Purpose: Our previous study showed that circulating tumor cell (CTC) count combined with gene mutation detection might help differentiate benign and malignant pulmonary nodules (PNs). Herein, we aimed to expand the study cohort and conduct further sequencing analysis. Materials and Methods: Patients with PNs were included, and CTCs were identified before operation. Low-coverage whole-genome sequencing (LC-WGS) and lung cancer-related targeted gene sequencing were performed on CTCs. The diagnostic efficacy was evaluated by receiver operating characteristic (ROC) curve. The differences in CTC counts among subgroups classified by demographic–clinical characteristics were analyzed. LC-WGS–based copy number variation (CNV) analysis and targeted gene mutation analysis were conducted. Results: A total of 172 patients were included. CTC count of 2.5 was identified by the ROC curves as the optimal diagnostic cutoff. The sensitivity and specificity of CTC count for differentiating benign and malignant PNs were 54.2% and 78.6%, respectively. The diagnostic sensitivity and specificity of combined CTC count, radiological nodule type, and any malignant imaging features were 84.7% and 71.4%, respectively. The CTC counts were significantly greater in patients with aggressive tumors, later stage, and spread through air spaces. CTCs from malignant cases had more CNVs than those from benign cases. Conclusion CTC count can be used in identifying malignant PNs. The diagnostic efficacy can be improved if combined with computed tomography imaging characteristics. Further CNV analysis might help differential diagnosis. Greater CTC count might suggest more aggressive tumors. CTC detection can provide important information and guidance for subsequent management of PNs.

BACKGROUND: Low-density computed tomography (LDCT) improved early lung cancer diagnosis but introduces an excess of false-positive pulmonary nodules data. Hence, accurate diagnosis of early-stage lung cancer remains challenging. The purpose of the study was to assess the feasibility of using circulating tumour cells (CTCs) to differentiate malignant from benign pulmonary nodules. METHODS: 122 patients with suspected malignant pulmonary nodules detected on chest CT in preparation for surgery were prospectively recruited. Peripheral blood samples were collected before surgery, and CTCs were identified upon isolation by size of epithelial tumour cells and morphological analysis. Laser capture microdissection, MALBAC amplification, and whole-exome sequencing were performed on 8 samples. The diagnostic efficacy of CTCs counting, and the genomic variation profile of benign and malignant CTCs samples were analysed. RESULTS: Using 2.5 cells/5 mL as the cut-off value, the area under the receiver operating characteristic curve was of 0.651 (95% confidence interval: 0.538-0.764), with a sensitivity and specificity of 0.526 and 0.800, respectively, and positive and negative predictive values of 91.1% and 30.3%, respectively. Distinct sequence variations differences in DNA damage repair-related and driver genes were observed in benign and malignant samples. TP53 mutations were identified in CTCs of four malignant cases; in particular, g.7578115T>C, g.7578645C>T, and g.7579472G>C were exclusively detected in all four malignant samples. CONCLUSIONS CTCs play an ancillary role in the diagnosis of pulmonary nodules. TP53 mutations in CTCs might be used to identify benign and malignant pulmonary nodules.
Humans ; Lung Neoplasms ; Exome Sequencing ; Multiple Pulmonary Nodules ; Carcinoma ; DNA Repair

TSCI have dyskinesia and sensory disturbance that can cause various life-threaten complications. The patients with traumatic spinal cord injury (TSCI), seriously affecting the quality of life of patients. Based on the epidemiology of TSCI and domestic and foreign literatures as well as expert investigations, this expert consensus reviews the definition, injury classification, rehabilitation assessment, rehabilitation strategies and rehabilitation measures of TSCI so as to provide early standardized rehabilitation treatment methods for TSCI.

