[Expression of DARS2 in colorectal cancer and its clinical significance].

Objective: To investigate the expression of DARS2 and its clinical significance in colorectal cancer. Methods: In this study, bioinformatics tools, especially gene expression profile interactive analysis 2 (GEPIA2), were used to conduct an in-depth analysis of DARS2 expression in colorectal cancer tissues. Immunohistochemical staining was carried out in 108 colorectal cancer specimens and 30 normal colorectal tissues obtained from the First Affiliated Hospital of Nanchang University, Nanchang, China. Colorectal cancer cell lines (HCT116 and SW480) were transfected with small interfering RNA (siRNA) and DARS2 overexpression plasmid to examine the effects of DARS2 knockdown and overexpression on cell function. To assess the effects on cell function, CCK8 and transwell migration assays were used to assess proliferation and cell motility, respectively. Additionally, protein immunoblotting was employed to scrutinize the expression of proteins associated with the epithelial-mesenchymal transition of colorectal cancer cells. Results: DARS2 exhibited a pronounced upregulation in expression within colorectal cancer tissues compared to their normal epithelial counterparts. Furthermore, DARS2 expression was higher in colorectal cancer of stage Ⅲ-Ⅳ than those of stage Ⅰ-Ⅱ, exhibiting a significant correlation with N staging, M staging, and pathological staging (P<0.05). Kaplan-Meier analyses showed a decreased overall survival rate in colorectal cancer with DARS2 expression compared to those without DARS2 expression (P<0.05). In the siRNA transfection group, there was a significant reduction in cell proliferation and migration (P<0.01 and P<0.05, respectively). Conversely, the transfection of DARS2 overexpression plasmids substantially increased both cell proliferation and migration (P<0.05). Additionally, immunoblotting revealed that DARS2 knockdown led to an upregulation of E-cadherin expression and a downregulation of N-cadherin and vimentin expression. In contrast, DARS2 overexpression resulted in increased N-cadherin and vimentin expression, coupled with reduction in E-cadherin expression. Conclusions: There is a strong association between DARS2 expression and colorectal cancer progression. Silencing DARS2 inhibits cell proliferation and migration, exerting a discernible influence on the epithelial-mesenchymal transition process.

Pooled Rate of Pseudoprogression, Patterns of Response, and Tumor Burden Analysis in Patients Undergoing Immunotherapy Oncologic Trials for Different Malignancies.

AIMS: Assess rates of true pseudoprogression in unconfirmed progressive disease (iUPD) in a pool of immunotherapy clinical trials for different cancers, analyze tumor characteristics that drive iUPD classification, and investigate potentials predictors of pseudoprogression. MATERIALS AND METHODS: Retrospective interpretation of prospectively acquired data. Patients from 18 immunotherapy clinical trials with two arms (RECIST 1.1, iRECIST), of 10 cancer types were selected. Pooled rate of true pseudoprogression among iUPD was estimated using a common effect meta-analysis. Target, Non-target, and new lesions as the trigger of confirmed-vs pseudo-progression were compared using Chi-Square and Fisher exact tests. Conditional logistic regression was used to investigate the association between age, sex, tumor burden at baseline, and number of follow ups and pseudoprogression. RESULTS: 60/287 (21%) patients (17 women) were classified as iUPD with at least one subsequent confirmatory timepoint. The overall pooled estimate of pseudoprogression was 15% (95%CI: 8%--26%). Nontarget lesions were significantly more frequent the cause of iUPD than change in Target lesions size (p< 0.001). Most observations of true pseudoprogression occurred in the first follow-up (77%), whereas confirmed progression occurred in later time points during the trial. Pseudoprogression was not significantly associated with age, sex, tumor burden at baseline, or number of timepoints. CONCLUSION: In a pool of immunotherapy trials, the rate of true pseudoprogression was 15%, most often in the first timepoint after baseline than later in treatment. iUPD categorization was mostly driven by changes in NT lesions rather than objective changes in measurements of target lesions.

[Analysis of prognostic factors of extranodal NK/T-cell lymphoma treated with pegaspargase/L-asparaginase: a multicenter retrospective study].

