ABSTRACT:Objective To construct V-set and immunoglobulin domain containing 4 (Vsig4)nanobodies (Nbs) as specific macrophage probes so as to use them as molecular probes of macrophagocytes.Methods A nanobody phage library was generated by using peripheral blood lymphocytes isolated from an alpaca immunized with recombinant Vsig4 protein.After three rounds of selection against recombinant Vsig4.The Nbs were subjected to sequencing and genome alignment to obtain VHH sequence.Nbs were isolated and tested for Vsig4 specificity in an ELISA using recombinant Vsig4.The affinity capacity of Nbs was verified by the cell line stably expressing Vsig4. Results A nanobody phage library with an estimated 7.27 × 107 clones with 70% insertion was successfully constructed.Totally 1 3 6 Vsig4-positive clones were sequenced and aligned according to different CDR3 sequences. In summary,1 5 Vsig4 nanobodies were obtained and grouped into 3 different CDR3 epitopes.The affinity of representing nanobody and Vsig4 was analyzed via ELISA;Nb1 1 9 showed the highest affinity against both recombinant and native Vsig4.Conclusion We successfully constructed and screened Vsig4 specific nanobody number 1 1 9 with high affinity and specificity.It can help with macrophage detection and in vivo monitoring.

Research collaboration and interdisciplinary integration is anecessary trend of the development of science and technology,and is especially true in biomedical field.This paper describes the interdisciplinary practice of the projects at Peking University Health Science Center in recent years.Problems and the efficiency of the management is also analysed.

Anti-Arrhythmia Agents ; therapeutic use ; Cardiomyopathies ; drug therapy ; etiology ; Child, Preschool ; Female ; Humans ; Moricizine ; therapeutic use ; Tachycardia ; complications ; drug therapy ; Treatment Outcome

Air ; analysis ; Chromatography, Gas ; methods ; Ethylene Oxide ; analysis ; Furans ; analysis ; Methylene Chloride ; analysis ; Workplace

Objective To discuss the effects of pressure control (PC) and positive end-expiratory pressure (PEEP) incremental method lung recruitment maneuver (RM) on haemodynamics in piglets with acute lung injury (ALI) induced by paraquat (PQ) poisoning. Methods The ALI/acute respiratory distress syndrome (ARDS) model was reproduced by intraperitoneal injection of 20% PQ (20 mL) in 10 healthy female piglets, and they were randomly divided into PC lung RM group (RM1 group) and PEEP incremental method lung RM group (RM2 group), with 5 piglets in each group. Heart rate (HR), mean arterial pressure (MAP), and cardiac index (CI) were monitored by pulse-indicated continuous cardiac output (PiCCO) monitoring before model reproduction (baseline), on the time of successfully set up of model and at 5, 15 and 30 minutes after RM. At the same time the arterial partial pressure of oxygen (PaO2) and arterial partial pressure of carbon dioxide (PaCO2) were recorded, and oxygenation index was calculated. Lung tissues were collected before model reproduction, on the time of successfully set up of model, and at 30 minutes after RM respectively, and pulmonary pathology changes were observed after hematoxylin and eosin (HE) staining under light microscopy. Results The HR, MAP, and PaCO2 on the time of successfully set up of model in both groups were increased obviously while CI, PaO2, and oxygenation index were decreased obviously as compared with those at baseline, all of which conformed to the expression of ALI/ARDS. With RM time extended, the HR in both groups was declined while MAP and CI were increased gradually. The HR and MAP at 5 minutes after RM of RM1 group were significantly lower than those of the RM2 group [HR (bpm): 126.8±5.2 vs. 134.0±3.8, MAP (mmHg, 1 mmHg = 0.133 kPa): 98.4±3.3 vs. 102.8±2.6, both P < 0.05]. The CI at 5 minutes and 15 minutes after RM of RM1 group was significantly higher than that of the RM2 group (mL·s-1·m-2: 56.7±5.0 vs. 46.7±6.7, 65.0±5.0 vs. 56.7±5.0, both P < 0.05). PaO2 and oxygenation index at 5 minutes after RM in both groups were significantly higher than those on the time of sucessfully set up of model. The PaO2 and oxygenation index were gradually decreased, and PaCO2 was increased with RM time extended, but no statistically significant differences at all time points were found between the two groups (all P > 0.05). The lung tissue in both groups showed a variety of pathological changes at 30 minutes after RM. The main performances were the loss of alveolar epithelial cells, the further wideness of alveolar interval and the distension of alveolar, and the part breakage of alveolar interval. The wideness of alveolar interval was more significant in RM2 group than that of RM1 group, and alveolar cleft was more common too. Conclusion Both PC and PEEP incremental method lung RM can improve the oxygenation of the piglets with ALI/ARDS induced by PQ, and the PC lung RM has less impact on haemodynamics.