Objective To investigate the correlation between decoy receptor (DcR) 1,DcR2,osteoprotegerin (OPG) gene polymorphisms and susceptibility of Crohn's disease (CD) in Han population in Zhejiang province.Methods From April 2008 to July 2017,at the Department of Gastroenterology of The Second Affiliated Hospital of Wenzhou Medical University,The First Affiliated Hospital of Wenzhou Medical University,Central Hospital of Wenzhou and Renmin Hospital of Wenzhou,285 patients diagnosed as having CD were enrolled,and during the same period 572 healthy individuals who received health checkup at the Second Affiliated Hospital of Wenzhou Medical University were collected as healthy control.The single nucleotide polymorphism (SNP) of DcR1 (rs12549481),DcR2 (rs1133782) and OPG (rs3102735) were examined by SNaPshot technique.An unconditional logistic regression analysis was performed to analyze the differences in each SNP mutation alleles and genotype frequencies between CD group and control group.Furthermore,their correlation with clinicopathological features of CD and the efficacy of corticosteroid and infliximab was also evaluated.Results The frequencies of mutant allele A and genotype GA + AA of DcR2 (rs1133782) of CD group were 11.93％ (68/570) and 22.81％ (65/285),respectively,which were higher than those of healthy control group (8.22％,94/1 144;and 15.91％,91/572;odds ratio (OR) =1.513,95％ confidence interval (CI) 1.088 to 2.104,P =0.013;OR =1.562,95％ CI 1.094 to 2.230,P =0.014).However there was no statistically significant difference in the mutant allele and genotype frequencies of DcR1 (rs12549481) and OPG (rs3102735) between two groups (all P ＞ 0.05).The frequencies of mutant allele C and genotype TC + CC of DcR1 (rs12549481) in patients with stricturing CD were 13.89％ (25/180)and 27.78％ (25/90),respectively,which were lower than those of patients with non-stricturing,non-penetrating CD (27.68％,62/224 and 48.21％,54/112),and the differences were statistically significant (OR =0.421,95％ CI 0.252 to 0.705,P =0.001;OR =0.413,95％ CI 0.229 to 0.747,P =0.003).Besides,the frequencies of mutant allele A and genotypes GA + AA of DcR2 in patients with penetrating CD were 7.23％ (12/166) and 13.25％ (11/83),which were lower than those of patients with non-stricturing,non-penetrating CD (15.62％,35/224 and 30.36％,34/112),and the differences were statistically significant (OR =0.407,95％ CI 0.205 to 0.809,P =0.009;OR =0.350,95％ CI 0.165 to 0.743,P =0.005).In addition,there was no statistically significant difference in the frequencies of mutant allele and genotypes of OPG (rs3102735) among subtypes of CD with different features (all P ＞ 0.012 5).Moreover,the DcR1 (rs12549481),DcR2 (rs1133782) and OPG (rs3102735) polymorphisms were not correlated with the efficacy of corticosteroid and infliximab (all P ＞ 0.05).Conclusions DcR1 (rs12549481) mutation may be correlated with stricturing CD.DcR2 (rs1133782) mutation may be correlated with CD,especially with penetrating CD.However,the gene polymorphism of OPG (rs3102735) is not correlated with the risk of CD susceptibility.And the above gene SNP may be independent of the efficacy of corticosteroid and infliximab.

Objective To investigate the clinical application of self-made snare in laparoscopic appendectomy. Methods The clinical datas of 197 cases who were acute appendicitis under laparoscopic appendectomy in the first people's hospital of Fuyang in Hangzhou from June 2015 to June 2017 were collected, then they were divided into self-made snare group (85) and Hem-o-lok group (112). Variations of operation time, operation bleeding, first postoperative anal exhaust time and length of stay above two groups were analyzed. Variations of incision infection, abdominal abscess and intestinal obstructionabove two groups were analyzed. Results Two groups of patients were compared with operation time [self-made snare group(43. 71±5. 54)min: Hem-o-lok group(42. 29±5. 34)min], operation bleeding[self-made snare group(l 1. 60±2. 44)mL: Hem-o-lok group(l 1. 00±2. 56)mL],first postoperative anal exhaust time [self-made snare group (10. 01±2. 27)h: Hem-o-lok group(9. 60±2. 32)h] and length of stay[self-made snare group(5. 19±0. 95)d: Hem-o-lok group (4. 99±0. 85)d], the differences were not statistically significant(P＞0. 05). Two groups of patients were compared with incision infection[self-made snare group 2 cases(2. 35％): Hem-o-lok group 3 cases(2. 68％)], abdominal abscess [self-made snare group 1 case(1. 18％): Hem-o-lok group 2 cases (1. 79％)] and intestinal obstructionabove [self-made snare group 3 cases(3. 53％): Hem-o-lok group 5 cases(4. 46％)], the differences were not statistically significant(P＞0. 05). Conclusion Self-made snare in laparoscopic appendectomy is safe and feasible.

OBJECTIVETo explore the central mechanism of postoperative fatigue syndrome by detecting the expression of NMDA receptor and tryptophan metabolism.