Objective: To explore the prognostic factors of extracellular NK/T cell lymphoma (ENKTL) treated with pegaspargase/L-asparaginase. Methods: The clinical data of 656 ENKTL patients diagnosed at 11 medical centers in the Huaihai Lymphoma Working Group from March 2014 to April 2021 were retrospectively analyzed. The patients were randomly divided into two groups: a training set (460 cases) and a validation set (196 cases) at 7∶3, and the prognostic factors of the patients were analyzed. A prognostic scoring system was established, and the predictive performance of different models was compared. Results: Patients' median age was 46 (34, 57) years, with 456 males (69.5% ) and 561 nasal involvement (85.5% ). 203 patients (30.9% ) received a chemotherapy regimen based on L-asparaginase combined with anthracyclines, and the 5-year overall survival rate of patients treated with P-GEMOX regimen (pegaspargase+gemcitabine+oxaliplatin) was better than those treated with SMILE regimen (methotrexate+dexamethasone+cyclophosphamide+L-asparaginase+etoposide) (85.9% vs 63.8% ; P=0.004). The results of multivariate analysis showed that gender, CA stage, the Eastern Cooperative Oncology Group performance status (ECOG PS) score, HGB, and EB virus DNA were independent influencing factors for the prognosis of ENKTL patients (P<0.05). In this study, the predictive performance of the prognostic factors is superior to the international prognostic index, Korean prognostic index, and prognostic index of natural killer lymphoma. Conclusion: Gender, CA stage, ECOG PS score, HGB, and EB virus DNA are prognostic factors for ENKTL patients treated with pegaspargase/L-asparaginase.

[Risk factors of pancreatitis after percutaneous transhepatic biliary drainage in patients with pancreatic cancer and obstructive jaundice].

Objective: To explore the risk factors and preventive strategies of pancreatitis after percutaneous transhepatic biliary drainage (PTBD) in patients with pancreatic cancer and obstructive jaundice. Methods: A total of 241 patients were retrospectively analyzed from May 2001 to October 2014 in Tianjin Medical University Cancer Institute and Hospital. The possibly correlated 9 factors were analyzed, including gender, age, hemoglobin level, total bilirubin level, degree of pancreatic duct dilatation, degree of pancreatic atrophy, degree of biliary stenosis, the pancreatic duct visualization, and drainage mode. Results: Univariate analysis suggested that pancreatic duct dilatation, pancreatic atrophy, visualized pancreatic duct and drainage mode were associated with the incidence of pancreatitis after PTBD (P<0.05). Logistic regression analysis showed that visualization of pancreatic duct (OR=6.33) was a risk factor for pancreatitis, while pancreatic duct dilatation (OR=0.14), pancreatic atrophy (OR=0.12) and external drainage (OR=0.11) were protective factors for pancreatitis. Conclusion: In pateints with pancreatic cancer and obstructive jaundice, pancreatic duct dilatation and pancreatic atrophy predict low risk of pancreatitis after PTBD,while intraoperative pancreatic duct visualization and internal or external drainage may increase the incidence of postoperative pancreatitis.

[Clinical prognostic analysis of 124 adult patients with hemophagocytic lymphohistiocytosis: a multicenter retrospective study of the Huaihai Lymphoma Working Group].

