Abstract: This study is to improve the affinity of scFv-AK404R against VEGFR2. The secondary mutational library was constructed by hydrophilic shuffling in CDR3 region of the heavy chain. VEGFR2-specific screening was performed by phage display technology and the protein of mutants was expressed in periplasm of E.coli HB2151 and purified by affinity chromatography. The affinity constant of scFvs was measured by competitive ELISA, and the structure of scFvs was analyzed by bioinformatics. The result showed that a library with 6.4x10(5) scFv members was established by electro-transformation. Two mutated clones with high absorbance value were isolated after screening. After purification by affinity chromatography, electrophoretically pure scFv proteins were obtained. The competitive ELISA showed that the affinities of WZ01 and WZ02 were three times higher than that of the parental AK404R, and bioinformatics analysis showed that the enlarged contact surface and fitted closely with KDR3 surface may be the reasons for improved affinity. These results suggest that introducing hydrophilic amino acids to the heavy chain CDR3 region is an effective approach to improve the affinity of scFv.

Abstract: This study is to improve the affinity of scFv-AK404R against VEGFR2. The secondary mutational library was constructed by hydrophilic shuffling in CDR3 region of the heavy chain. VEGFR2-specific screening was performed by phage display technology and the protein of mutants was expressed in periplasm of E.coli HB2151 and purified by affinity chromatography. The affinity constant of scFvs was measured by competitive ELISA, and the structure of scFvs was analyzed by bioinformatics. The result showed that a library with 6.4x10(5) scFv members was established by electro-transformation. Two mutated clones with high absorbance value were isolated after screening. After purification by affinity chromatography, electrophoretically pure scFv proteins were obtained. The competitive ELISA showed that the affinities of WZ01 and WZ02 were three times higher than that of the parental AK404R, and bioinformatics analysis showed that the enlarged contact surface and fitted closely with KDR3 surface may be the reasons for improved affinity. These results suggest that introducing hydrophilic amino acids to the heavy chain CDR3 region is an effective approach to improve the affinity of scFv.
Amino Acid Sequence ; Antibody Affinity ; Chromatography, Affinity ; Computational Biology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; metabolism ; Hydrophobic and Hydrophilic Interactions ; Peptide Library ; Single-Chain Antibodies ; genetics ; immunology ; isolation & purification ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; immunology ; metabolism

Objective To investigate the clinical features, diagnosis, differential diagnosis, treatment, and prognosis of adrenal lymphangioma. Methods Three cases of adrenal lymphangioma were reported, and the clinical features, treatment and prognosis were analyzed. Results Three cases were incidentally discovered, laboratory tests and endocrine hormone examinations were normal, CT or MRI showed lesions with low density, no reinforced or mild enhancement. All 3 cases underwent laparoscopic adrenalectomy, postoperative pathology supported the diagnosis of adrenal lymphangioma. They were followed up for 8-months, 1-year, and 4-years respectively, with no recurrence. Conclusions Adrenal lymphangioma is a rare benign adrenal leison, with no typical clinical manifestations. Preoperative diagnosis depends on imaging examinations. Histopathological examination is essential in making final diagnosis. Surgery is the preferred treatment option. The prognosis is relatively good.

OBJECTIVETo investigate the effects of ulinastatin on lung injury in hemorrhagic shock rats.

METHODSTwenty-four normal SD rats were randomly divided into 3 groups (n=8), namely the control group, hemorrhagic shock group (group H) and ulinastatin group (group U). In group H and group U, blood was drawn from the femoral artery over a period of 10 min until a mean arterial pressure of 40 mmHg was obtained. Controlled hypotension was then maintained at 40-/+5 mmHg for 60 min by blood drawing or infusion when necessary. All the blood drawn and an equivalent volume of Ringer lactate solution were subsequently infused for resuscitation. Four hours after the resuscitation, the activity of superoxidedismutase (SOD), content of malondialdehyde (MDA), expression of heme oxygenase-1 (HO-1), wet to dry weight ratio (W/D), and pathologic changes of the lung tissues were measured or observed.

RESULTSCompared with those in the control group, the content of MDA, expression of HO-1 and W/D increased significantly in both group H and group U (P<0.05); these indexes in group U were significantly lower than those in group H (P<0.05). The activity of SOD in group U was significantly lower than that in the control group (P<0.05) but higher than that in group H (P<0.05). Optical microscopy demonstrated milder inflammatory cell infiltration and interstitial edema in the lung tissues in group U than in group H.

CONCLUSIONUlinastatin can lower the content of MDA, W/D and the expression of HO-1, increase the activity of SOD, and reduce histological lung injury in rats with hemorrhagic shock.

Animals ; Glycoproteins ; pharmacology ; Heme Oxygenase-1 ; metabolism ; Lung Injury ; etiology ; prevention & control ; Male ; Malondialdehyde ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Shock, Hemorrhagic ; complications ; metabolism ; Superoxide Dismutase ; metabolism

BACKGROUNDInoperable chronic thromboembolic pulmonary hypertension (CTEPH) is a severe clinical syndrome characterized by right cardiac failure and possibly subsequent liver dysfunction. However, whether serum markers of liver dysfunction can predict prognosis in inoperable CTEPH patients has not been determined. Our study aimed to evaluate the potential role of liver function markers (such as serum levels of transaminase, bilirubin, and gamma-glutamyl transpeptidase [GGT]) combined with 6-min walk test in the prediction of prognosis in patients with inoperable CTEPH.