METHODSAfter being numbered according to the weight, ninety-six male SD rats were randomly divided into control group (bowel loop was flipped after laparotomy and received intraperitoneal injection of saline at a dose of 1 ml/kg), POFS model(70% of the length of small intestine was resected and received intraperitoneal injection of saline at a dose of 1 ml/kg), and NMDA antagonist groups(70% of the length of small intestine was resected and received intraperitoneal injection of MK801 at a dose of 1 ml/kg). Each group was divided into subgroups by postoperative 1, 3, 5 and 7 d, with 8 rats in each subgroup. The hippocampus was removed at each time point after open field test (OFT) to detect the mRNA expression levels of NMDA receptor 1 and kynurenine aminotransferase III((KATIII() by real-time PCR. Protein level of NMDA receptor 1 was detected by Western blot. High performance liquid chromatography (HPLC) was used to measure the concentrations of tryptophan (TRP), kynurenine (KYN) and kynurenic acid(KYNA). Ultra-structural changes of hippocampal neurons were observed by transmission electron microscopy(TEM).

RESULTSAs compared to control group, exercise score decreased(P<0.05), rest time and central panel residence time prolonged, periphery/central panel ratio increased (all P<0.05), mRNA and protein expressions of NMDA receptor 1 increased (P<0.05), mRNA expression of KAT III( decreased (P<0.05), KYN/TRP ratio and KYN/KYNA ratio decreased (all P<0.05) in POFS group on postoperative day 1 and 3. As compared to POFS group, central panel residence time and periphery/central panel ratio decreased on postoperative day 1, and mRNA and protein expressions of NMDA receptor 1 decreased on postoperative day 1 and 3 (all P<0.05) in antagonist group. TEM revealed that degenerated neuron was found in the hippocampus of POFS rats, while such damage was improved in antagonist group.

CONCLUSIONThe increased expression level of NMDA receptor may play an important role in POFS. NMDA receptor antagonist MK801 may improve the POFS.

Animals ; Fatigue ; Hippocampus ; Humans ; Injections, Intraperitoneal ; Male ; Postoperative Period ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate ; Signal Transduction ; Transaminases

Objective To explore the central mechanism of postoperative fatigue syndrome by detecting the expression of NMDA receptor and tryptophan metabolism. Methods After being numbered according to the weight, ninety-six male SD rats were randomly divided into control group (bowel loop was flipped after laparotomy and received intraperitoneal injection of saline at a dose of 1 ml/kg ), POFS model (70% of the length of small intestine was resected and received intraperitoneal injection of saline at a dose of 1 ml/kg), and NMDA antagonist groups (70% of the length of small intestine was resected and received intraperitoneal injection of MK801 at a dose of 1 ml/kg ). Each group was divided into subgroups by postoperative 1, 3, 5 and 7 d, with 8 rats in each subgroup. The hippocampus was removed at each time point after open field test (OFT) to detect the mRNA expression levels of NMDA receptor 1 and kynurenine aminotransferase Ⅲ (KATⅢ) by real-time PCR. Protein level of NMDA receptor 1 was detected by Western blot. High performance liquid chromatography (HPLC) was used to measure the concentrations of tryptophan (TRP), kynurenine (KYN) and kynurenic acid(KYNA). Ultra-structural changes of hippocampal neurons were observed by transmission electron microscopy (TEM). Results As compared to control group, exercise score decreased (P<0.05), rest time and central panel residence time prolonged, periphery/central panel ratio increased (all P<0.05), mRNA and protein expressions of NMDA receptor 1 increased (P<0.05), mRNA expression of KAT Ⅲ decreased (P<0.05), KYN/TRP ratio and KYN/KYNA ratio decreased (all P<0.05) in POFS group on postoperative day 1 and 3. As compared to POFS group, central panel residence time and periphery/central panel ratio decreased on postoperative day 1 , and mRNA and protein expressions of NMDA receptor 1 decreased on postoperative day 1 and 3 (all P<0.05) in antagonist group. TEM revealed that degenerated neuron was found in the hippocampus of POFS rats , while such damage was improved in antagonist group. Conclusion The increased expression level of NMDA receptor may play an important role in POFS. NMDA receptor antagonist MK801 may improve the POFS.