Objective: Factors influencing the prognosis of hemophagocytic lymphohistiocytosis (HLH) in adults were analyzed based on multicentric data. Methods: Clinical data of 124 adult patients with HLH diagnosed in eight medical centers in the Huaihai Lymphoma Working Group from March 2014 to July 2020 were collected. The optimal truncation value of continuous variables was obtained based on the Maxstat algorithm, X-Tile software, and restricted cubic spline. Cox proportional risk regression model was used to construct the adult HLH risk prediction model, and the visualization of the model was realized through the histogram. The bootstrap resampling method was used to verify the model, C-index and calibration curve was used to verify the histogram, and the prediction accuracy was checked. Kaplan-Meier analysis was used to calculate the survival rate and draw the survival curve. Furthermore, the differences between groups were tested by log-rank. Results: The median age of the 124 patients was 55 (18-84) years, including 61 (49.19%) males. The most common etiology was infection. Serum ferritin increased in 110 cases (88.71%) , hepatosplenomegaly in 57 cases (45.97%) . Of the 124 patients, 77 (62.10%) died, and the median survival time of the patients was 7.07 months. Univariate results showed that the prognosis of adult HLH was influenced by sex, age, fibrinogen, serum creatinine, alanine aminotransferase, and albumin (P<0.05) . The results of multivariate analysis showed that gender, platelet, albumin, alanine aminotransferase, and treatment regimens were independent influencing factors for prognosis. Based on the above five risk factors, the prediction model of the histogram was established, and the C-index of the model was 0.739. Finally, the calibration chart showed good consistency between the observed and predicted values of HLH. Conclusion: The prognosis of the adult hemophagocytic syndrome is influenced by many factors. Gender, platelet, albumin, alanine aminotransferase, and treatment regimens are independent risk factors. Therefore, the established histogram provides a visual tool for clinicians to evaluate the prognosis of adult HLH.

NAT2 knockdown inhibits the development of colorectal cancer and its clinical significance.

OBJECTIVE: This study aims to explore the correlation between N-acetyltransferase 2 (NAT2) expression in colorectal cancer (CRC) tissues and the progression and prognosis of CRC. Through in vitro and in vivo experiments, the biological functions of NAT2 in the occurrence and development of CRC were explored. PATIENTS AND METHODS: Immunohistochemical (IHC) staining, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot were used to detect the difference of NAT2 expression in CRC tissues and normal tissues. The role of NAT2 in the cell proliferation, apoptosis, migration, invasion, and tumorigenesis and development of CRC was analyzed by cell counting kit-8 (CCK-8), colony formation, flow cytometry, transwell cell invasion, wound-healing assays and construction of nude mouse xenograft model. The correlation between the expression level of NAT2, and the overall survival and clinicopathological characteristics of CRC patients were statistically analyzed to preliminarily determine the clinical significance of NAT2 in the diagnosis and prognosis of CRC. RESULTS: The expression level of NAT2 was notably upregulated in CRC. NAT2 knockdown inhibited the proliferation, migration, invasion and in vivo tumor formation of CRC cells, and promoted cell apoptosis. High NAT2 expression was associated with TNM stage, differentiation degree, tumor size, distant metastasis, lymph node metastasis and poor prognosis in CRC patients. CONCLUSIONS: This study showed that the expression level of NAT2 in CRC tissues was increased and closely related to the metastasis and prognosis of CRC. In addition, NAT2 can be used as a new prognostic biomarker and therapeutic target for CRC.

Study of the predictors for radiation pneumonitis in patient with non-small cell lung cancer received radiotherapy after pneumonectomy.

PURPOSE: To identify the valuable predictors of grade≥2 radiation pneumonitis (RP) in patient treated with radiotherapy after pneumonectomy for non-small cell lung cancer (NSCLC); and to construct a nomogram predicting the incidence of grade≥2 RP in such patients. PATIENTS AND METHODS: We reviewed 82 patients with NSCLC received radiotherapy after pneumonectomy from 2008 to 2018. The endpoint was grade≥2 RP. Univariate and multivariate regression analysis were conducted to evaluate significant factors of grade≥2 RP. Receiver operating characteristic (ROC) curve was used to establish optimal cutoff values and the nomogram was built to make the predictive model visualized. Descriptive analysis was performed on 5 patients with grade 3 RP. RESULTS: A total of 22(26.8%) patients developed grade 2 RP and 5(6.1%) patients were grade 3 RP. V5, V10, V20, V30, MLD, PTV, and PTV/TLV were associated with the occurrence of grade≥2 RP in univariate analysis, while none of the clinical factors was significant; V5(OR,1.213;95%CI,1.099-1.339; P<0.001) and V20(OR,1.435;95%CI,1.166-1.765; P=0.001) were the independent significant predictors by multivariate analysis and were included in the nomogram. The ROC analysis for the cutoff values for predicting grade≥2 RP were V5>23% (AUC=0.819, sensitivity:0.701, specificity:0.832) and V20>8% (AUC=0.812, sensitivity:0.683, specificity:0.811). Additionally, grade≥3 RP did not occur when V5<30%, V20<13% and MLD<751.2cGy, respectively. CONCLUSIONS: Our study showed that V5 and V20 were independent predictors for grade≥2 RP in NSCLC patients receiving radiotherapy after pneumonectomy. Grade 3 RP did not occur whenV5<30%, V20<13% and MLD<751.2cGy, respectively. In addition, patient underwent right pneumonectomy may have a lower tolerance to radiation compared to left pneumonectomy.

