Objective To investigate the effects of down-regulation of Netrin-1 expression on cell proliferation,apoptosis and migration of a cutaneous squamous cell carcinoma(CSCC)cell line SCL-1.Methods Unspecific(control)small interference RNA(siRNA)and Netrin-1-targeting siRNA were designed and constructed.SCL-1 cells were divided into 3 groups to remain untreated(control group 1),be transfected with control siRNA(control group 2)and Netrin-l-targeting siRNA(experiment group)by using Lipofectamine 2000 respectively.After 48 hours of additional culture,flow cytometry was performed to detect the apoptosis of cells,Western blot to determine the protein expression of caspase-3 and-9,Netrin-1 and matrix metalloproteinases (MMP)in these cells,and Boyden chamber was utilized to evaluate the migration of cells.Enzyme linked immunosorbent assay(ELISA)was performed with cholecystokinin-8(CCK-8)kit to estimate the proliferation of cells at 24,48,72 and 96 hours after the transfection.Results The expression of Netrin-1 protein was significantly downregulated in SCL-1 cells in the experiment group compared with the control group 1 and 2 (0.207 ± 0.044 vs.0.741 ± 0.111 and 0.716 ± 0.109,F =31.43,P ＜ 0.05).The Netrin-1-targeting siRNA significantly downregulated the proliferation of SCL-1 cells,but induced the apoptosis in SCL-1 cells with significant differences in the percentage of early apoptotic cells and late apoptotic cells between the control group 1,2 and experiment group(11.74 ± 0.33 vs.11.55 ± 1.22 vs.24.74 ± 2.43,F=68.63,P＜ 0.01;3.82 ± 0.32 vs.4.69 ± 0.68 vs.5.33 ± 0.18,F =8.58,P ＜ 0.05).The experiment group showed a significant increase in the expression of caspase-3 and-9(1.458 ± 0.111 vs.0.398 ± 0.057 and 0.412 ± 0.049,F =183.99,P ＜ 0.01;0.967 ± 0.099 vs.0.234 ± 0.036 and 0.205 ± 0.033,F =138.11,P ＜ 0.01),but a statistical decrease in the expression of MMP-2(0.235 ± 0.038 vs.0.862 ± 0.040 and 0.892 ± 0.035,F =291.07,P ＜ 0.01)compared with the control group 1 and 2.Conclusions The down-regulation of Netrin-1 expression can suppress the proliferation and migration of SCL-1 cells,but promote the apoptosis in SCL-1 cells.

Objective To investigate whether mixed venous-arterial carbon dioxide difference (Pv-aCO2) combined with passive leg raising (PLR) could better guide volume management for patients post off-pump coronary artery bypass grafting (OPCABG).Methods Eighty patients experienced OPCABG, and admitted to Tianjin Chest Hospital from June 1st to October 1st in 2016 were enrolled. They were randomly divided into two groups: observation group given Pv-aCO2 combined with PLR directed therapy and control group given central venous pressure (CVP) directed therapy, 40 cases in each group. The difference in body temperature (T), heart rate (HR), mean arterial pressure (MAP), CVP, oxygenation index (PaO2/FiO2), mixed venous oxygen saturation (SvO2), Pv-aCO2, blood lactate (Lac), fluid intake, scores of the vasoactive agents at 6 hours post-operation, sequential organ failure assessment (SOFA) of 24 hours, mechanical ventilation time, the length of intensive care unit (ICU) stay, and hospitalization time were compared. The correlation between Pv-aCO2 and cardiac index (CI), Pv-aCO2 and Lac were analyzed by Spearman analysis in observation group.Results The T, HR, MAP, CVP, PaO2/FiO2, SvO2 and Lac at 6 hours post-operation were higher than those at admission in two groups, and Pv-aCO2 were significantly decreased. The SvO2 and fluid intake in observation group were higher than those in control group [SvO2: 0.671±0.068 vs. 0.634±0.052, fluid intake (mL): 454±151 vs. 304±106, bothP < 0.05], Pv-aCO2, Lac and scores of the vasoactive agents were lower than those in control group [Pv-aCO2 (mmHg, 1 mmHg = 0.133 kPa): 6.1±1.8 vs. 7.0±1.8, Lac (mmol/L): 1.7±0.5 vs. 2.8±0.6, scores of the vasoactive agents: 3.18±1.01 vs. 4.48±1.50, allP < 0.05], mechanical ventilation time and the length of ICU stay were less than those in the control group (hours: 16.52±6.41 vs. 21.96±9.00, 45.51±9.36 vs. 51.76±13.66, bothP< 0.05). There was no significant difference in SOFA, hospitalization time between the two groups. There was negative correlation with Pv-aCO2 and CI (r = -0.752,P < 0.01), and no correlation with Pv-aCO2 and Lac (r = -0.154,P = 0.171).Conclusion Pv-aCO2 combined with PLR can better guide volume management in the patients post OPCABG, reduce the usage of vasoactive agents and decrease the mechanical ventilation time and the length of ICU stay.