METHODSFrom June 2005 to May 2013, 77 consecutive patients with inoperable CTEPH without confounding co-morbidities were recruited for this prospective cohort study. Baseline clinical characteristics and 6-min walk distance (6MWD) results were collected. Serum biomarkers of liver function, including levels of aspartate aminotransferase, alanine aminotransferase, GGT, uric acid, and serum bilirubin, were also determined at enrollment. All-cause mortality was recorded during the follow-up period.

RESULTSDuring the follow-up, 22 patients (29%) died. Cox regression analyses demonstrated that increased serum concentration of total bilirubin (hazard ratio [HR] = 7.755, P < 0.001), elevated N-terminal of the prohormone brain natriuretic peptide (HR = 1.001, P = 0.001), decreased 6MWD (HR = 0.990, P < 0.001), increased central venous pressure (HR = 1.074, P = 0.040), and higher pulmonary vascular resistance (HR = 1.001, P = 0.018) were associated with an increased risk of mortality. Serum concentrations of total bilirubin (HR = 4.755, P = 0.007) and 6MWD (HR = 0.994, P = 0.017) were independent prognostic predictors for CTEPH patients. Patients with hyperbilirubinemia (≥23.7 μmol/L) had markedly worse survival than those with normobilirubinemia.

CONCLUSIONElevated serum bilirubin and decreased 6MWD are potential predictors for poor prognosis in inoperable CTEPH.

Aged ; Antihypertensive Agents ; therapeutic use ; Bilirubin ; blood ; Exercise Test ; Female ; Humans ; Hypertension, Pulmonary ; blood ; drug therapy ; pathology ; Male ; Middle Aged ; Prognosis ; Prospective Studies

Objective To improve the core competency and comprehensive quality of fresh nurse, and lay the foundation for future development. Methods Three stages standardized core competency training were employed for 2006-2007 year new graduate nurses, and compared to traditional training methods applied for 2004-2005 year new graduate nurses. Results The service attitude evaluation, professional knowledge, skills evaluation, and comprehensive quality evaluation scores of 2006-2007 year new graduate nurses were significantly higher than the 2004-2005 year new graduate nurses ( P ＜ 0. 05 ). Conclusions A planned and standardized core competency training program was more effective than traditional training methods, and helpful to promote stability and comprehensive quality of nursing staff.

Objective:To analyze the mechanism of Jindan Tablets, Xiaoyan Lidan Tablets and ursodeoxycholic acid in the treatment of gallstone and cholecystitis based on network pharmacology; To conduct a comparative analysis.Methods:The chemical components of Jindan Tablets, Xiaoyan Lidan Tablets and ursodeoxycholic acid and their drug targets were collected from Traditional Chinese Medicine Database and Analysis Platform (TCMSP). DAVID 6.8 database was used to search for the associated diseases of the drug targets. The disease targets of gallstone and cholecystitis were collected from GeneCards and other databases. The protein-protein interactions network was established based on the intersecting targets of three drugs and two diseases. KEGG enrichment analysis was performed based on the DAVID 6.8 database. Cytoscape 3.7.1 software was used to construct a complex network and topology analysis of component- target- disease between three drugs and diseases.Results:222 chemical components and 3 133 drug targets were collected for Jindan Tablets. 104 chemical components and 1 425 action targets were collected for Xiaoyan Lidan Tablets. 1 chemical component and 119 action targets were collected for ursodeoxycholic acid. The three drugs were associated with 31 diseases. 1 382 disease targets for gallstones and cholecystitis were collected. There were 237, 163 and 33 targets for gallstones and cholecystitis in the three drugs, of which 17 were shared by the three drugs and 20 were shared by Jindan Tablets and Xiaoyan Lidan Tablets. Based on the DAVID database, 113, 74 and 10 significant KEGG enrichment pathways were obtained for the three drugs respectively.Conclusions:The three drugs shared many targets and pathways in the treatment of gallstones and cholecystitis, which all had the function of regulating metabolism and inhibiting inflammatory response, while participating in apoptosis, oxidative stress and cancer pathology process. However, they had their own special effects, with Jindan Tablets favoring involving in the cancer process and inhibition of inflammation, and promoting angiogenesis. Xiaoyan Lidan Tablets and ursodeoxycholic acid focused on regulating cholesterol metabolism, and Xiaoyan Lidan Tablets also regulated steroid metabolism and inhibit inflammation, while ursodeoxycholic acid regulated bile acid metabolism.