[Closed percutaneous reduction and minimally invasive treatment of spastic hammery deformity].

Objective: To investigate the efficacy of percutaneous extensor tendon reconstruction in treating spastic hammery deformity. Methods: From February 2009 to July 2018, the clinicaldata of 36 patients with fresh sputum hammer fingers treated in Jinan People's Hospital were analyzed retrospectively. The tendon was percutaneously sutured with PDS Ⅱmonofilament suture and the distal end of the tendon was fixed to the base of the distal phalanx through the bone hole. Removal of the K-wire 6 weeks after the operation, the brace was used to fix the affected finger in the dorsal extension. For 8-10 weeks, only the brace was worn at night and the flexion and extension of the affected finger was gradually strengthened.The extension and flexion function of the interphalangeal joint of the finger was compared pre and post the operation with t test. Results: A total of 36 patients were enrolled but only 33 patientswere followed up for 6 to 15 months. The hammer-shaped deformity was corrected and there was no pain when moving fingers after the operation.The straightening angle of the interphalangeal joint of the finger improved from 46.2°±6.3° before surgery to 7.5°±0.6° after (t=35.12, P<0.05). The passive straightening angle decreased from 3.2°±0.3° before surgery to 0.9°±0.2° after (t=37.11, P<0.05). According to the Crawford functional assessment: excellent in 19 fingers, good in 10 fingers, can be in 4 fingers. The excellent rate was 87.9%. There was no knot exposure, skin necrosis and other complications. Conclusions: Percutaneous resection of the extensor tendon is fixed in the basal phalanx. It is a simple and feasible minimally invasive surgery for hammer-shaped deformity. It can obviously correct the hammer-shaped deformity and has fewer complications.

[Phenotype and genotype analysis of 55 children patients with Wilson's disease].

Objective: To understand the clinical phenotype and spectrum of ATP7B gene mutation in children with Wilson's disease (WD). Methods: A total of 55 cases diagnosed with WD at the Children's Hospital Affiliated to Nanjing Medical University from June 2012 to June 2018 were taken as the research subject. ATP7B gene point mutation was detected by direct sequencing after PCR amplification. Heterozygous mutation in children was discovered by sequencing. Furthermore, the long segment mutation of exon was analyzed by multiplex ligation-dependent probe amplification (MLPA). Results: All 55 WD children had varying degree of liver damage symptoms. Among them, 2 cases had combined neurological symptoms. The positive rates of K-F ring (21%), 24-hour urine copper (97.7%), and ceruloplasmin were all abnormal. The results of ATP7B gene had identified 8 homozygous, 41 compound heterozygous and 6 heterozygous in 55 cases. Direct sequencing method had detected ten cases of ATP7B heterozygotes. In addition, MLPA analysis showed that other allele in four cases had a deletion of the ATP7B gene exon. In all cases, 35 different ATP7B gene mutations were detected, including 23 missense mutations, 3 frameshift mutations, 4 nonsense mutations, 3 exon deletions and 2 splicing changes. The most common allele mutation was c.2333G > T/p.R778L in exon 8, with an allele frequency of 36.54%, followed by c.2975C > T/p.P992L in exon 13, with an allele frequency of 14.42%. Conclusion: ATP7B gene c.2333G > T/p.R778L and c.2975C > T/p.P992L mutations are the most common mutations in children with WD in China. WD patients report shows that there are three long deletion mutations in the exon of the ATP7B gene. For WD children whose DNA sequencing is heterozygous ATP7B gene, it is suggested to further use MLPA method to detect deletion mutations of exons.