Objective To determine the value of oxygenation index in assessing the outcome of mechanical ventilated patients with acute respiratory distress syndrome (ARDS).Methods From September 2008 to September 2013,patients meeting the Berlin definition of ARDS were evaluated in this retrospective study.Data included oxygenation index (PaO2/FiO2) on day before,and day 1 and day 3 after mechanical ventilation.The levels of PaO/FiO2 on day before and day 1 after mechanical ventilation were compared between 28-day survivors and non-survivors.Results There were 236 patients meeting the criteria of the Berlin Definition for diagnosis and treated with mechanical ventilation.The mean score of APACHIE Ⅱ and sequential organ failure assessment (SOFA) at the beginning were (19.1 ± 3.0) and (10.8 ±2.5),respectively,while oxygenation index on day before mechanical ventilation was (150.3 ± 62.6) mmHg.According to the hypoxemia grade,patients were divided into mild (n =36),moderate (n =122) and severe (n =78) ARDS,and their levels of PaO2/FiO2 were (80.1 ± 8.1),(162.3 ± 19.9) and (261.6 ± 22.3) mmHg,respectively.There were 92 non-survivors and 144 survivors.No obvious difference in oxygenation index of non-survivors among on day before、and day 1 and day 3 after mechanical ventilation.There was statistically significant difference in oxygenation index between on day before and day 1 after mechanical ventilation in survivors (P ＜ 0.05).Compared with the survivors,the score of APACHE Ⅱ,SOFA,and oxygenation index on day 3 were associated with increased mortality in the non-survivors,respectively (P ＜ 0.05).In respect to the mortality,the cut-off point of score of oxygenation index set at ＜ 180 mmHg on Day 3,an area under the receiver operating curve (AUC) was 0.749 with statistically significance (P ＜ 0.05),leading to sensitivity 61.7％ and specificity 93.2％.The relationship between prognosis and antibiotic resistance did not have statistically significance.Conclusions Data of oxygenation index on early phage of ARDS may be valuable to predict the outcome.A strong predictor of adverse outcome in such conditions was the score of oxygenation index on Day 3 ≤ 180 mmHg.

ObjectiveTo assess the relationship between the estimated glomerular filtration rate (eGFR) and the severity of coronary artery stenosis in elder people. MethodsSix hundred and fifty-seven patients who underwent selective coronary artery angiography were divided into 3 groups according to age: group Ⅰ (202 patients aged <60 years), group Ⅱ (181 patients aged from 60 to 69 years) and group Ⅲ (274 patients aged ≥70 years). According to the simplified MDRD equation,impaired renal function and severe renal dysfunction, respectively. Clinical features and the severity of coronary artery stenosis were compared among the 3 groups. ResultsThe level of average eGFR tended to he decreased with the narrowed coronary arteries. The difference of average eGFR between patients with muhivessel versus two-vessel coronary artery disease was of statistical significance in Group Ⅲ(P = 0. 035). After controlling for the effects of smoking, hypertension, dyslipidemia,aging, and diabetes, the Gensini score which reflected the severity of coronary artery stenosis was negatively correlated with the level of eGFR (r =-0. 110, P = 0. 005). ConclusionsIt suggests that the eGFR decrease is associated with the severity of coronary artery stenosis, and there is a linear correlation between eGFR and Gensini score in elder people, eGFR can be used to evaluate the severity of coronary artery disease and prognosis.

