had renal failure, 1 case had pulmonary embolism, 1 case had deep vein thrombosis, 1 case had urinary infection and 1 case had urine retention. Of the 33 cases with postoperative early complications, there were 3 cases with grade Ⅰ complications, 22 cases with grade Ⅱ complications, 5 cases with grade Ⅲa complications, 2 cases with grade Ⅲb complications and 1 case with grade Ⅳ complica-tions of Clavien-Dindo classification. Cases with postoperative early complications, cases with local complications, cases with systemic complications were 22, 13, 9 in the low ACCI group, versus 11, 7, 7 in the high ACCI group, respectively. There were significant differences in cases with postoperative early complications and cases with systemic complications between the two groups ( χ2=4.38, 4.66, P<0.05), and there was no significant difference in cases with local complications between the two groups ( χ2=2.20, P>0.05). Results of Logistic regression analysis showed that ACCI was a related factor for postoperative early complications of gastric cancer patients undergoing laparoscopic radical gastrectomy [ odds ratio=2.32, 95% confidence interval ( CI) as 1.04-5.21, P<0.05]. (4) Follow-up. All the 242 patients were followed up for 36(range,1?46)months. During the follow-up, 53 patients died and 13 patients survived with tumor. The 3-year recurrence-free survival rate of the 242 patients was 73.5%. The follow-up time, cases died and cases survived with tumor during follow-up, the 3-year recurrence-free survival rate were 36(range, 2-46)months, 29, 10, 80.0% for the low ACCI group, versus 35(range, 1-42)months, 24, 3, 47.4% for the high ACCI group. There was a significant difference in the 3-year recurrence-free survival rate between the two groups ( χ2=30.49, P<0.05). (5) Analysis of factors affecting the 3-year recurrence-free survival rate of patients. Results of univariate analysis showed that preoperative comorbidities, ACCI, tumor diameter, histological type, vascular invasion, lymphatic invasion, neural invasion, tumor pathological TNM staging, postoperative early complications were related factors for postoperative 3-year recurrence-free survival rate of gastric cancer patients undergoing laparoscopic radical gastrectomy [ hazard ratio ( HR)=2.52, 3.64, 2.62, 0.47, 2.87, 1.90, 1.86, 21.77, 1.97, 95% CI as 1.52-4.17, 2.22-5.95, 1.54-4.46, 0.27-0.80, 1.76-4.70, 1.15-3.12, 1.10-3.14, 3.01-157.52, 1.11-3.50, P<0.05]. Results of multivariate analysis showed that ACCI, tumor pathological TNM staging, adjuvant chemotherapy were indepen-dent influencing factors for postoperative 3-year recurrence-free survival rate of gastric cancer patients undergoing laparoscopic radical gastrectomy ( HR=3.65, 11.00, 40.66, 0.39, 95% CI as 2.21-6.02, 1.40-86.73, 5.41-305.69, 0.22-0.68, P<0.05). Conclusions:ACCI is a related factor for post-operative early complications of gastric cancer patients undergoing laparos-copic radical gastrectomy. ACCI, tumor pathological TNM staging, adjuvant chemotherapy are indepen-dent influencing factors for postoperative 3-year recurrence-free survival rate of gastric cancer patients undergoing laparoscopic radical gastrectomy.

Ubiquitination is a post-translational modification of proteins in eukaryotes, which mediates the specific degradation and signal transduction of proteins to regulate a variety of life processes and thus affects functions of the body. The disorder and imbalance of ubiquitination network is a major cause of serious human diseases. Ubiquitin molecules can form eight homogeneous ubiquitin chains with different topological structures, which vary greatly in abundance and function. At present, the classical ubiquitin chains K48 and K63 with high abundance and rich substrates have been intensively studied, while other atypical ubiquitin chains with low content remain to be studied. However, it has been proved that atypical ubiquitin chains play a key role in intracellular regulation. K6 is an important atypical ubiquitin chain, which is similar to K48 chain and has a tight spatial structure. It plays a role in DNA damage repair, mitochondrial quality control, the occurrence and development of tumor, and the pathogenesis of Parkinson's disease. Due to the lack of specific antibodies and effective enrichment methods for K6, little is known about its substrate and regulatory mechanism. This paper systematically reviews the structural characteristics, regulatory mechanism, biological functions, and relevant diseases of atypical K6 linkages, aiming to provide reference for the functional study of K6.
Humans ; Protein Processing, Post-Translational ; Signal Transduction ; Ubiquitin/chemistry* ; Ubiquitination

Ubiquitination, one type of the most common post-translational modification, mediates the regulation of protein homeostasis in vivo. Since ubiquitin itself contains multiple lysine residues and one N-terminal free amino group, eight types of ubiquitin chains can be formed. The K27 ubiquitin chain is formed through the ubiquitination of the ubiquitin Lys27 (K27), which adopts a compact conformation. In recent years, biological function of the K27 ubiquitin chain in innate immunity, protein homeostasis and DNA damage has been discovered, but the molecular mechanisms of K27 ubiquitin chain assembly, recognition and hydrolysis are still poorly understood. Here we review the structural features and biological functions of K27 ubiquitin chain, to provide a reference for future studies.