[Pathological characteristics and molecular diagnosis of non-tuberculosis Mycobacterium lung disease].

Objective: To investigate the clinicopathological features of non-tuberculosis mycobacterial lung disease and the role of molecular pathology in diagnosis. Methods: Forty-five formalin-fixed, paraffin embedded (FFPE) specimens were collected from the Department of Pathology, Beijing Chest Hospital from February 2016 to August 2019. The clinical, imaging and histopathologic features, bacteriologic data and morphologic characteristics of acid fast bacilli (AFB) were analyzed retrospectively. Specific gene sequence IS6110 of Mycobacterium tuberculosis (MTB) was detected by fluorescence PCR. Identification of Mycobacteria was by melting curve method. Fifty cases of pulmonary tuberculosis were selected in the same period as control. Results: The NTM lung cases included 18 cases (40.0%, 18/45) of M. intracellulare, eight cases (17.8%, 8/45) of M. xenopi, six cases (13.3%, 6/45) of M. avium, six cases (13.3%, 6/45) of M. kansasii, six cases (13.3%, 6/45) of M. chelonae and one case (2.2%, 1/45) of M. simiae. Histopathologically, there were necrotizing granulomas in 34 cases (75.6%, 34/45), non-necrotizing granuloma in one case (2.2%, 1/45) and non-granulomatous lesions in 10 cases (22.2%, 10/45). The necrosis was pink necrosis, basophilic necrosis rich in nuclear fragments and suppurative necrosis. Pulmonary TB showed more pink necrosis and basophilic necrosis, the difference was statistically significant (χ(2)=10.270, P=0.001; χ(2)=7.449, P=0.006). Seventeen cases (37.8%, 17/45) of NTM lung disease showed giant multinucleated giant cells, which were significantly different from those in pulmonary tuberculosis group (χ(2)=13.446, P<0.01). The number and morphology of AFB were also different. More AFB were found in M. intracellular cases and significant AFB were easily seen in M. kansasii infection. Conclusions: M. tuberculosis and NTM cannot be reliably differentiated by histologic features or by AFB morphology. Molecular assays are important to distinguish tuberculosis from NTM lung disease.

[Application of multidisciplinary team (MDT) in the treatment of severe trauma].

OBJECTIVE: To explore the effect of multi-disciplinary team (MDT) in general hospitals on severe trauma patients. METHODS: This study reviewed the treatment of patients with severe trauma in trauma center of Peking University People's Hospital from March 2017 to April 2019. The baseline information: the patients' gender, age, injury mechanism, etc.; the start indicators: the Glasgow coma scale (GCS), trauma index (TI), injury severity score (ISS); the start related indicators: time for activation, time for MDT to arrive, time for CT scan, time for damage control surgery; patient treatment and prognosis: ICU (intensive care unit) length of stay, number of cured and discharged patients, number of dead cases, number of patients transferred to rehabilitation hospital, were all analyzed. It discussed the composition of MDT, the initiation scheme, the indicators of initiation of MDT for severe trauma, and analyzed the correlation between the application of MDT and the prognosis of patients. RESULTS: From March 2017 to April 2019, 112 trauma patients were treated by MDT in Peking University People's Hospital. There were 69 males and 43 females. The minimum age was 15 years, the maximum age was 89 years, most of them were 36-55 years old. The main injury mechanism was traffic accident injury. The GCS, TI, ISS were 13.0±2.9, 13.0±2.8, and 21.5±11.9, respectively. It took 3.7±0.8 minutes to start the call, 6.1±0.9 minutes for MDT personnel to arrive at the emergency rescue area, 23.8±3.0 minutes for fast CT and 92.6±15.4 minutes for injury control operation. All the hospitalized patients were treated effectively. ICU (Intensive care unit) hospitalization time was 12.6±6.7 days. 55 discharged patients were cured, 5 died (1 died of hemorrhagic shock, 4 died of severe brain injury) and 52 transferred to rehabilitation hospital. CONCLUSION: The treatment of severe trauma patients by MDT in trauma center of general hospitals can greatly improve the ability and level of treatment of severe trauma patients, make up for the lack of treatment of severe trauma especially multiple trauma patients in large general hospitals, and improve the treatment effect of severe trauma patients. It provides a reference model for large general hospitals to treat patients with severe trauma and multiple trauma and for the construction of trauma centers.