Objective To investigate the relationship between the genetic predisposition to type 2 diabetes mellitus(T2 DM)and mitochondrial DNA base variants in elderly patients.Methods PCR restriction fragment length polymorphism(PCR-RFLP)analysis was used to screen base variants at position 3243 and 3426 of mitochondrial DNA in 186 elderly cases with T2 DM and 170 healthy controls,and DNA sequence was confirmed.Results No carrier of 3243 A→G variant or 3426 A→G variant was found in both groups,however there was 1 case with 3290 T→C variant in diabetic group.Conclusions No significantly association was found between mitochondrial DNA 3243 or 3426 base variants and the predisposition of type 2 diabetes mellitus in elderly patients.

Objective To investigate the relationship between the expression level of platelet membrane glycoprotein?3(GPⅢa,CD61)and the severity of disease in patients with hemorrhagic fever with renal syndrome(HFRS).Methods One hundred and four patients with HFRS and 30 healthy individuals were recruited.The percentage of CD61 positive platelets and the mean fluores- cence intensities(MFI)of platelet membrane glycoprotein?3 were determined by flow cytometry (FCM).The 104 patients studied were divided into three groups based on their expression levels of platelet membrane glycoprotein?3 at oligurie phase.Clinical data and laboratory parameters in different groups were compared and analyzed.Results The expression levels of CD61 in patients with HFRS were significantly higher than those in control group,although no significant difference in the percentage of CD61 positive platelets between patients with HFRS and controls was detected.The MFI of CD61 expression in patients with HFRS at fever phase,oliguric phase and polyuric phase was 19.75?2.57,17.46?1.48 and 15.55?0.60,respectively,which was significantly higher than that in control group(3 20?0.12).The expression level of CD61 in patients with HFRS at oliguric phase was negatively correlated with platelet count and serum albumin(r=-0.637 and-0.695,respec- tively)and positively correlated with white blood cell count,blood urea nitrogen,serum creatinine and alanine aminotransferase(r=0.945,0.904,0.956 and 0.891,respectively).When the patients were compared according to the expression levels of CD61,it was indicated that the higher the expression level of CD61,the higher the incidence of uremia,hypoalbuminemia,abnormal liver func- tion and leukocytosis.Conclusions The expression levels of platelet membrane glycoprotein?3 in patients with HFRS are different in different clinical phases and are significantly correlated with the severity of the disease in the patients.It suggests that the expression levels of platelet?3 integrin are dramatically increased in patients with HFRS,which may be an indicator for the severity of disease and be helpful for monitoring the state of the patients' diseases and evaluating the severity of the disease.

Objective To develop a nylon membrane chip for rapid and systematic detection of the diabetes-associated 45 mutant loci in mitochondrial DNA(mtDNA).Methods The mutant-and wild-type probes were designed for detection of 45 mutant loci in mtDNA with Primer Premier 5.0 and NCBI BLAST softwares and the 90 probes with 8 poly T were immobilized on the Hybond N~+ nylon membranes which were treated with 5?SSC Buffer by UV-crosslinking;Then asymmetric PCR was employed to obtain the target single strand DNA(ssDNA).The PCR products were labeled with biotin after purification.NBT/BCIP was used as substrate that yields a very intense purple signal followed by AP-avidin,and the signals were observed in 24 samples with known sequences to evaluate the chips,each sample was repeatedly measured three times.Results The specific target fragments of 45 loci can be amplified under the same condition with nine sets of primers.The annealing temperatures of the wild-type [(59.01?1.42)℃] and mutant-type [(59.34?1.29)℃ ] probes are so close(t=1.046,P =0.301)that hybridization can be performed at the same temperature.The spots on the membrane chip are distinct,regular and well-distributed.The results of positive-and negative-control are perfect.The signals of negative probes and the background are similar.The results of chip were nearly concordant with that of DNA sequences(?~2=113.132,Kappa value =0.888,P = 0.000)except for T16189C mutant.Conclusions We have successfully developed a nylon membrane chip for rapid and systematic detection of the diabetes-associated 44 mutant loci in mtDNA.It could be used for screening for diabetic patients and high-risk people.

Objective To study correlation between systemic sclerosis(SSc)and plasma D-dimer and to reveal the probably rules of the fibrinolytic systems in SSc.Methods In 2013 January to 2014 January,cellected 32 patients with SSc and 35 with healthy controls to detected level of plasma D-dimer.Logistic and t student test were used for statistical analysis for correlation between SSc and pulmonary artery hypertension.Results When compared to healthy controls (0.28±0.04μg/ml),the level of plasma D-dimer were significantly increased in SSc patient (0.31±0.05μg/ml,t=1.997,P=0.008).After stratifying SSc patients according to disease subset,whereas patients with diffuse subset displayed substantially increased values (0.41±0.06μg/ml,t=2.051,P<0.001).The level of plasma D-dimer was associated with pulmonary artery hyper-tension (OR=4.38,95%CI=2.59~8.91,P=0.008).Conclusion Demonstrated that SSc patients with diffuse subset are characterized by increased plasma D-dimer values,reflecting a potential activation of fibrinolytic cascaded,which might finally predispose these patients to thrombotic complications and pulmonary artery hypertension.