Objective To explore the analgesic effect of intra-articular botulinum neurotoxin type A (BoNTA) injection in rats with adjuvant-arthritis pain,to quantify the expression of calcitonin gene-related peptide (CGRP) in the dorsal root ganglia (DRG) associated with arthritis pain,and to investigate the retrograde axonal transport of BoNT-A into the DRG after peripheral injection.Methods Ninety Sprague-Dawley rats were randomly divided into groups A,B,C,D and E,each of 18.A murine model of adjuvant-arthritis pain was established by injecting 50 μL of complete Freund's adjuvant into the left ankle in all the mice except those in group A.The control group A was treated with intra-articular injection of 50 μL of saline solution.Three weeks later,groups A and B were treated with a 20 μL intra-articular saline injection,while groups C,D and E received an intra-articular injection of BoNT-A at 1 U/20 μL,3 U/20 μL or 10 U/20 μL respectively.Pain threshold and muscle strength were graded before and 1,5,15 and 21 days after the modelling,as well as at 1,3,5 and 14 days after the BoNT-A treatments.Protein expression and the CGRP-positive cell number were observed,as well as any BoNT-A-cleaved synaptosomal-associated 25 kDa protein (cl-SNAP-25) in the DRG using Western blotting and immunofluorescence.Results Compared with group A,there was a significant decrease in the average mechanical withdrawal threshold and muscle strength and a significant increase in the protein expression and the CGRP-positive cell number in the other 4 groups.Compared with group B,the mechanical withdrawal threshold had increased significantly more in groups D and E at 5 days after the BoNT-A injection and in group C at 14 days after the treatment.Compared with group B,the protein expression and the number of CGRP-positive cells were significantly lower in groups D and E at 3 days after the BoNT-A injection.The decrease in group C was significant after 14 days.No significant differences were found between groups D and E in any measurement at any time point.There was no significant difference among groups B,C and D in terms of muscle strength.Five days after the BoNT-A injection,significantly decreased muscle strength was observed in group E.In addition,BoNT-A cleaved-SNAP-25 was detected in the DRG.Conclusion BoNT-A can reduce arthritis pain through inhibiting the expression of CGRP in the DRG.Its analgesic effect has a dose response.A peripheral injection of BoNT-A can arrive at the DRG through retrograde axonal transport.

OBJECTIVETo clone and characterize the DNA and cDNA sequences of phenylalanine ammonia-lyase gene (PAL) from Fagopyrum dibotrys, and investigate the biological activity of the obtained PAL.

METHODUsing homology cloning and RT-PCR techniques, the DNA and full-length cDNA sequences of PAL gene were amplified from F. dibotrys. The obtained sequences were analyzed by bioinformatics software. The ORF of PAL gene was cloned into expression vector pET-30b(+) and transformed into Escherichia coli BL21 (DE3) for expression the recombined protein. The catalytic activity of the recombined protein was determined by Spectrophotometer and thin layer chromatography (TLC) methods.

RESULTThe DNA sequence of PAL gene (designated as FdPAL, GenBank accession number: HM628904) was 2 583 bp in size, of which consisted two extrons and a single intron, and the full-length cDNA of FdPAL was 2 169 bp in size, which contained an ORF. The deduced protein of FdPAL contained 722 amino acids with calculated molecular weight (MW) of 78.31 kDa and an isoelectric point (pI) of 5.94. The SDS-PAGE results showed that the molecular weight of recombinant FdPAL protein was 75.37 kDa, which is consistent with the predictions. After 4 hours of induction, the enzymatic specific activity of FdPAL reached the summit, up to 4 386 nmol x g(-1) x min(-1). The reaction products were also identified by TLC, using L-Phe and trans-cinnamic acid as the internal standard.

CONCLUSIONThe PAL gene (both DNA sequence and full-length cDNA sequence) was cloned from F. dibotrys, and it has the same classic characters as other PALs in plants. The recombinant FdPAL was efficiently expressed in E. coli and had the activity for catalyzing the conversion from L-phenylalanine to cinnamic acid.