[Phylogenetic analysis of Echinococcus granulosus genotypes based on the GenBank database].

OBJECTIVE: To analyze the sequences of the cytochrome C oxidase subunit I (Cox1) gene of various Echinococcus granulosus genotypes that are currently recorded in the GenBank database, so as to investigate the genetic variation and differentiation of the E. granulosus genotypes across the world. METHODS: The sequences of the Cox1 gene of various E. granulosus genotypes that are currently recorded in the GenBank database were collected, and the same sequences of the Cox1 gene identified from a region were excluded. The mutation sites among the Cox1 gene sequences were identified and a phylogenetic tree was created based on the Cox1 gene. RESULTS: Transversion mutation was the predominant type of mutation in the Cox1 gene of E. granulosus. The same Cox1 gene sequence was found in E. granulosus G1, G6 and G7 genotypes isolated from various geographical locations across the world, with the corresponding GenBank accession numbers of KY766891, MH300971 and MH301007, respectively. Phylogenetic analysis revealed that E. granulosus G10 genotype had a remarkable geographical aggregation. CONCLUSIONS: E. granulosus G1, G6 and G7 genotypes have primitive Cox1 gene sequences. There is a geographical aggregation of the E. granulosus G10 genotype in the phylogenetic tree, which has a tendency towards reproductive isolation.

Long noncoding RNA SNHG14 enhances migration and invasion of ovarian cancer by upregulating DGCR8.

OBJECTIVE: Ovarian cancer is the most common fatal gynecologic malignancy in females all over the world. Recently, long noncoding RNAs (lncRNAs) have been reported to exert pivotal functions in tumorigenesis. In this research, lncRNA SNHG14 was studied to identify its role in the metastasis of ovarian cancer. PATIENTS AND METHODS: SNHG14 expression was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) in ovarian cancer specimens. Functional assays including wound healing assay, transwell assay, and Matrigel assay were performed to detect the effect of SNHG14 on the migration and invasion of ovarian cancer cells. In addition, the underlying mechanism was further explored through qRT-PCR and Western blot assay. RESULTS: SNHG14 level was dramatically higher in ovarian cancer specimens. Moreover, cell migration and invasion were significantly attenuated via the inhibition of SNHG14, while enhanced via the SNHG14 overexpression. Besides, the expression of DGCR8 mRNA and protein was markedly downregulated after the knockdown of SNHG14, while upregulated after SNHG14 overexpression. Furthermore, the expression level of DGCR8 was increased in cancer tissues and positively related to the expression of SNHG14 in ovarian cancer tissues. CONCLUSIONS: In summary, SNHG14 could enhance cell migration and invasion via upregulating DGCR8 in ovarian cancer.

[Expression of FATS in non-small cell lung cancer and its relationship with prognosis].

Objective: To investigate the expression of fragile-site associated tumor suppressor (FATS) in non-small cell lung cancer and its relationship with clinicopathological features and prognosis. Methods: A total of 140 non-small cell lung cancer (NSCLC) cases and 30 adjacent normal tissues were used to detect the expression level of FATS protein, and to analyze the relationship of FATS protein expression and clinicopathological features and prognosis of NSCLC. Results: Western blot showed that the expression of FATS in adjacent normal tissues was significantly higher than that in non-small cell lung cancer tissues. The results of immunohistochemistry showed that the high expression rate of FATS in 140 cases of NSCLC was 40.0%, and the high expression rate of FATS in 30 cases of adjacent tissues was 73.3%. The difference was statistically significant (P=0.01). Further analysis showed that the TNM stage (P=0.044) and lymph node metastasis (P=0.022) were significant difference between FATS high expression group and low expression group. The 6-year overall survival (OS) rates of NSCLC patients with FATS high-expression and low-expression were 57.1% and 23.8%, respectively, and the 6-year disease-free survival (DFS) rates were 53.6% and 21.4%, respectively, with statistically significant differences (P=0.001). In Cox multivariate analysis, we found gender (HR=1.658, P=0.028; HR=1.684, P=0.023), TNM staging (HR=2.327, P=0.019; HR=2.332, P=0.013) and FATS expression (HR=0.532, P=0.010; HR=0.538, P=0.009) were independent prognostic factors for both OS and DFS of NSCLC patients. Conclusions: The expression of FATS protein is associated with the development and is an independent prognostic factor of NSCLC patients. The detection of FATS protein is expected to be a new biomarker for evaluating the prognosis of NSCLC patients.