Objective To compare the clinical effect of neoadjuvant chemoradiotherapy (nCRT) and neoadjuvant chemotherapy (nCT) in the treatment of locally advanced esophageal squamous cell carcinoma.Methods The retrospective cohort study was conducted.The clinicopathological data of 156 patients with local advanced esophageal squamous cell carcinoma who were admitted to the Zhongshan Hospital of Fudan University from January 1,2010 to December 31,2015 were collected.Among 156 patients,59 undergoing nCRT were allocated into the nCRT group and 97 undergoing nCT were allocated into the nCT group.Patients in the nCRT group and nCT group respectively received 2 cycles chemotherapy by the TP regimen+40 Gy radiotherapy (2 Gy/d) and 2 cycles chemotherapy by the TP regimen.Patients were evaluated by imaging examinations after 6 weeks neoadjuvant therapy completion,and then underwent abdominal and right chest-left cervico three-incision thoracoscopic surgery.Observation indicators:(1) treatment situations;(2) postoperative pathological examination;(3) follow-up and survival situations.Follow-up using outpatient examination and telephone interview was performed once every 3 months within 2 years and once every 6 months after 3 years up to January 2017.Follow-up included levels of tumor markers [carcinoembryonic antigen (CEA) and SCC-Ag],thoracic or abdominal computed tomography (CT),neck and abdominal ultrasonography and gastroscopy or PET/CT examination if necessary.Measurement data with normal distribution were represented as (x)±s and comparison between groups was analyzed using the t test.Measurement data with skewed distribution were described as M (range) and comparison between groups was analyzed using the nonparametric test.Count data were analyzed using the chi-square test or Fisher exact probability.Comparison of ordinal data was done by the nonparametric test.The survival rate was calculated using the life table method and survival was analyzed by the Log-rank test.Results (1) Treatment situations:all the patients in the 2 groups were able to burden neoadjuvant therapy and thoracic esophagectomy.Six patients in the nCRT group and 15 in the nCT group had conversion to open surgery.Operation time,volume of intraoperative blood loss,cases with postoperative readmission of ICU,cases with complications,cases with perioperative death and duration of hospital stay were (201 ± 25) minutes,(137± 66)mL,5,24 (10 with pulmonary complications,8 with anastomotic leakage,3 with hoarseness,2 with cardiovascular complications and 1 with chylopleura),0,12 days (range,9-93 days) in the nCRT group and (195±20) minutes,(133±58) mL,8,30 (11 with anastomotic leakage,10 with pulmonmy complications,4 with hoarseness,2 with cardiovascular complications,1 with postoperative hemorrhage,1 with delayed gastric emptying and 1 with chylopleura),1,11 days (range,9-78 days) in the nCT group,respectively,with no statistically significant difference between the 2 groups (x2 =0.883,t =0.102,0.692,x2 =0.048,1.541,Z =0.225,P＞ 0.05).(2) Postoperative pathological examination:R0 resection rate was 96.6％ in the nCRT group and 93.8％ in the nCT group,with no statistically significant difference between the 2 groups (x2 =0.589,P＞0.05).Results of postoperative pathological examination showed that G0,G1,G2 and G3 of tumor regression grade were respectively detected in 18,16,7,18 patients in the nCRT group and 4,5,4,84 patients in the nCT group,with a statistically significant difference between the 2 groups (Z=-7.151,P＜0.05).Stage 0,Ⅰ,Ⅱ,ⅢA,Ⅲ B and ⅣA of postoperative ypTNM stage were respectively detected in 16,9,23,4,6,1 patients in the nCRT group and 4,9,37,6,34,7 in the nCT group,with a statistically significant difference between the 2 groups (Z=-4.890,P＜0.05).The down-staging was detected in 48 patients of the nCRT group and 50 patients of the nCT group,with a statistically significant difference between the 2 groups (x2=13.957,P＜0.05).(3) Follow-up and survival situations:of 156 patients,153 were followed up for 12-82 months,with a median time of 36 months.The 1-,3-,5-year overall survival rates were 88.1％,61.4％,34.9％ in the nCRT group and 81.4％,43.8％,23.1％ in the nCT group,with a statistically significant difference between the 2 groups (x2=4.336,P＜0.05).Conclusion The nCRT in the treatment of locally advanced esophageal squamous cell carcinoma can enhance postoperative pathological response rate,down-staging rate and overall survival rate compared with nCT,without increasing incidence of perioperative complications.