Objective:To investigate the prognostic significance of DTA(DNMT3A,TET2,ASXL1)gene mutations in patients with non-M3 acute myeloid leukemia(AML).Methods:The clinical data of 180 newly diagnosed AML patients hospitalized in the First People's Hospital of Changzhou from January 2018 to April 2022 were retrospectively analyzed.Next-generation sequencing technology was used to detect 150 gene mutations in the patients,and log-rank tests and Cox regression models were used to analyze the prognostic factors.Results:DTA gene mutations were detected in 83(46.1％)of 180 AML patients.Compared to patients without DTA mutations,patients with DTA mutations were significantly older(P＜0.001).The median overall survival(OS)time and disease-free survival(DFS)time in the DTA mutation group were significantly shorter than those in the group without DTA mutation(both P＜0.05).Multivariate analysis showed that age ≥ 60 years(P＜0.001),with DTA mutation(P=0.018),and intermediate-risk(relative to favorable-risk)(P=0.005)were independent risk factors for OS in AML patients.Conclusion:AML patients with DTA mutations are relatively older,with shorter median OS time and DFS time,and poor prognosis.

ObjectiveOur recent study has demonstrated that extracellular acidification promotes lipid accumulation in macrophages via the activation of acid sensing ion channel 1 (ASIC1), but the underlying mechanism remains unclear. This study aims to explore the effect of extracellular acidification on macrophage lipophagy and the underlying mechanism. MethodsRAW264.7 macrophages were incubated with 25 mg/Lox-LDL in a pH 6.5 culture medium for 24 h to build macrophage-derived foam cell models induced by extracellular acidification. Then, RAW264.7 macrophages were cultured in the acidic medium of pH 6.5 with or without PcTx-1 (ASIC1 specific blocker, 10 μg/L) or Nec-1 (RIP1 specific inhibitor, 20 μmol/L) for 24 h, intracellular lipid accumulation was observed by oil red O staining. The expressions of total ASIC1, plasma membrane ASIC1, RIP1, p-RIP1 Ser166, TFEB, p-TFEB Ser142, LC3 and p62 were measured by Western blot. The co-localization of lipids (indicated by Bodipy) with LC3II (autophagosomes) and LAMP1 (lysosomes) was analyzed by a confocal laser scanning microscopy, respectively. Morphological changes of lipophagy in the cells were observed by using transmission electron microscopy. ABCA1-mediated cholesterol efflux was determined by cholesterol fluorescence kits. ResultsCompared with pH 7.4+ox-LDL group, the intracellular lipid accumulation in the pH 6.5+ox-LDL group was significantly increased. Meanwhile, the expressions of plasma membrane ASIC1, p-RIP1 Ser166, p-TFEB Ser142, and p62 proteins were elevated significantly, while LC3II protein level and LC3II/LC3I ratio were decreased. Accordingly, compared with pH 7.4+ox-LDL group, the macrophage lipophagy of the pH 6.5+ox-LDL group was inhibited as indicated by the decreased localization of lipid droplets with LC3 and LAMP1, a decrease in the number of lipophagosomes as well as an increase in lipid droplets. Furthermore, ATP binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux from the macrophages of pH 6.5+ox-LDL group reduced dramatically. However, these above effects of extracellular acidification on RAW264.7 macrophages were abolished by PcTx-1 and Nec-1, respectively. ConclusionThese findings suggest extracellular acidification promotes the phosphorylation of TFEB at Ser142 via activating ASIC1/RIP1 pathway, thereby impeding lipophagy in RAW 264.7 macrophages, and that ASIC1 may be a new potential target for preventing aberrant lipid accumulation diseases including atherosclerosis.

This scoping review aimed to summarize the application information and clinical studies of oral Chinese patent medicines. The oral Chinese patent medicines in treating lung cancer were screened out by searching pf the drug directory， related guidelines， and medical information websites. The data including functions， application， ingredients， and prices of these medicines were collected. Six public databases were searched with the time interval of establishment to August 22， 2021 for collection of the clinical studies of oral Chinese patent medicines in the treatment of lung cancer. The expert consensuses， systematic reviews， randomized controlled trials， non-randomized controlled trials， and non-controlled trials were selected for analysis. A total of 104 oral Chinese patent medicines were screened out， including 31 capsules， 16 granules， 20 oral liquids， 17 tablets， 17 pills， and 3 ointments， in which altogether 198 herbal medicines were involved. The single-dose prices of 2， 36， and 66 medicines were > CNY 100， CNY 10-100， and < CNY 10， respectively. There were 410 clinical studies associated with 48 oral Chinese patent medicines， which were published from 1986 to 2021. These publications included 1 expert consensus， 21 systematic reviews， 277 randomized controlled trials， 87 non-randomized controlled trials， and 24 non-controlled trials. In the clinical studies， the Chinese patent medicines were usually applied in combination with radiotherapy and chemotherapy. The evaluation of primary outcomes focused on 9 indicators including clinical efficacy， quality of life， and incidence of side effects. In conclusion， the oral Chinese patent medicines demonstrated significant advantages in the treatment of lung cancer， and the relevant clinical trials were increasing year by year， with multiple outcome indicators being evaluated. More comprehensive and standardized clinical studies need to be designed for oral Chinese patent medicines in treating lung cancer in the future.