Expression of CD63 in Lung Tissue of Guinea Pigs Dying of Anaphylactic Shock.

ABSTRACT: Objective To study the protein expression of cluster of differentiation 63 （CD63） in lung tissues of guinea pigs that died of anaphylactic shock and discuss the diagnostic value of CD63 for death from anaphylactic shock. Methods Twenty guinea pigs were randomly divided into control group, anaphylactic shock immediate death group, cold storage group （4 ℃ for 48 h） and frozen group （-20 ℃ for 7 d）. The animal model of guinea pigs that died of anaphylactic shock was established with human mixed serum injection. The expression changes of CD63 protein and CD63 mRNA in lung tissues were detected by hematoxylin-eosin （HE） staining, immunohistochemical staining, Western blotting, enzyme-linked immunosorbent assay （ELISA） and real-time RT-PCR. Results HE staining results showed congestion, and edema of lung tissues, and eosinophil infiltration in the anaphylactic shock groups. Western blotting analysis results showed that the expression of CD63 protein in the lung tissues of guinea pigs that died of anaphylactic shock was significantly higher than that in the control group （P<0.05）. Comparison between the anaphylactic shock groups was made, and the differences had no statistical significance. The results of immunohistochemical staining and real-time RT-PCR were consistent with that of Western blotting. ELISA results showed that CD63 protein expression in the immediate death group was higher than that in the control group （P<0.05）. Conclusion The expression of CD63 protein and CD63 mRNA in the lung tissues of guinea pigs that died of anaphylactic shock is significantly enhanced. Animal carcasses which were put in cold storage for 48 h and frozen for 7 d do not affect the examination of the above indicators. CD63 protein is expected to become an auxiliary diagnostic indicator of death from anaphylactic shock.

[Double Labeling and Simultaneous Monitoring for Hsp70 and Hsf-1 Kinetics in SCC-25 Cells with a Short-Term Dietary Restriction of Leucine Following Heat Shock].

To develop a quantum-dot-based multiplexed imaging system for the simultaneous monitoring of Hsf- 1/Hsp70 after heat shock, and to evaluate the effects of combined thermotherapy and leucine deprivation therapy on Hsf-1 inactivation. SCC-25 cells were leucine starved for 0, 1, 2, 3 or 4 days following which the cells underwent heat shock at 42°C for 30 min. At 6 h after heat shock, Hsf-1 activation and translocation to the nucleus was observed in cells that were leucine starved for 0, 1 and 2 days, and the synthesis of Hsp70 and Hsf-1 reached their maximum values and had a tendency to gather in the nucleus. However, in cells that were leucine starved for 3 and 4 days, Hsf-1 activity and Hsp70 synthesis level was dramatically decreased. Dietary restriction of leucine for at least three days could result in the inactivation of Hsf-1, leading to a reduction in Hsp70 synthesis. The combination of thermotherapy and short-term leucine deprivation therapy may become effective approach for the treatment of oral tumors.

A novel polysaccharide obtained from Craterellus cornucopioides enhances immunomodulatory activity in immunosuppressive mice models via regulation of the TLR4-NF-κB pathway.