Amino Acid Sequence ; Base Sequence ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Fagopyrum ; classification ; genetics ; Molecular Sequence Data ; Phenylalanine Ammonia-Lyase ; chemistry ; genetics ; metabolism ; Phylogeny ; Protein Conformation ; Recombinant Proteins ; chemistry ; genetics ; metabolism

OBJECTIVE: To determine whether diffusion kurtosis imaging (DKI) is effective in monitoring tumor response to neoadjuvant chemotherapy in patients with osteosarcoma. MATERIALS AND METHODS: Twenty-nine osteosarcoma patients (20 men and 9 women; mean age, 17.6 ± 7.8 years) who had undergone magnetic resonance imaging (MRI) and DKI before and after neoadjuvant chemotherapy were included. Tumor volume, apparent diffusion coefficient (ADC), mean diffusivity (MD), mean kurtosis (MK), and change ratio (ΔX) between pre- and post-treatment were calculated. Based on histologic response, the patients were divided into those with good response (≥ 90% necrosis, n = 12) and those with poor response (< 90% necrosis, n = 17). Several MRI parameters between the groups were compared using Student's t test. The correlation between image indexes and tumor necrosis was determined using Pearson's correlation, and diagnostic performance was compared using receiver operating characteristic curves. RESULTS: In good responders, MDpost, ADCpost, and MKpost values were significantly higher than in poor responders (p < 0.001, p < 0.001, and p = 0.042, respectively). The ΔMD and ΔADC were also significantly higher in good responders than in poor responders (p < 0.001 and p = 0.01, respectively). However, no significant difference was observed in ΔMK (p = 0.092). MDpost and ΔMD showed high correlations with tumor necrosis rate (r = 0.669 and r = 0.622, respectively), and MDpost had higher diagnostic performance than ADCpost (p = 0.037) and MKpost (p = 0.011). Similarly, ΔMD also showed higher diagnostic performance than ΔADC (p = 0.033) and ΔMK (p = 0.037). CONCLUSION: MD is a promising biomarker for monitoring tumor response to preoperative chemotherapy in patients with osteosarcoma.
Bone Neoplasms ; Diffusion ; Drug Therapy ; Female ; Humans ; Magnetic Resonance Imaging ; Male ; Necrosis ; Osteosarcoma ; ROC Curve ; Tumor Burden

Based on the method of regulating qi and resolving toxins， this paper discussed the core pathogenesis of “inflammation-cancer” transformation of ulcerative colitis. It is believed that the disorder of qi movement， endogenous pathogenic factors of “heat， stasis and dampness” are cemented in the large intestine， and the pathogenic factors are too excessive to be solved， which will become toxic after a long time and lead to cancerous changes. Clinical prevention and treatment applies the method of regulating qi and resolving toxins， and the method of regulating qi was proposed as clearing internal qi， regulating blood qi and strengthening spleen qi， so as to clear heat， dissipate blood stasis and dissolve dampness； different methods of regulating qi and resolving toxins were flexibly combined according to the pathogenic characteristics of different stages of toxicity， in order to interrupt the process of “inflammation-cancer” transformation of ulcerative colitis.

Primary intrahepatic stones （PIS） is a refractory disease with a high incidence rate in Southwest China， which greatly affects the life of patients. Metabolites， such as β-glucuronidase produced by chronic biliary tract infection， play an important role in the formation of pigmented stones. In addition to exogenous β-glucuronidase produced by bacteria， endogenous β-glucuronidase produced by intrahepatic bile duct cells also plays an important role in the formation of stones. This article analyzes the research advances in the role of β-glucuronidase in the pathogenesis of PIS， in order to provide a possible method for the prevention and treatment of PIS.

Primary intrahepatic stones (PIS) is a refractory disease with a high incidence rate in southwest China, and some patients still require surgery again or even more times after initial treatment. Many studies in recent years have shown that some specific flora can colonize in the intrahepatic bile duct, leading to chronic infection and inflammation of the biliary system, and these specific types of flora, called "stone-causing flora", can produce metabolites such as β-glucuronidase and play an important role in the formation of pigmented stones. This article analyzes the role of stone-causing flora in the pathogenesis of PIS, so as to provide more treatment options for PIS patients.