Objective To explore the central mechanism of postoperative fatigue syndrome by detecting the expression of NMDA receptor and tryptophan metabolism. Methods After being numbered according to the weight, ninety-six male SD rats were randomly divided into control group (bowel loop was flipped after laparotomy and received intraperitoneal injection of saline at a dose of 1 ml/kg ), POFS model (70% of the length of small intestine was resected and received intraperitoneal injection of saline at a dose of 1 ml/kg), and NMDA antagonist groups (70% of the length of small intestine was resected and received intraperitoneal injection of MK801 at a dose of 1 ml/kg ). Each group was divided into subgroups by postoperative 1, 3, 5 and 7 d, with 8 rats in each subgroup. The hippocampus was removed at each time point after open field test (OFT) to detect the mRNA expression levels of NMDA receptor 1 and kynurenine aminotransferase Ⅲ (KATⅢ) by real-time PCR. Protein level of NMDA receptor 1 was detected by Western blot. High performance liquid chromatography (HPLC) was used to measure the concentrations of tryptophan (TRP), kynurenine (KYN) and kynurenic acid(KYNA). Ultra-structural changes of hippocampal neurons were observed by transmission electron microscopy (TEM). Results As compared to control group, exercise score decreased (P<0.05), rest time and central panel residence time prolonged, periphery/central panel ratio increased (all P<0.05), mRNA and protein expressions of NMDA receptor 1 increased (P<0.05), mRNA expression of KAT Ⅲ decreased (P<0.05), KYN/TRP ratio and KYN/KYNA ratio decreased (all P<0.05) in POFS group on postoperative day 1 and 3. As compared to POFS group, central panel residence time and periphery/central panel ratio decreased on postoperative day 1 , and mRNA and protein expressions of NMDA receptor 1 decreased on postoperative day 1 and 3 (all P<0.05) in antagonist group. TEM revealed that degenerated neuron was found in the hippocampus of POFS rats , while such damage was improved in antagonist group. Conclusion The increased expression level of NMDA receptor may play an important role in POFS. NMDA receptor antagonist MK801 may improve the POFS.

Objective To assess the condition of left ventricular outflow tract obstruction (LVOTO) under resting conditions and physiological exercise in hypertrophic cardiomyopathy (HCM) patients.Methods A total of 60 patients with HCM and left ventri cular outflow tract gradient (LVOTG) ＜ 50 mm Hg (1 mm Hg =0.133 kPa) at rest were enrolled consecutively,and LVOTG at rest and exercise were measured by echocardiography.Of 51 patients with gradients ＜ 30 mm Hg at rest,26 were latent LVOTO with exercise peak value LVOTG ≥ 30 mm Hg,25 were non LVOTO with exercise peak value LVOTG ＜ 30 mm Hg,and 9 were resting obstruction with LVOTG 30-49 mm Hg.The morphological characteristics of different types of obstruction were analyzed.Results Patients with latent LVOTO were more likely to have SAM(73.1％ vs 8.0％),narrow of LVOT(46.2％ vs 4.0％),higher resting gradients [(16.9 ±7.2) mm Hg vs (7.1 ± 4.3) mm Hg] and mitral regurgitation grade at rest than patients with non-obstructive (all P values ＜ 0.05).The distribution of septal hypertrophy were different in the two groups (P ＜ 0.05).Multivariate logistic regression analysis showed independent predictors of latent LVOTO were SAM (OR 6.431,95 ％ CI 2.323-291.112,P =0.002) at rest and distribution of septal hypertrophy (OR 0.011,95％ CI 0.001-0.179,P =0.008).Conclusions Approximately half of patients with nonobstructive HCM at rest have latent LVOTO.SAM and distribution of septal hypertrophy may be useful to identify patients with latent obstruction.

Objective:To observe the expression of BTLA on T cells during activation and further analyze its inhibitory effects on T cell activation in different phases.Methods: T cells from PBMC were enriched by negative selection using magnetic beads.Expression of BTLA,CTLA-4 and PD-1 on freshly isolated human T cells and kinetics expression of BTLA,CILA-4 and PD-1 on CD3 mAb stimulated T cells were examined by flow cytometry.T cells were stimulated by anti-CD3 mAb combined with anti-CD28 mAb in the presence of anti-BTLA mAb 8H9,then T cell proliferation was tested by MTT assay in the different culture time.Immature DCs were generated from monocytes cultured in the medium containing GM-CSF and IL-4, and further driven to maturation by anti-CD40 mAb.Expression of HVEM on DCs was measured by flow cytometry.T cells were co-cultured with DCs in the presence of soluble 8H9 or anti-HVEM antibody to block HVEM-BTLA interaction,T cell proliferation was measured by MTT assay.Results:Freshly isolated T cells exhibited high levels of BTLA expression, but not CTLA-4 and PD-1.After T cell activation, BTLA expression decreased on first 2 days, with rapidly increasing to high levels.Unlike BTLA, expression of CTLA-4 and PD-1 was gradually increased during T cell activation.8H9 significantly inhibited the proliferation of T cell stimulated by CD3 mAb and CD28 mAb.8H9 could still exhibit inhibitory effect on T cell proliferation after 24 h or 48 h of preactivation by CD3 mAb plus CD28 mAb stimulation.HVEM was highly expressed on immature DCs, and down-regulated on mature DCs.Blockade of BTLA by soluble 8H9 or anti-HVEM antibody enhanced DC-mediated T cell proliferation within 48 h.Conclusion: BTLA signal enhances the threshold of T cell activation and plays importantly negative regulatory role in the initiation and early phase of T cell activation.