The immunoregulatory effect of a novel Craterellus cornucopioides polysaccharide (CCP) with a triple-helix structure on immunosuppressive BALB/c mice models was investigated; moreover, the immune response of BALB/c mice models in the preventive and therapeutic treatment groups treated with CCP was explored, and its molecular mechanism was elucidated. It was found that the BALB/c mice models in the preventive groups treated with CCP (120 and 240 mg kg-1 d-1) had better immunoregulatory activity. The spleen and thymus weight indices of the BALB/c mice models were significantly increased, and the histopathological analysis indicated a protective function of CCP against the immunosuppression induced by cyclophosphamide (CTX). Moreover, CCP displayed definite and clear synergistic effects on the T- or B-lymphocyte proliferation induced by ConA or LPS, respectively, promoted the natural killer (NK) cell activity and significantly increased phagocytic activity to activate peritoneal macrophages in immunosuppressive mice. The western blot and quantitative real-time polymerase chain reaction (qRT-PCR) results provided comprehensive evidence that CCP could upregulate the protein expression of the G-protein-coupled cell membrane receptor TLR4 and the production of its downstream protein kinases (TRAF6, TK1, p-IKKα/ß and NF-κB p50); this, in turn, enhanced the production of cytokines (IL-2, IL-6, TNF-α and IFN-α) through both preventive and therapeutic treatments via regulation of the TLR4-NFκB pathway in the peritoneal macrophage of immunosuppressive mice.

Biological background of the genomic variations of cf-DNA in healthy individuals.

BACKGROUND: Cell-free DNA (cf-DNA)-based liquid biopsy is emerging as a revolutionary new method in individualized cancer treatment and prognosis monitoring, although detecting early-stage cancers using cf-DNA remains challenging, partially because of the undefined biological background of cf-DNA. MATERIALS AND METHODS: We investigated somatic mutations in the cf-DNA of 259 cancer-free individuals with a median age of 47 years using an endogenous barcoding duplex method with an ultralow base error rate (2 × 10-7) and compared the variant allele frequencies (VAFs) of these mutations between the cf-DNA and the corresponding blood cell DNA. RESULTS: Sixty percent (155/259) of the samples showed at least one nonsynonymous mutation on either of two similar target panels covering 508 and 559 cancer-related genes. For individuals older than 50 years of age, the positive rate increased to 76%. Most cf-DNA mutations were also present at similar VAFs in the paired blood cell DNA. The most frequently mutated genes were driver genes of hematologic malignancies, including DNMT3A, TET2, AXSL1, and JAK2. However, the other 58.4% (192/329) of the mutations were likely 'passenger mutations' of clonal hematopoiesis, including mutations in NOTCH2, FAT3, EXT2, ERBB4, and ARID2, which are driver genes of solid tumors. CONCLUSION: Hematopoietic clone-derived mutations, including 'driver mutations' and 'passenger mutations', are prevalent in the cf-DNA of both healthy individuals and cancer patients and may be a potential source of false positives in the liquid biopsy. Our results also suggest the ineffectiveness for distinguishing clonal hematopoietic mutations of low VAF (≤0.1%) from tumor-derived mutations using conventional next-generation sequencing of blood cell DNA. However, an error correction model with an ultralow error rate and high coverage depth is required for blood cell DNA sequencing, which is difficult and costly to achieve with current technologies.

Structure characterization, physicochemical property and immunomodulatory activity on RAW264.7 cells of a novel triple-helix polysaccharide from Craterellus cornucopioides.

In the study, a new triple-helix polysaccharide with favorable stability was purified from C. cornucopioides. Its structural characterization, stability and solution behavior were investigated by the GC-MS, periodate oxidation-smith degradation, FT-IR, 1D and 2D NMR spectroscopy, methylation analysis, Scanning electron microscope, Congo-red, CD, TGA and DSC analysis. The results showed that Craterellus cornucopioide polysaccharide (CCP) possessed the molecular weight of 1.97 × 103 kDa, is mainly composed of mannose (48.73%), galactose (17.37%), glucose (15.97%) and xylose (17.93%), respectively. It was a heteroglycan with (1 → 3)­linked­ß­d­Manp­(1 → 6)­linked α­d­Galp backbone distributed by (1 → 4)­linked­α­d­Xylp­t­α­d­Manp and t­ß­d­Glup units at O-6. The result of TGA and DSC assay indicated that CCP has a favorable thermal stability. MTT and Scanning electro microscopy (SEM) assay showed that CCP could significantly improve the proliferation activity and induce cells activation of RAW264.7 in a certain range of concentrations and period.